KR20220093719A - Method for preparing soybean extract with bone metabilic function improvement - Google Patents
Method for preparing soybean extract with bone metabilic function improvement Download PDFInfo
- Publication number
- KR20220093719A KR20220093719A KR1020200184735A KR20200184735A KR20220093719A KR 20220093719 A KR20220093719 A KR 20220093719A KR 1020200184735 A KR1020200184735 A KR 1020200184735A KR 20200184735 A KR20200184735 A KR 20200184735A KR 20220093719 A KR20220093719 A KR 20220093719A
- Authority
- KR
- South Korea
- Prior art keywords
- soybean extract
- hours
- cells
- weight
- preparing
- Prior art date
Links
- 235000020712 soy bean extract Nutrition 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000006872 improvement Effects 0.000 title claims description 4
- 210000000988 bone and bone Anatomy 0.000 title abstract description 8
- 230000000694 effects Effects 0.000 claims abstract description 47
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 35
- 244000068988 Glycine max Species 0.000 claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- 239000007864 aqueous solution Substances 0.000 claims abstract description 12
- 108091005804 Peptidases Proteins 0.000 claims abstract description 10
- 239000004480 active ingredient Substances 0.000 claims abstract description 10
- 102000035195 Peptidases Human genes 0.000 claims abstract description 9
- 235000013305 food Nutrition 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 60
- 210000002997 osteoclast Anatomy 0.000 claims description 24
- 210000000963 osteoblast Anatomy 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 239000004365 Protease Substances 0.000 claims description 11
- 239000008213 purified water Substances 0.000 claims description 11
- 240000006439 Aspergillus oryzae Species 0.000 claims description 10
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims description 10
- 230000004097 bone metabolism Effects 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 241000122821 Aspergillus kawachii Species 0.000 claims description 7
- 230000001582 osteoblastic effect Effects 0.000 claims description 7
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 7
- 230000004151 fermentation Effects 0.000 abstract description 7
- 150000001720 carbohydrates Chemical class 0.000 abstract description 2
- 230000007102 metabolic function Effects 0.000 abstract 2
- 244000005700 microbiome Species 0.000 abstract 1
- 230000004069 differentiation Effects 0.000 description 29
- 230000001965 increasing effect Effects 0.000 description 24
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 description 17
- 235000008696 isoflavones Nutrition 0.000 description 17
- 239000006143 cell culture medium Substances 0.000 description 16
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 15
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 15
- 102000008186 Collagen Human genes 0.000 description 15
- 108010035532 Collagen Proteins 0.000 description 15
- 229920001436 collagen Polymers 0.000 description 15
- 150000002515 isoflavone derivatives Chemical class 0.000 description 13
- 150000008442 polyphenolic compounds Chemical class 0.000 description 12
- 235000013824 polyphenols Nutrition 0.000 description 12
- 102000004067 Osteocalcin Human genes 0.000 description 11
- 108090000573 Osteocalcin Proteins 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 11
- 102000007469 Actins Human genes 0.000 description 10
- 108010085238 Actins Proteins 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- ZQSIJRDFPHDXIC-UHFFFAOYSA-N daidzein Chemical compound C1=CC(O)=CC=C1C1=COC2=CC(O)=CC=C2C1=O ZQSIJRDFPHDXIC-UHFFFAOYSA-N 0.000 description 8
- 230000004072 osteoblast differentiation Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 7
- 108010004032 Bromelains Proteins 0.000 description 7
- 102000005158 Subtilisins Human genes 0.000 description 7
- 108010056079 Subtilisins Proteins 0.000 description 7
- 229960005309 estradiol Drugs 0.000 description 7
- 229930182833 estradiol Natural products 0.000 description 7
- 229930182470 glycoside Natural products 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 235000019750 Crude protein Nutrition 0.000 description 6
- 235000019835 bromelain Nutrition 0.000 description 6
- 150000002338 glycosides Chemical class 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 231100000002 MTT assay Toxicity 0.000 description 5
- 238000000134 MTT assay Methods 0.000 description 5
- 230000033558 biomineral tissue development Effects 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 4
- 235000007240 daidzein Nutrition 0.000 description 4
- 230000008021 deposition Effects 0.000 description 4
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 4
- 235000006539 genistein Nutrition 0.000 description 4
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 description 4
- 229940045109 genistein Drugs 0.000 description 4
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 239000007758 minimum essential medium Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 235000019419 proteases Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000007363 ring formation reaction Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 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 4
- 230000035899 viability Effects 0.000 description 4
- 208000006386 Bone Resorption Diseases 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003149 assay kit Methods 0.000 description 3
- 230000024279 bone resorption Effects 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 description 3
- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- 230000011164 ossification Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- BVPWJMCABCPUQY-UHFFFAOYSA-N 4-amino-5-chloro-2-methoxy-N-[1-(phenylmethyl)-4-piperidinyl]benzamide Chemical compound COC1=CC(N)=C(Cl)C=C1C(=O)NC1CCN(CC=2C=CC=CC=2)CC1 BVPWJMCABCPUQY-UHFFFAOYSA-N 0.000 description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 230000037180 bone health Effects 0.000 description 2
- 230000002308 calcification Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 229940074391 gallic acid Drugs 0.000 description 2
- 235000004515 gallic acid Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- -1 sapomin Chemical compound 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 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
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 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 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 101000883515 Homo sapiens Chitinase-3-like protein 1 Proteins 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
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108010009711 Phalloidine Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 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
- 102000014128 RANK Ligand Human genes 0.000 description 1
- 108010025832 RANK Ligand Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- HFVAFDPGUJEFBQ-UHFFFAOYSA-M alizarin red S Chemical compound [Na+].O=C1C2=CC=CC=C2C(=O)C2=C1C=C(S([O-])(=O)=O)C(O)=C2O HFVAFDPGUJEFBQ-UHFFFAOYSA-M 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000013527 bean curd Nutrition 0.000 description 1
- 239000003150 biochemical marker Substances 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 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 1
- 244000013123 dwarf bean Species 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 102000054350 human CHI3L1 Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 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
- 210000003041 ligament Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- LWJKSVUJZBEXCH-UHFFFAOYSA-N n-[5-[2-chloro-5-(trifluoromethyl)phenyl]pyrazin-2-yl]-2,6-difluorobenzamide Chemical compound FC1=CC=CC(F)=C1C(=O)NC1=CN=C(C=2C(=CC=C(C=2)C(F)(F)F)Cl)C=N1 LWJKSVUJZBEXCH-UHFFFAOYSA-N 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 230000024121 nodulation Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 235000002949 phytic acid Nutrition 0.000 description 1
- 229940068041 phytic acid Drugs 0.000 description 1
- 239000000467 phytic acid Substances 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000013424 sirius red staining Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000003687 soy isoflavones Nutrition 0.000 description 1
- 235000013322 soy milk Nutrition 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; 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
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/05—Mashed or comminuted pulses or legumes; Products made therefrom
- A23L11/07—Soya beans, e.g. oil-extracted soya bean flakes
-
- 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
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/08—Denaturation, e.g. denaturation of protein
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/14—Extraction
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Botany (AREA)
- Agronomy & Crop Science (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Medicines Containing Plant Substances (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
본 발명은 골 대사 기능 개선 효과를 갖는 대두 추출물의 제조방법 및 상기 대두 추출물을 유효성분을 포함하는 골대사 기능 개선용 식품 조성물에 관한 것이다. The present invention relates to a method for producing a soybean extract having an effect of improving bone metabolism, and to a food composition for improving bone metabolism comprising the soybean extract as an active ingredient.
콩(Glycine max)은 아시아, 아프리카, 오스트레리아 등에 널리 분포하는 한해살이 풀로서, 식용작물이다. 콩은 여러 품종이 있는데, 용도에 따라 성숙자실용, 청예사료용, 녹비용 등으로 구분한다. 대두는 두부, 간장, 된장과 같은 전통식품의 원료로 사용되어 왔다. 대두는 35~40%의 단백질, 15~20%의 지방 및 20~25%의 당을 포함한다. 대두에는 필수아미노산과 불포화지방산이 포함되어 있으며, 식이섬유, 레시틴, 사포민, 피틴산, 이소플라본과 같은 생리활성물질이 함유되어 있다. Soybean ( Glycine max ) is an annual grass widely distributed in Asia, Africa, and Australia, and is an edible crop. There are several varieties of soybeans, and depending on the use, they are divided into mature fruiting use, cheongye feed, and green bean. Soybeans have been used as a raw material for traditional foods such as tofu, soy sauce, and soybean paste. Soybeans contain 35-40% protein, 15-20% fat and 20-25% sugar. Soybeans contain essential amino acids and unsaturated fatty acids, and contain physiologically active substances such as dietary fiber, lecithin, sapomin, phytic acid, and isoflavones.
대두 이소플라본은 배당체 형태로 존재하는데, 위산과 장내 미생물에 의해 유리 상태의 게니스테인(genistein), 다이드제인(daidzein) 등의 대사산물로 전환되어 장에서 흡수된다. 따라서, 발효식품을 섭취하는 것이 이소플라본을 더 효과적으로 섭취하는 것이라 할 수 있다. Soy isoflavones exist in the form of glycosides, which are converted into metabolites such as free genistein and daidzein by gastric acid and intestinal microbes and absorbed in the intestine. Therefore, it can be said that eating fermented foods is a more effective intake of isoflavones.
이소플라본의 체내 흡수를 용이하게 하기 위해 비배당체 이소플라본을 대두에서 추출하는 방법에 대한 다수의 연구가 공개되어 있다. Numerous studies have been published on how to extract non-glycolysate isoflavones from soybeans to facilitate the absorption of isoflavones in the body.
한국등록특허 제10-1302376호에서는 대두를 원적외선을 처리하여 이소플라본 함량이 증가된 대두분말의 제조방법을 개시한다.Korean Patent No. 10-1302376 discloses a method for producing soybean powder with an increased content of isoflavones by treating soybeans with far-infrared rays.
한국등록특허 제10-188152호에서는 비배당체 이소플라본이 강화된 전두유를 제조하고, 고속원심분리, 에탄올추출, 필터여과, 농축, 혼합 및 동결 건조 단계를 거처 대두 이소플라본 제제를 제조하는 방법을 개시한다. Korean Patent No. 10-188152 discloses a method for preparing soybean isoflavone preparations by preparing whole soymilk fortified with non-glycoside isoflavones, high-speed centrifugation, ethanol extraction, filter filtration, concentration, mixing, and freeze-drying steps. start
본 발명은 총 폴리페놀 함량이 증가되고, 게니스테인(genistein), 다이드제인(daidzein), 이소플라본(비배당체(aglycone) 및 비당체(glycoside) 포함) 함량이 증가되고, 골 대사 증진 효과 및 파골세포 활성 억제 효과를 갖는 대두 추출물을 제조하는 방법을 제공하고자 한다. 또한, 총 폴리페놀 함량과 게니스테인(genistein), 다이드제인(daidzein) 등의 유효성분 함량이 증가된 대두 추출물을 유효성분으로 포함하는 골대사 기능 개선용 식품 조성물을 제공하고자 한다. The present invention increases the total polyphenol content, genistein, daidzein, isoflavone (including aglycone and glycoside) content increases, bone metabolism enhancing effect and An object of the present invention is to provide a method for preparing a soybean extract having an osteoclast activity inhibitory effect. In addition, an object of the present invention is to provide a food composition for improving bone metabolism function, comprising as an active ingredient a soybean extract having an increased content of total polyphenols and active ingredients such as genistein and daidzein.
상기한 과제는, 1~2일간 정제수에 침지한 대두를 100℃에서 10~30분간 가열한 후 냉각하는 단계; 냉각된 대두를 분쇄하고, 고형분 함량이 5~10 중량%인 수용액을 제조하는 단계; 상기 수용액에 고형분 중량 대비 1 내지 10중량%의 탄수화물 분해효소를 넣고 1 내지 3시간 동안 처리하여 1차 가수분해물을 제조하는 단계; 상기 1차 가수분해물에 고형분 중량 대비 1 내지 10중량%의 단백질 분해효소를 넣고 5 내지 7시간 동안 처리하여 2차 가수분해물을 제조하는 단계: 및 상기 2차 가수분해물에 아스퍼질러스 오리자에(Aspergillus oryzae) 또는 아스퍼질러스 카와키(Aspergillus kawachii)를 접종하여 발효시키는 단계를 포함하는, 대두 추출물의 제조 방법에 의해 달성된다. The above-described task comprises the steps of heating soybeans immersed in purified water for 1 to 2 days at 100° C. for 10 to 30 minutes and then cooling; pulverizing the cooled soybeans and preparing an aqueous solution having a solid content of 5 to 10% by weight; preparing a primary hydrolyzate by adding 1 to 10% by weight of a carbohydrate-degrading enzyme to the aqueous solution based on the solids weight and treating it for 1 to 3 hours; Preparing a secondary hydrolyzate by adding 1 to 10% by weight of a protease to the primary hydrolyzate based on the solids weight and treating for 5 to 7 hours: and Aspergillus oryzae ( Aspergillus oryzae ) or Aspergillus kawachii ( Aspergillus kawachii ) is achieved by a method for producing a soybean extract, comprising the step of inoculation and fermentation.
바람직하게는, 상기 탄수화물 분해효소는 AMG, 테르마밀, 덱스트로자임, 비스코자임 및 셀룰클라스트로 이루어진 군에서 선택된 1종 이상일 수 있다.Preferably, the carbohydrate-degrading enzyme may be at least one selected from the group consisting of AMG, thermamyl, dextrozyme, biscozyme, and cellulose clast.
또한 바람직하게는, 상기 단백질 분해효소는 알카라아제, 플라보르자임 및 프로타멕스로 이루어진 군에서 선택된 1종 이상일 수 있다.Also preferably, the protease may be at least one selected from the group consisting of alkalase, flavorzyme and protamex.
또한 본 발명의 과제는, 상기 방법으로 제조된 대두 추출물을 유효성분으로 포함하고, 조골 세포인 MC3T3-E1 세포의 조골 활성을 촉진하고, 파골 세포 활성을 억제하는, 골대사 기능 개선용 식품 조성물에 의해 달성된다.Another object of the present invention is to provide a food composition for improving bone metabolism, comprising the soybean extract prepared by the above method as an active ingredient, promoting osteoblast activity of MC3T3-E1 cells, which are osteoblasts, and inhibiting osteoclast activity. is achieved
본 발명에 따른 대두 추출물의 제조방법에 따르면, 총 폴리페놀 함량 및 게니스테인(genistein), 다이드제인(daidzein) 등의 유효성분 함량이 증가되고, 골 대사 기능 증진 효과를 갖는 대두 추출물을 제조할 수 있다. According to the method for producing a soybean extract according to the present invention, the total polyphenol content and the content of active ingredients such as genistein and daidzein are increased, and a soybean extract having an effect of enhancing bone metabolism can be prepared. can
도 1은 대두의 복합효소처리에 따른 총 폴리페놀 및 조단백질 함량을 측정한 결과 그래프이다.
도 2는 대두 발효물의 총 폴리페놀 함량을 측정한 결과 그래프이다.
도 3은 대두 발효물의 이소플라본(비배당체)의 함량을 측정한 결과 그래프이다.
도 4는 본 발명에 따른 대두추출물 (SBE)이 파골세포의 actin ring 형성에 미치는 영향을 보여주는 사진이다.1 is a graph showing the results of measuring total polyphenol and crude protein content according to complex enzyme treatment of soybeans.
2 is a graph showing the result of measuring the total polyphenol content of fermented soybeans.
3 is a graph showing the result of measuring the content of isoflavones (non-glycosides) in fermented soybeans.
4 is a photograph showing the effect of the soybean extract (SBE) according to the present invention on the actin ring formation of osteoclasts.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 하기의 정의를 가지며 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미에 부합된다. 또한, 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다.All technical terms used in the present invention, unless otherwise defined, have the following definitions and are consistent with the meanings commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention.
용어 "약"이라는 것은 참조 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이에 대해 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 또는 1% 정도로 변하는 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이를 의미한다.The term "about" means 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varying by 4, 3, 2 or 1%.
본 명세서를 통해, 문맥에서 달리 필요하지 않으면, "포함하다" 및 "포함하는"이란 말은 제시된 단계 또는 구성요소, 또는 단계 또는 구성요소들의 군을 포함하나, 임의의 다른 단계 또는 구성요소, 또는 단계 또는 구성요소들의 군이 배제되지는 않음을 내포하는 것으로 이해하여야 한다.Throughout this specification, unless the context requires otherwise, the terms "comprises" and "comprising" include, but are not limited to, a given step or element, or group of steps or elements, but any other step or element, or It should be understood to imply that a step or group of elements is not excluded.
본 발명은 총 폴라보노이드 및 이소플라본 함량이 증가되고 골대사 기능 개선 효과를 갖는 대두 추출물의 제조방법에 관한 것이다. 상기 방법은, 정제수에 일정 시간동안 침지한 대두를 가열한 후 냉각하는 단계(S11); 냉각된 대두를 분쇄하고, 고형분 함량이 5~10 중량%인 수용액을 제조하는 단계(12); 상기 수용액에 고형분 중량 대비 1 내지 10중량%의 탄수화물 분해효소를 넣고 처리하여 1차 가수분해물을 제조하는 단계(S13); 상기 1차 가수분해물에 고형분 중량 대비 1 내지 10중량%의 단백질 분해효소를 넣고 처리하여 2차 가수분해물을 제조하는 단계(S14): 및 상기 2차 가수분해물에 아스퍼질러스 오리자에(Aspergillus oryzae) 또는 아스퍼질러스 카와키(Aspergillus kawachii)를 접종하여 발효시키는 단계(S15)를 포함한다.The present invention relates to a method for producing a soybean extract having an increased content of total flavonoids and isoflavones and an effect of improving bone metabolism. The method comprises the steps of heating and cooling the soybeans immersed in purified water for a certain time (S11); pulverizing the cooled soybeans and preparing an aqueous solution having a solid content of 5 to 10% by weight (12); preparing a primary hydrolyzate by adding 1 to 10% by weight of a carbohydrate-degrading enzyme to the aqueous solution based on the solids weight (S13); Preparing a secondary hydrolyzate by adding 1 to 10% by weight of a protease to the primary hydrolyzate based on the solids weight (S14): and Aspergillus oryzae in the secondary hydrolyzate ) or Aspergillus kawachii ( Aspergillus kawachii ) and inoculated and fermented (S15).
이하 각 단계를 구체적으로 설명한다. Hereinafter, each step will be described in detail.
먼저, 정제수에 일정 시간동안 침지한 대두를 가열한 후 냉각한다(S11). 구체적으로서, 세척한 대두를 1~2일간 정제수에 침지한 후 100℃에서 10~30분간 가열한 후 냉각한다. 이때 정제수는 대두 중량의 10~100배를 사용할 수 있으며, 보다 바람직하게는 100배 중량의 정제수를 사용할 수 있다. First, the soybeans immersed in purified water for a certain time are heated and then cooled (S11). Specifically, the washed soybeans are immersed in purified water for 1 to 2 days, heated at 100° C. for 10 to 30 minutes, and then cooled. In this case, purified water may be used in an amount of 10 to 100 times the weight of soybeans, and more preferably purified water of 100 times the weight of soybeans may be used.
이어서, 냉각된 대두를 고속 믹서 등을 이용하여 분쇄한 다음, 고형분 함량이 5~10 중량%이 되도록 정제수를 추가하여 대두 분쇄 수용액을 제조한다(S12). Then, the cooled soybeans are pulverized using a high-speed mixer or the like, and purified water is added so that the solid content is 5 to 10% by weight to prepare an aqueous soybean pulverization solution (S12).
상기 수용액에 고형분 중량 대비 1 내지 10중량%의 탄수화물 분해효소를 넣고 1 내지 3시간 동안 처리하여 1차 가수분해물을 제조한다(S13). 1 to 10% by weight of a carbohydrate-degrading enzyme is added to the aqueous solution based on the weight of the solid content and treated for 1 to 3 hours to prepare a primary hydrolyzate (S13).
상기 탄수화물 분해효소는 노보자임사의 AMG, 테르마밀, 덱스트로자임, 비스코자임 및 셀룰클라스트로 이루어진 군에서 선택된 1종 이상일 수 있다. 보다 바람직하게는 AMG일 수 있다. 탄수화물 분해효소의 처리 시간은 1~3시간이 바람직하며, 보다 바람직하게는 2시간일 수 있다. The carbohydrate-degrading enzyme may be at least one selected from the group consisting of AMG, thermamyl, dextrozyme, biscozyme, and cellulose clast manufactured by Novozyme. More preferably, it may be AMG. The treatment time of the carbohydrate-degrading enzyme is preferably 1 to 3 hours, and more preferably 2 hours.
이어서, 상기 1차 가수분해물에 고형분 중량 대비 1 내지 10중량%의 단백질 분해효소를 넣고 5 내지 7시간 동안 처리하여 2차 가수분해물을 제조한다(S14), Then, 1 to 10% by weight of a protease is added to the primary hydrolyzate based on the solids weight and treated for 5 to 7 hours to prepare a secondary hydrolyzate (S14),
상기 단백질 분해효소는 알카라아제, 플라보르자임 및 프로타멕스로 이루어진 군에서 선택된 1종 이상일 수 있다. 보다 바람직하게는 알카라아제일 수 있다. 단백질 분효소의 처리 시간은 5~7시간이 바람직하며, 보다 바람직하게는 6시간일 수 있다. 2차 가수분해물은 발효 공정 전에 100℃에서 20분간 처리하여 효소를 실활시킬 수 있다.The proteolytic enzyme may be at least one selected from the group consisting of alkalase, flavorzyme, and protamex. More preferably, it may be an alkalase. The treatment time of the proteinase is preferably 5 to 7 hours, more preferably 6 hours. The secondary hydrolyzate can be treated at 100° C. for 20 minutes before the fermentation process to inactivate the enzyme.
이어서, 상기 2차 가수분해물에 아스퍼질러스 오리자에(Aspergillus oryzae) 또는 아스퍼질러스 카와키(Aspergillus kawachii)를 접종하여 발효시킨다(S15). 보다 바람직하게는 아스퍼질러스 오리자에(Aspergillus oryzae)를 사용할 수 있으며, 25~35℃에서 12시간 내지 24시간 동안 발효시킬 수 있다. 발효가 완료되면, 100℃에서 20분간 살균 처리하고, 여과하고 농축 및 동결 건조 과정을 거칠 수 있다. Then, the secondary hydrolyzate is inoculated with Aspergillus oryzae or Aspergillus kawachii and fermented (S15). More preferably, Aspergillus oryzae ( Aspergillus oryzae ) may be used, and may be fermented at 25-35° C. for 12 hours to 24 hours. When fermentation is complete, it can be sterilized at 100° C. for 20 minutes, filtered, and subjected to concentration and freeze-drying processes.
또한, 본 발명은 상기 방법으로 제조된 대두 추출물을 유효성분으로 포함하고, 조골 세포인 MC3T3-E1 세포의 조골 활성을 촉진하고, 파골 세포 활성을 억제하는, 골대사 기능 개선용 식품 조성물을 제공한다. In addition, the present invention provides a food composition for improving bone metabolism, comprising the soybean extract prepared by the above method as an active ingredient, promoting osteoblast activity of MC3T3-E1 osteoblasts, and inhibiting osteoclast activity.
이하에서 실시예를 들어서 본 발명을 상세하게 설명하지만, 이들 실시예에 의해 본 발명의 권리범위가 제한되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited by these Examples.
실시예 1: 대두의 효소처리물의 제조 및 유효성분 분석 Example 1 : Preparation of soybean enzyme-treated product and analysis of active ingredients
대두를 1%(w/w) 상태로 정제수에 23시간 침지한 후 이를 분쇄한 수용액을 100℃에서 30분간 가열(살균)하여 실온까지 냉각시켜 대조구 및 시료로 사용하였다. 사용된 효소는 노조자임사의 AMG, 셀룰클라스트(Cellulclast), 덱스트로자임(Dextrozyme), 테르마밀(Termamyll), 비스코자임(Viscozyme), 알카라아제(Alcalase). 브로멜라인(Bromelain)이다. 효소 처리 조건은 아래 표 1과 같다.Soybeans were immersed in purified water in a 1% (w/w) state for 23 hours, and then an aqueous solution of the pulverized solution was heated (sterilized) at 100° C. for 30 minutes, cooled to room temperature, and used as a control and sample. The enzymes used were Nozozyme's AMG, Cellulclast, Dextrozyme, Termamyll, Viscozyme, Alcalase. It is Bromelain. Enzyme treatment conditions are shown in Table 1 below.
1%enzyme star
One%
추가처리
+:
복합처리▶:
further processing
+:
Complex processing
알카라아제+브로멜라인Cellullast ▶
Alcalase + Bromelain
알카라아제+브로멜라인Biscozyme▶
Alcalase + Bromelain
알카라아제+브로멜라인Dextrozyme▶
Alcalase + Bromelain
냉각된 시료를 효소의 최적의 pH로 조정한 후, 원료 중량 대비 0.5 중량%의 효소를 첨가한 후 진탕배양기에서 가수분해를 진행하였다. 각 처리구의 총 폴리페놀, 조단백질 함량을 분석하였다. After the cooled sample was adjusted to the optimum pH of the enzyme, 0.5% by weight of the enzyme was added based on the weight of the raw material, and then hydrolysis was performed in a shaker incubator. Total polyphenol and crude protein content of each treatment group were analyzed.
총 폴리페놀 함량은 각 효소량 및 시간별로 제조된 수용액 20uL와 7.5% 탄산나트륨 용액 80uL 혼합 후, Folin-ciocalteu's phenol 용액 100uL을 넣어 30분간 반응시킨 후 725nm에서 흡광도를 측정하였다. 갈산(Gallic acid)을 표준용액으로 사용하여, 시료 중 갈산 함량으로 결과를 나타냈다.The total polyphenol content was measured by mixing 20uL of the prepared aqueous solution and 80uL of 7.5% sodium carbonate solution for each enzyme amount and time, 100uL of Folin-ciocalteu's phenol solution was added, reacted for 30 minutes, and absorbance was measured at 725nm. Gallic acid was used as a standard solution, and the result was shown as the gallic acid content in the sample.
조단백질 함량은 시료액 1g에 분해촉진제로 황산칼륨 2g, 진한 황산 12mL을 넣고 킬달 분해장치에서 시료액이 연한 미색에서 무색이 될 때까지 탄화하고, 탄화물을 단백질 증류 정량기기를 활용하여 증류, 중화 및 적가하여 조단백질 함량을 측정하였다. 분석결과를 도 1에 나타냈다.To determine the crude protein content, add 2 g of potassium sulfate and 12 mL of concentrated sulfuric acid as a decomposition accelerator to 1 g of the sample solution, carbonize the sample solution in a Kjeldahl decomposition device from light off-white to colorless, and distill, neutralize and It was added dropwise to measure the crude protein content. The analysis results are shown in FIG. 1 .
도 1을 보면, AMG로 처리한 후 알카라아제와 브로멜라인으로 처리한 처리구에서 총 폴리페놀과 조단백질 함량이 가장 높았다. 이소플라본은 단백질의 일종이므로 조단백질 함량이 높을수록 이소플라본의 함량이 높을 수 있으며, 항산화 물질의 지표가 될 수 있는 총 폴리페놀 함량의 결과로 AMG가 적합하다고 판단하였다. Referring to Figure 1, after treatment with AMG, the total polyphenol and crude protein content was the highest in the treatment group treated with alkalinease and bromelain. Since isoflavones are a type of protein, the higher the crude protein content, the higher the content of isoflavones. As a result of the total polyphenol content, which can be an indicator of antioxidant substances, AMG was judged to be suitable.
반복된 상기 실험의 결과로 최종적으로는 알카라아제(Alcalase)와 AMG가 가격 및 효율성에서 가장 적합한 효소라고 판단하였다. As a result of the repeated experiment, it was finally determined that Alcalase and AMG were the most suitable enzymes in terms of price and efficiency.
실시예 2: 대두 발효 추출물의 제조 및 유효성분 분석 Example 2 : Preparation of fermented soybean extract and analysis of active ingredients
실시예 1에서 확립된 효소 2종을 활용하여 제조된 효소가수분해물의 10%(W/W) 수용액으로 만들어 100℃, 20분간 살균, 냉각 후 대조구 및 시료로 사용하였다. 냉각 시료에 아스퍼질러스 카와키(Aspergillus kawachii), 아스퍼질러스 오리자에(Asp. oryzae)를 접종하여 12시간, 각 균 별 적정 온도에서 발효시켰다. 발효 완료된 처리구를 100℃, 20분간 살균하고 여과지로 여과하여 각각 처리구의 총 폴리페놀, 이소플라본(비배당체)의 함량을 분석하였다. It was made into a 10% (W/W) aqueous solution of the enzymatic hydrolyzate prepared by utilizing the two enzymes established in Example 1, sterilized at 100° C. for 20 minutes, cooled, and used as a control and sample. The cooled sample was inoculated with Aspergillus kawachii and Aspergillus oryzae and fermented for 12 hours at an appropriate temperature for each bacteria. The fermented treated group was sterilized at 100° C. for 20 minutes and filtered through filter paper to analyze the content of total polyphenols and isoflavones (non-glycoside) in each treatment group.
이소플라본(비배당체)의 함량은 아래 방법으로 측정하였다. 먼저, 각 효소량 및 시간별로 제조된 수용액을 70% 에탄올을 시료액 대비 20배 가하여 환류 추출 및 감압 여과하였다. 여과 완료된 용액은 농축하여 에탄올을 증발시키고, 농축물을 10배 희석한 후 0.45um Syringe Filter로 여과하여 바이알에 담은 후 HPLC 기기 및 아래 표 2의 조건으로 이소플라본 함량을 정량하였다. 그 결과를 도 2 및 도 3에 나타냈다. The content of isoflavones (non-glycosides) was measured by the following method. First, 70% ethanol was added 20 times to the prepared aqueous solution for each enzyme amount and time compared to the sample solution, followed by reflux extraction and filtration under reduced pressure. The filtered solution was concentrated to evaporate ethanol, and the concentrate was diluted 10-fold, filtered through a 0.45um Syringe Filter, put in a vial, and then the content of isoflavones was quantified using an HPLC instrument and the conditions shown in Table 2 below. The results are shown in FIGS. 2 and 3 .
B: Acetonitril 60: Water 40: Formic acid 0.1 A: Acetonitril 10: Water 90: Formic acid 0.1
B: Acetonitril 60: Water 40: Formic acid 0.1
도 2를 보면, 발효 균주에 따른 대두 발효물의 총폴리페놀 함량은 대조구가 340.9 ppm이었으며, Aspergillus oryzae 발효구는 505.7 ppm, kawachii 발효구는 506.6 ppm 으로 발효에 의한 총폴리페놀 증대 효과가 확인되었으며, 발효구간 차이는 없는 것으로 확인되었다.Referring to FIG. 2 , the total polyphenol content of the soybean fermented product according to the fermentation strain was 340.9 ppm in the control group, 505.7 ppm in the Aspergillus oryzae fermented group, and 506.6 ppm in the kawachii ferment group, confirming the effect of increasing the total polyphenols by fermentation. No difference was found.
도 3을 보면, 발효 균주에 따른 대두 발효물의 비배당체 기준 총이소플라본 함량은 대조구에서 28.2 ppm 검출되었으며, oryzae 발효구에서 68.7 ppm, kawachii 발효구에서는 11.9 ppm 검출되어 이소플라본 총량 기준으로는 oryzae 발효구가 이소플라본 증대에 가장 효과적인 것으로 나타났다. Referring to FIG. 3 , the total isoflavone content based on the non-glycoside of the soybean fermented product according to the fermentation strain was detected at 28.2 ppm in the control group, 68.7 ppm in the oryzae fermenter, and 11.9 ppm in the kawachii fermenter. Gu was found to be the most effective in increasing isoflavones.
실시예 3: 대두 추출물의 제조Example 3: Preparation of Soybean Extract
대두를 정제수에 1일간 침지(대두가 잠길 정도)한 후 대두를 100℃에서 20분간 가열(살균)하여 실온까지 냉각시켰다. 냉각된 대두를 고속믹서를 이용하여 분쇄하고, 고형분 함량이 10 중량%가 되도록 정제수를 첨가하였다. 이어서 고형분 중량 대비 6중량%의 탄수화물 분해효소(AMG, 노보자임사)를 넣고 2.17 시간 동안 가수분해하였다. 이어서, 고형분 중량 대비 9중량%의 단백질 분해효소(알카라아제, 노보자임사)를 넣고 6시간 동안 가수분해하였다. 상기 가수분해물에 아스퍼질러스 오리자에(Aspergillus oryzae)를 접종(1 cyrovial(108/g)하여 30℃에서 18시간 동안 발효시켰다. 이어서 100℃에서 20분간 살균처리한 후, 여과(Advantec No. 20)하고, 농축 및 동결 건조시켜 대두 추출물(SBE)을 제조하였다. Soybeans were immersed in purified water for one day (soybeans were submerged), and then the soybeans were heated (sterilized) at 100° C. for 20 minutes and cooled to room temperature. The cooled soybeans were pulverized using a high-speed mixer, and purified water was added so that the solid content was 10% by weight. Then, 6% by weight of carbohydrate degrading enzyme (AMG, Novozyme) was added based on the solid weight and hydrolyzed for 2.17 hours. Then, 9% by weight of a proteolytic enzyme (Alcalase, Novozyme) was added based on the solid weight and hydrolyzed for 6 hours. The hydrolyzate was inoculated with Aspergillus oryzae (1 cyrovial (10 8 /g) and fermented at 30° C. for 18 hours. Then, after sterilization at 100° C. for 20 minutes, filtration (Advantec No. 20), concentrated and freeze-dried to prepare soybean extract (SBE).
실험예 1: 조골세포 활성 평가Experimental Example 1: Evaluation of osteoblast activity
골 형성 단계에서 조골세포는 골 전구세포의 증식, 세포외기질의 성숙, 석회화 유도의 과정을 거쳐 분화하며, 조골세포의 활성을 반영하는 대표적인 생화학적 마커로는 ALP, 콜라겐(collagen), 골 석회화 결절 형성이 있다. 그중 ALP와 콜라겐(collagen)은 세포증식 말기와 세포외기질형성 단계에서 높게 발현되므로 조골세포의 초기분화 마커로 이용되고, 골 석회화 결절의 형성은 조골세포 분화의 최종 단계에서 나타나므로 후기분화 마커로 이용된다.In the bone formation stage, osteoblasts differentiate through the processes of proliferation of bone progenitors, maturation of extracellular matrix, and induction of calcification. there is formation Among them, ALP and collagen are highly expressed at the end of cell proliferation and extracellular matrix formation, so they are used as early differentiation markers of osteoblasts. used
본 실험에서는, 실시예 3에서 제조된 대두 추출물의 뼈 건강 개선 효과를 평가하기 위하여, 대두 추출물이 조골세포(MC3T3-E1 세포)의 초기분화 마커인 ALP 활성과 콜라겐(collagen) 형성, 후기분화 마커인 석회화 결절 형성에 미치는 영향을 평가하였다. In this experiment, in order to evaluate the bone health improvement effect of the soybean extract prepared in Example 3, the soybean extract was used as an early differentiation marker of ALP activity, collagen formation, and late differentiation marker of osteoblasts (MC3T3-E1 cells). The effect on phosphorus calcified nodule formation was evaluated.
생쥐의 뼈에서 유래한 MC3T3-E1 osteoblastic cells은 American Type Culture Collection(ATCC)에서 구입하여 사용하였다. MC3T3-E1 세포는 α-minimum essential medium (α-MEM, Gibco-Invitrogen)에 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin을 첨가한 세포배양액을 사용하여 37℃ 습윤한 CO2 배양기 (5% CO2/95% air)에서 배양하였다. 세포가 배양 접시의 80% 정도 차면 phosphate-buffered saline (PBS, pH 7.4)으로 세포의 단층을 씻어내고 0.25% trypsin-2.65 mM EDTA를 처리하여 계대 배양하였고 배지는 2일마다 교환하였다. MC3T3-E1 osteoblastic cells derived from mouse bone were purchased from the American Type Culture Collection (ATCC) and used. MC3T3-E1 cells were prepared using a cell culture medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin in α-minimum essential medium (α-MEM, Gibco-Invitrogen) at 37°C. Incubated in a humidified CO 2 incubator (5% CO 2 /95% air). When the cells were filled to about 80% of the culture dish, the monolayer of the cells was washed with phosphate-buffered saline (PBS, pH 7.4) and subcultured with 0.25% trypsin-2.65 mM EDTA, and the medium was changed every 2 days.
MC3T3-E1 세포가 배양접시의 90% 정도 찼을 때 조골세포로 분화를 유도하기 위해 α-MEM 배지에 10 mM β-glycerophosphate(Sigma-Aldrich Co.)와 50 μg/mL ascorbic acid (Sigma-Aldrich Co.)를 첨가한 조골세포 분화배양액으로 세포배양액을 교환하여 세포를 배양하였고, 조골세포 분화배양액은 3일마다 교환하였다.When MC3T3-E1 cells are about 90% full of the culture dish, 10 mM β-glycerophosphate (Sigma-Aldrich Co.) and 50 μg/mL ascorbic acid (Sigma-Aldrich Co.) were added to α-MEM medium to induce differentiation into osteoblasts. .) was added to the cell culture medium by exchanging the cell culture medium, and the osteoblast differentiation culture medium was exchanged every 3 days.
(1) 대두추출물이 MC3T3-E1 세포의 세포 생존율(cell viabillty)에 미치는 영향 펑가(1) Effect of soybean extract on cell viability of MC3T3-E1 cells
대두 추출물의 독성에 의한 세포증식 감소는 조골세포의 분화 및 활성에 영향을 미치므로, 이를 배제하기 위해 MC3T3-E1 세포의 증식에 영향을 미치지 않은 대두 추출물의 처리농도를 설정하고자 하였다. MC3T3-E1 세포를 5 × 104 cells/well로 24-well plate에 분주하여 24시간 배양한 후 다양한 농도(0, 5, 10, 25, 50, 100, 200, 400, 600, 800, 1000 μg/mL)의 대두 추출물(실시예 3)을 세포배양액에 처리하여 72시간 배양한 후 MTT assay를 실시하였다. MTT assay는 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Amresco) 용액으로 세포배양액을 교환하여 2시간 동안 추가 배양한 후 살아있는 세포에서 형성된 formazan을 isopropanol로 용출하여 SpectraMaxM2 Microplate reader(Molecular Devices)를 사용하여 570 nm에서 흡광도를 측정하여 세포의 증식능을 측정하였다. MC3T3-E1 세포의 생존율은 시험물질을 처리하지 않은 대조군(0 μg/mL)에 대한 백분율로 나타내었다. 그 결과를 표 3에 나타냈다. 표 3에 나타낸 바와 같이 대두 추출물을 800 μg/mL 이상의 농도로 처리하였을 때 MC3T3-E1 세포의 생존률이 유의적으로 감소하였다. 이후 시험물질의 처리농도를 0, 100, 200, 400 μg/mL로 정하여 시험을 수행하였다.Since the decrease in cell proliferation due to the toxicity of the soybean extract affects the differentiation and activity of osteoblasts, in order to exclude this, the treatment concentration of the soybean extract that does not affect the proliferation of MC3T3-E1 cells was set. MC3T3-E1 cells were seeded in a 24-well plate at 5 × 10 4 cells/well and cultured for 24 hours at various concentrations (0, 5, 10, 25, 50, 100, 200, 400, 600, 800, 1000 μg). /mL) of soybean extract (Example 3) was treated with a cell culture medium and cultured for 72 hours, followed by MTT assay. The MTT assay is performed by exchanging the cell culture medium with a 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Amresco) solution, culturing for 2 hours, and then removing the cells from the living cells. The formed formazan was eluted with isopropanol, and the cell proliferation ability was measured by measuring the absorbance at 570 nm using a SpectraMaxM2 Microplate reader (Molecular Devices). The viability of MC3T3-E1 cells was expressed as a percentage of the control group (0 μg/mL) not treated with the test substance. The results are shown in Table 3. As shown in Table 3, when the soybean extract was treated at a concentration of 800 μg/mL or higher, the viability of MC3T3-E1 cells was significantly reduced. Thereafter, the test was performed by setting the treatment concentration of the test substance to 0, 100, 200, and 400 μg/mL.
(실시예 3)(Example 3)
(% of control)(% of control)
Values are expressed as mean ± SEM.Values are expressed as mean ± SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of 0 μg/mL group. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of 0 μg/mL group.
(2) 대두 추출물이 ALP(Alkaline phosphatase) 활성에 미치는 영향 측정(2) Measurement of the effect of soybean extract on ALP (Alkaline phosphatase) activity
ALP 활성을 평가하기 위해 MC3T3-E1 세포를 1 × 104 cells/well이 되도록 96- well plate에 분주하고, 24시간 동안 안정화 시켰다. 실시예 3의 대두 추출물을 다양한 농도(0, 100, 200, 400 μg/mL)로 함유한 조골세포 분화배양액으로 세포배양액을 교환하여 5일간 배양하였다. 5일간 분화 유도된 세포의 ALP 활성은 TRACP & ALP assay kit (Takara Bio Inc.)를 사용하여 제조회사가 제시한 방법에 따라 측정하였다. 그 결과를 표 4에 나타냈다.To evaluate ALP activity, MC3T3-E1 cells were aliquoted in a 96-well plate to 1 × 10 4 cells/well, and stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation culture medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 5 days. ALP activity of cells induced to differentiate for 5 days was measured using the TRACP & ALP assay kit (Takara Bio Inc.) according to the method suggested by the manufacturer. The results are shown in Table 4.
표 4에 나타난 바와 같이 MC3T3-E1 세포의 분화를 유도하였을 때 ALP 활성은 21.3 ± 0.6 U/L로 미분화하였을 때의 0.8 ± 0.1 U/L과 비교하여 현저히 증가하였다. 대두추출물을 100, 200, 400 μg/mL의 농도로 처리하였을 때 ALP 활성은 각각 22.1 ± 0.8 U/L, 27.4 ± 2.6 U/L, 31.0 ± 2.0 U/L로 조골분화대조군 (G1)과 비교하여 200 μg/mL의 농도부터 농도가 증가함에 따라 ALP 활성이 유의적으로 증가하였다. 양성대조물질인 estradiol을 100 nM로 처리하였을 때 ALP 활성이 2.53 ± 0.3 U/L로 조골분화대조군 (G1)과 비교하여 ALP 활성이 유의적으로 증가하였다As shown in Table 4 When differentiation of MC3T3-E1 cells was induced, ALP activity was significantly increased at 21.3 ± 0.6 U/L, compared to 0.8 ± 0.1 U/L when undifferentiated. When the soybean extract was treated at concentrations of 100, 200, and 400 μg/mL, the ALP activity was 22.1 ± 0.8 U/L, 27.4 ± 2.6 U/L, and 31.0 ± 2.0 U/L, respectively, compared to the osteoblastic differentiation control group (G1). Thus, from the concentration of 200 μg/mL, the ALP activity significantly increased as the concentration increased. When estradiol, a positive control, was treated with 100 nM, ALP activity was 2.53 ± 0.3 U/L, which significantly increased ALP activity compared to the osteoblast differentiation control group (G1).
Values are expressed as mean ± SEM.Values are expressed as mean ± SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; †p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.
(3) 콜라겐(collagen) 합성 측정(Sirius red 염색)(3) Collagen synthesis measurement (Sirius red staining)
콜라겐은 인체 각 결합 조직에 분포하는 중요한 단백질로서, 제 1형 콜라겐은 피부, 골 및 인대에 풍부하게 존재하며 조골세포에 의해 주로 합성되고 전체 골단백의 85~90%를 차지한다. 콜라겐 분자에 강하게 결합하는 anionic dye인 Sirius Red를 사용하여 세포를 염색하여 콜라겐 합성 정도를 측정하였다. MC3T3-E1 세포를 5 × 104 cells/well이 되도록 24-well plate에 분주한 후 24시간 동안 안정화 시켰다. 실시예 3의 대두 추출물을 다양한 농도(0, 100, 200, 400 μg/mL)로 함유한 조골세포 분화배양액으로 세포배양액을 교환하여 8일간 배양하였다. 8일간 분화 유도된 세포의 콜라겐 합성은 Sirius red collagen detection kit (Chondrex)을 사용하여 제조회사가 제시한 방법에 따라 측정하였다. 대두 추출물이 조골세포의 콜라겐 합성에 미치는 영향을 조사하여 표 5에 나타내었다. Collagen is an important protein distributed in each connective tissue of the human body. Type 1 collagen is abundantly present in the skin, bone, and ligaments, is synthesized mainly by osteoblasts, and accounts for 85-90% of the total bone protein. The degree of collagen synthesis was measured by staining the cells using Sirius Red, an anionic dye that strongly binds to collagen molecules. MC3T3-E1 cells were aliquoted in a 24-well plate so as to become 5 × 10 4 cells/well, and then stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation culture medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 8 days. Collagen synthesis of cells induced for differentiation for 8 days was measured using a Sirius red collagen detection kit (Chondrex) according to the method suggested by the manufacturer. Table 5 shows the effect of soybean extract on collagen synthesis in osteoblasts.
표 5에 나타난 바와 같이 MC3T3-E1 세포의 분화를 유도하였을 때 (G1) 합성된 콜라겐은 466.5 ± 20.6 μg/mL로 미분화하였을 때 (G0)의 82.8 ± 8.0 μg/mL와 비교하여 5.63배 증가하였다. 분화유도에 의해 증가한 콜라겐 합성은 대두추출물의 처리에 의해 200 μg/mL의 농도부터 처리농도가 증가함에 따라 콜라겐 합성이 유의적으로 증가하였고, 400 μg/mL을 처리하였을 때 (G4) 546.4 ± 16.6 μg/mL로 조골분화대조군 (G1)과 비교하여 17.1% 콜라겐 합성이 증가하였다. 양성대조물질인 estradiol은 100 nM로 처리하였을 때 (G5) 526.9 ± 5.1 μg/mL로 12.9% 증가하였다.As shown in Table 5, when differentiation of MC3T3-E1 cells was induced (G1), the synthesized collagen was increased 5.63 times compared to 82.8 ± 8.0 μg/mL of (G0) when undifferentiated at 466.5 ± 20.6 μg/mL (G0). . As for the collagen synthesis increased by differentiation induction, from the concentration of 200 μg/mL by the treatment of soybean extract, the collagen synthesis increased significantly as the treatment concentration increased, and when treated with 400 μg/mL (G4) 546.4 ± 16.6 At μg/mL, collagen synthesis was increased by 17.1% compared to the osteoblast differentiation control group (G1). When estradiol, a positive control, was treated with 100 nM (G5), it was increased by 12.9% to 526.9 ± 5.1 μg/mL.
Values are expressed as mean ± SEM.Values are expressed as mean ± SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; †p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.
(4) 무기질 침착(mineralization) 측정(Alizarin red 염색)(4) Mineralization measurement (Alizarin red staining)
안트라퀴노이드(Anthraquinonoid) 유도체인 Alizarin Red S를 사용하여 조골세포에 침착된 칼슘 함량을 측정하여 조골세포의 무기질 침착(mineralization) 정도를 조사하였다. Alizarin은 무기질화된 세포의 기질에 염색되므로 석회화된 양과 염색 정도가 상호 비례한다. 따라서 Alizarin red 염색을 하여 대두 추출물 처리에 따른 무기질 침착을 조사하였다. MC3T3-E1 세포를 5 × 104 cells/well이 되도록 24 well plate에 분주한 후 24시간 동안 안정화 시켰다. 실시예 3의 대두 추출물을 다양한 농도(0, 100, 200, 400 μg/mL)로 함유한 조골세포 분화배양액으로 세포배양액을 교환하여 18일간 배양하였다. 세포의 무기질 침착은 Osteogenesis assay kit (Millipore)를 사용하여 제조회사가 제시한 방법에 따라 측정하였다. 그 결과를 표 6에 나타냈다. 표 6에 나타난 바와 같이 조골분화유도에 의해 증가한 무기질 침착은 SEB 100, 200, 400 μg/mL의 처리농도에서 각각 39.79 ± 1.45 μM, 42.50 ± 2.45 μM, 65.64 ± 5.97 μM로 조골분화대조군 (G1, 37.64 ± 1.78 μM)과 비교하여 증가하였으나, 400 μg/mL의 처리농도에서만 유의적으로 무기질 침착이 증가하였다. The degree of mineralization of osteoblasts was investigated by measuring the calcium content deposited in osteoblasts using Alizarin Red S, an anthraquinonoid derivative. Alizarin stains the mineralized cell matrix, so the amount of calcification and the degree of staining are proportional to each other. Therefore, Alizarin red staining was used to investigate mineral deposition following treatment with soybean extract. MC3T3-E1 cells were aliquoted in a 24-well plate so as to become 5 × 10 4 cells/well, and then stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 18 days. Cell mineral deposition was measured using the Osteogenesis assay kit (Millipore) according to the method suggested by the manufacturer. The results are shown in Table 6. As shown in Table 6, mineral deposition increased by induction of osteoblastic differentiation was 39.79 ± 1.45 µM, 42.50 ± 2.45 µM, and 65.64 ± 5.97 µM at the treatment concentrations of
Values are expressed as mean ± SEM.Values are expressed as mean ± SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; †p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.
(5) Osteocalcin(OCN) 함량 측정(5) Osteocalcin (OCN) content measurement
OCN은 주로 조골세포에 의하여 생성되며 활성이 활발한 조골세포의 특이 표지자로 알려진 기질 단백질이다. OCN는 골형성의 생화학적 표지자로 조골세포에서 합성된다. MC3T3-E1 세포를 5 × 104 cells/well이 되도록 24-well plate에 분주한 후 24시간 동안 안정화 시켰다. 실시예 3의 대두 추출물을 다양한 농도(0, 100, 200, 400 μg/mL)로 함유한 조골세포 분화배양액으로 세포배양액을 교환하여 18일간 배양하였다. 세포가 24시간 동안 condition한 세포배양액을 수거하였다. 세포가 condition한 세포배양액 내의 osteoblast-secreted osteocalcin (OCN) 수준은 OCN ELISA Kit (Takara Bio Inc.)를 사용하여 제조사의 제시 방법에 따라 측정하였다. 그 결과를 표 7에 나타냈다. 표 7을 보면, 조골분화대조군 (G1)의 OCN 함량은 0.804 ± 0.044 ng/mL이었으며, SBE을 100, 200, 400 μg/mL의 농도로 처리 시 각각 1.218 ± 0.068 ng/mL, 1.361 ± 0.092 ng/mL, 1.830 ± 0.158 ng/mL으로 100 μg/mL의 농도부터 OCN의 생성이 유의적으로 증가하였다.OCN is mainly produced by osteoblasts and is a matrix protein known as a specific marker of active osteoblasts. OCN is synthesized in osteoblasts as a biochemical marker of bone formation. MC3T3-E1 cells were aliquoted in a 24-well plate so as to become 5 × 10 4 cells/well, and then stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 18 days. Cell cultures were collected in which cells were conditioned for 24 hours. The level of osteoblast-secreted osteocalcin (OCN) in the cell culture medium conditioned by the cells was measured using the OCN ELISA Kit (Takara Bio Inc.) according to the manufacturer's method. The results are shown in Table 7. Referring to Table 7, the OCN content of the osteoblastic differentiation control group (G1) was 0.804 ± 0.044 ng/mL, and when SBE was treated at a concentration of 100, 200, and 400 μg/mL, 1.218 ± 0.068 ng/mL and 1.361 ± 0.092 ng, respectively. OCN production was significantly increased from a concentration of 100 μg/mL to /mL, 1.830 ± 0.158 ng/mL.
(100 nM)Estradiol
(100 nM)
Values are expressed as mean ± SEM.Values are expressed as mean ± SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; †p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.
본 실험을 통해, 본 발명에 따른 대두 추출물이 MC3T3-E1 세포의 세포증식에 독성을 나타내지 않는 농도에서 조골분화지표인 ALP 활성, 콜라겐 합성, mineralization 침착 및 osteocalcin 생성을 유의적으로 증가시킨 것을 확인하였다. Through this experiment, it was confirmed that the soybean extract according to the present invention significantly increased ALP activity, collagen synthesis, mineralization deposition, and osteocalcin production, which are indicators of osteoblast differentiation, at a concentration that was not toxic to MC3T3-E1 cell proliferation. .
실험예 2: 파골세포 활성 평가Experimental Example 2: Evaluation of osteoclast activity
실시예 3에서 제조된 대두 추출물의 뼈 건강 개선 효과를 평가하기 위하여, 대두 추출물의 파골세포 활성에 미치는 영향을 측정하였다. In order to evaluate the bone health improvement effect of the soybean extract prepared in Example 3, the effect of the soybean extract on osteoclast activity was measured.
생쥐에서 유래한 대식세포인 RAW 264.7 세포는 American Type Culture Collection (ATCC)에서 구입하여 사용하였다. RAW 264.7 세포는 Dulbecco’s Modified Eagle’s Medium (DMEM, Welgene)에 10% FBS, 100 units/mL penicillin과 100 μg/mL streptomycin을 첨가한 세포 배양액을 사용하여 37℃ 습윤한 CO2 배양기 (5% CO2/95% air)에서 배양하였다. 세포가 배양접시의 80% 정도 찼을 때, phosphate buffer saline (PBS, pH 7.4)으로 세포 단층을 씻어낸 후 세포배양액을 첨가하여 세포를 떼어내어 계대 배양하였고, 배지는 2일마다 교환하였다. RAW 264.7 cells, which are macrophages derived from mice, were purchased from the American Type Culture Collection (ATCC) and used. RAW 264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Welgene) in a cell culture medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin in a 37°C humidified CO 2 incubator (5% CO 2 / 95% air). When the cells were about 80% full of the culture dish, the cell monolayer was washed with phosphate buffer saline (PBS, pH 7.4), the cell culture solution was added, the cells were removed and subcultured, and the medium was exchanged every 2 days.
RAW 264.7 세포를 파골세포로 분화를 유도하기 위해 α-MEM에 50 ng/mL RANKL (Sigma-Aldrich Co.)와 10 μM PD98059 (Santa Cruz)를 첨가한 파골세포 분화배양액으로 세포배양액을 교환하여 세포를 배양하였고, 파골세포 분화배양액은 3일마다 교환하였다. 이렇게 얻어진 파골세포를 아래 실험에 사용하였다. In order to induce the differentiation of RAW 264.7 cells into osteoclasts, the cell culture medium was exchanged with the osteoclast differentiation medium containing 50 ng/mL RANKL (Sigma-Aldrich Co.) and 10 μM PD98059 (Santa Cruz) added to α-MEM. was cultured, and the osteoclast differentiation culture medium was exchanged every 3 days. The osteoclasts thus obtained were used in the experiments below.
(1) 대두 추출물이 RAW 264.7 세포의 세포 생존율(cell viability) 에 미치는 영향 (1) Effect of soybean extract on cell viability of RAW 264.7 cells
RAW 264.7 세포의 세포 생존율은 MTT assay 방법으로 측정하였다. The cell viability of RAW 264.7 cells was measured by the MTT assay method.
RAW 264.7 세포를 5 × 104 cells/well로 24-well plate에 분주하여 24시간 배양한 후 다양한 농도(0, 5, 10, 25, 50, 100, 200, 400, 600, 800, 1,000 μg/mL)의 대두 추출물(실시예 3)을 세포배양액에 처리하여 72시간 배양한 후 MTT assay를 실시하였다. MTT assay는 1 mg/mL MTT (Amresco) 용액으로 세포배양액을 교환하고 2시간 동안 세포를 추가 배양한 후 살아있는 세포에서 형성된 formazan을 isopropanol로 용출하여 SpectraMaxM2 Microplate reader (Molecular Devices)를 사용하여 570 nm에서 흡광도를 측정하여 세포의 증식능을 측정하였다. RAW 264.7 세포의 생존율은 시험물질을 처리하지 않은 대조군(0 μg/mL)에 대한 백분율로 나타내었다. 그 결과를 표 8에 나타냈다. 표 8에 나타낸 바와 같이 시험물질의 처리는 RAW264.7 세포의 생존률에 유의적인 차이를 나타내지 않아 세포독성이 없음을 나타낸다. 이후 시험물질의 처리농도를 0, 100, 200, 400 μg/mL로 정하여 시험을 수행하였다. RAW 264.7 cells were seeded at 5 × 10 4 cells/well in a 24-well plate and cultured for 24 hours at various concentrations (0, 5, 10, 25, 50, 100, 200, 400, 600, 800, 1,000 μg/ mL) of soybean extract (Example 3) was treated with a cell culture medium and cultured for 72 hours, followed by MTT assay. MTT assay is performed by exchanging the cell culture medium with 1 mg/mL MTT (Amresco) solution and eluting formazan formed from living cells with isopropanol after culturing the cells for 2 hours at 570 nm using a SpectraMaxM2 Microplate reader (Molecular Devices). By measuring the absorbance, the proliferative capacity of the cells was measured. The viability of RAW 264.7 cells was expressed as a percentage relative to the control (0 μg/mL) not treated with the test substance. The results are shown in Table 8. As shown in Table 8, the treatment with the test substance did not show a significant difference in the viability of RAW264.7 cells, indicating that there is no cytotoxicity. Thereafter, the test was performed by setting the treatment concentration of the test substance to 0, 100, 200, and 400 μg/mL.
(% of control)(% of control)
Values are expressed as mean ± SEM.Values are expressed as mean ± SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; †p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.
(2) 대두추출물 (SBE)이 TRAP 활성에 미치는 영향 (2) Effect of soybean extract (SBE) on TRAP activity
TRAP은 파골세포의 특이 표지자로 골 흡수작용 시 분비가 증가하기 때문에 TRAP의 활성도를 측정하여 파골세포 분화 정도를 확인한다. TRAP 활성을 평가하기 위해 RAW 264.7 세포를 5 × 103 cells/well이 되도록 96- well plate에 분주하고, 24시간 동안 안정화 시켰다. 실시예 3의 대두 추출물을 다양한 농도(0, 100, 200, 400 μg/mL)로 함유한 파골세포 분화배양액으로 세포배양액을 교환하여 5일 배양하였다. 5일간 분화를 유도한 후 세포의 TRAP 활성은 TRACP & ALP assay kit (Takara Bio Inc.)를 사용하여 제조회사가 제시한 방법에 따라 측정하였다. 그 결과를 표 9에 나타냈다. 표 9에 나타난 바와 같이 RAW 264.7 세포를 파골세포로의 분화를 유도하였을 때 TRAP 활성은 6.71 ± 0.10 U/L로 미분화하였을 때의 2.47 ± 0.02 U/L와 비교하여 현저히 증가하였다. 증가된 TRAP 활성은 SBE를 100, 200, 400 μg/mL의 농도로 처리하였을 때 각각 5.69 ± 0.06 U/L, 5.64 ± 0.13 U/L, 4.36 ± 0.04 U/L로 파골분화대조군(G1, 0 μg/mL)과 비교하여 100 μg/mL의 농도부터 농도가 증가함에 따라 TRAP 활성이 유의적으로 감소하였다.Since TRAP is a specific marker of osteoclasts and its secretion increases during bone resorption, the degree of osteoclast differentiation is confirmed by measuring the activity of TRAP. To evaluate TRAP activity, RAW 264.7 cells were aliquoted in a 96-well plate to 5 × 10 3 cells/well, and stabilized for 24 hours. The cell culture medium was exchanged with the osteoclast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 5 days. After inducing differentiation for 5 days, TRAP activity of cells was measured using the TRACP & ALP assay kit (Takara Bio Inc.) according to the method suggested by the manufacturer. The results are shown in Table 9. As shown in Table 9 When RAW 264.7 cells were induced to differentiate into osteoclasts, TRAP activity was significantly increased at 6.71 ± 0.10 U/L, compared with 2.47 ± 0.02 U/L when undifferentiated. The increased TRAP activity was 5.69 ± 0.06 U/L, 5.64 ± 0.13 U/L, and 4.36 ± 0.04 U/L when SBE was treated at a concentration of 100, 200, and 400 μg/mL, respectively, in the osteoclast differentiation control group (G1, 0 μg/mL), the TRAP activity was significantly decreased as the concentration increased from the concentration of 100 μg/mL.
Values are expressed as mean ± SEM.Values are expressed as mean ± SEM.
* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; †p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.
(3) 대두추출물 (SBE)이 파골세포의 actin ring 형성에 미치는 영향(3) Effect of soybean extract (SBE) on actin ring formation of osteoclasts
골세포 표면의 actin ring 구조는 필수적이며 이를 통해 골기질을 흡수한다. 파골세포가 분화하면 그 크기가 거대해지고 표면이 actin ring을 형성하여 골흡수 과정에 관여하게 된다. 따라서 파골세포의 actin ring 형성은 세포가 뼈를 흡수할 수 있는 능력에 대한 중요 지표가 된다. RAW 264.7 세포를 1 × 104 cells/well이 되도록 cover glass가 포함된 24-well plate에 분주하였고, 24시간 동안 안정화 시켰다. 실시예 3의 대두 추출물을 다양한 농도(0, 100, 200, 400 μg/mL)로 함유한 파골세포 분화배양액으로 세포배양액을 교환하여 5일간 배양하였다. 배지를 제거하고 PBS로 세척한 후 4% paraformaldehyde와 0.1% Triton X-100으로 처리하여 세포를 고정하였다. 고정된 세포는 actin과 핵을 염색하기 위해 각각 Alexa Fluor 594 Phalloidin과 DAPI로 염색하였다. 염색된 세포는 광학현미경(Carl Zeiss)을 사용하여 분화된 파골세포의 액틴 링의 형태 변화를 관찰하였다. 그 결과를 도 4에 나타냈다. 파골분화대조군 (G1)의 경우 actin ring이 가장 선명하며 세포들이 밀집되어 있지만 대두 추출물(SBE)의 처리 농도가 증가함에 따라 actin ring이 현저하게 줄어들었다. 이는 대두 추출물의 처리에 의해 뼈 흡수가 가능한 단계로의 분화가 억제됨은 나타낸다. The actin ring structure on the bone cell surface is essential, and through this, the bone matrix is absorbed. When osteoclasts differentiate, their size becomes huge and the surface forms an actin ring, which is involved in the bone resorption process. Therefore, the actin ring formation of osteoclasts is an important indicator of the cell's ability to resorb bone. RAW 264.7 cells were aliquoted in a 24-well plate with a cover glass so that the concentration was 1 × 10 4 cells/well, and the cells were stabilized for 24 hours. The cell culture medium was exchanged with the osteoclast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 5 days. After removing the medium and washing with PBS, the cells were fixed by treatment with 4% paraformaldehyde and 0.1% Triton X-100. The fixed cells were stained with Alexa Fluor 594 Phalloidin and DAPI to stain actin and nuclei, respectively. The stained cells were observed for morphological changes in the actin ring of differentiated osteoclasts using an optical microscope (Carl Zeiss). The results are shown in FIG. 4 . In the case of the osteoclast differentiation control group (G1), the actin ring was the clearest and the cells were dense, but the actin ring was significantly reduced as the concentration of soybean extract (SBE) increased. This indicates that the treatment of soybean extract inhibited differentiation to a stage capable of bone resorption.
본 실험을 통해, 본 발명의 대두 추출물이 RAW 264.7 세포의 세포증식에 독성을 나타내지 않는 농도에서 파골분화지표인 TRAP 활성을 감소하고, actin ring 형성을 저해하는 것을 확인하였다. Through this experiment, it was confirmed that the soybean extract of the present invention reduced TRAP activity, an osteoclast differentiation index, and inhibited actin ring formation at a concentration that was not toxic to the cell proliferation of RAW 264.7 cells.
상기한 실험 결과들로부터, 본 발명에 따른 대두 추출물은 조골활성을 증진하고 파골활성을 억제하여 골대사를 개선하는 것을 확인할 수 있다. From the above experimental results, it can be confirmed that the soybean extract according to the present invention improves bone metabolism by enhancing osteoblastic activity and suppressing osteoclast activity.
Claims (4)
냉각된 대두를 분쇄하고, 고형분 함량이 5~10 중량%인 수용액을 제조하는 단계;
상기 수용액에 고형분 중량 대비 1 내지 10중량%의 탄수화물 분해효소를 넣고 1 내지 3시간 동안 처리하여 1차 가수분해물을 제조하는 단계;
상기 1차 가수분해물에 고형분 중량 대비 1 내지 10중량%의 단백질 분해효소를 넣고 5 내지 7시간 동안 처리하여 2차 가수분해물을 제조하는 단계: 및
상기 2차 가수분해물에 아스퍼질러스 오리자에(Aspergillus oryzae) 또는 아스퍼질러스 카와키(Aspergillus kawachii)를 접종하여 발효시키는 단계를 포함하는, 대두 추출물의 제조 방법.Heating soybeans immersed in purified water for 1 to 2 days at 100° C. for 10 to 30 minutes and then cooling;
pulverizing the cooled soybeans and preparing an aqueous solution having a solid content of 5 to 10% by weight;
preparing a primary hydrolyzate by adding 1 to 10% by weight of a carbohydrate-degrading enzyme to the aqueous solution based on the solids weight and treating it for 1 to 3 hours;
Preparing a secondary hydrolyzate by adding 1 to 10% by weight of a protease to the primary hydrolyzate based on the solids weight and treating it for 5 to 7 hours: And
A method for producing a soybean extract, comprising inoculating the secondary hydrolyzate with Aspergillus oryzae or Aspergillus kawachii and fermenting the same.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020200184735A KR20220093719A (en) | 2020-12-28 | 2020-12-28 | Method for preparing soybean extract with bone metabilic function improvement |
PCT/KR2021/018640 WO2022145793A1 (en) | 2020-12-28 | 2021-12-09 | Method for preparing glycine max extract having effect of improving bone metabolic functions |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020200184735A KR20220093719A (en) | 2020-12-28 | 2020-12-28 | Method for preparing soybean extract with bone metabilic function improvement |
Publications (1)
Publication Number | Publication Date |
---|---|
KR20220093719A true KR20220093719A (en) | 2022-07-05 |
Family
ID=82260605
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020200184735A KR20220093719A (en) | 2020-12-28 | 2020-12-28 | Method for preparing soybean extract with bone metabilic function improvement |
Country Status (2)
Country | Link |
---|---|
KR (1) | KR20220093719A (en) |
WO (1) | WO2022145793A1 (en) |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101239624B1 (en) * | 2010-12-30 | 2013-03-07 | 주식회사농심 | Preparation method of Hydrolyzed Vegetable Protein |
KR101881562B1 (en) * | 2017-08-28 | 2018-07-25 | 주식회사 유웰바이오 | Isoflavone formulation from soybean with effect of preventing osteoporosis, and manufacturing method thereof |
KR101949990B1 (en) * | 2017-08-28 | 2019-02-20 | 주식회사 유웰바이오 | Whole soy milk containing increased active isoflavone with effect of improving osteoporosis, and manufacturing method thereof |
KR102134365B1 (en) * | 2018-10-31 | 2020-07-16 | 씨제이제일제당 주식회사 | Method for producing soy protein concentrates hydrolysate |
KR102148099B1 (en) * | 2019-06-28 | 2020-08-25 | 푸른솔푸드 주식회사 | Enzyme food composite manufacture method using Isolated Soy Protein and brown rice |
-
2020
- 2020-12-28 KR KR1020200184735A patent/KR20220093719A/en not_active Application Discontinuation
-
2021
- 2021-12-09 WO PCT/KR2021/018640 patent/WO2022145793A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
WO2022145793A1 (en) | 2022-07-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Paucar-Menacho et al. | Effect of time and temperature on bioactive compounds in germinated Brazilian soybean cultivar BRS 258 | |
KR101824147B1 (en) | Soybean fermented composition having enhanced physiological activity and health functional food for alleviating climacteric or menopausal syndrome comprising the same | |
RU2693610C1 (en) | Method for preparation of rice paste with hot pepper and rice paste prepared in such way with hot pepper | |
KR102044602B1 (en) | Preparation method for soybean-fermented composition having enhanced GABA, Conjugated linoleic acid and Aglycone isoflavones, combined probiotics for its fermentation and functional food of anti-diabetic and anti-obesity effects containing the soybean-fermented composition | |
Xie et al. | Ganoderma lucidum inhibits tumour cell proliferation and induces tumour cell death | |
CN102648284B (en) | Polyphenol derivative and method for producing the same | |
KR101963978B1 (en) | Composition of fermented sprout ginseng having increased ginsenosides Rh2 and compound K, bioactive components, and preparation method thereof | |
Amarowicz et al. | Antioxidative and radical scavenging effects of phenolics from Vicia sativum | |
KR100844185B1 (en) | The manfacturing method of fermentation artemisia tea with lactic acid | |
Hong et al. | Improved functionality of soft soybean curd containing Monascus fermented soybean ethanol extract | |
JP2003238424A (en) | Biologically converted ginseng and method of producing the same | |
KR20190109308A (en) | Composition for preventing and treating of bone diseases comprising fermented extracts from aronia and black rice | |
KR102555674B1 (en) | Composition containing Tenebrio molitor and Protaetia larva brevitarsis larva enzymatic hydrolysate for preventing or treating menopausal symptom | |
KR20220093719A (en) | Method for preparing soybean extract with bone metabilic function improvement | |
KR101917534B1 (en) | Vegetable Brown Sauce with enhanced taste and anti-oxydant property and preparation method thereof | |
Hu et al. | Characterization of precipitation from citrus vinegar during ageing: chemical constituents, formation mechanism and anti-proliferative effect | |
KR100403565B1 (en) | a manufacturing process feeds which be made from a Tofuwast redkoji containing monacolin | |
KR20190030850A (en) | Platycodon radix composition containing high protocatechuic acid, epicatechin, and oleic acid and enhanced inhibition activities of alpha-glucosidase and pancreatic lipase, and preparation method thereof | |
Liyanage et al. | Boiled, sprouted, and raw cowpea‐incorporated diets modulate high‐fat diet‐induced hypercholesterolemia in rats | |
KR102169156B1 (en) | Method for increasing platycodin E content in Platycodon grandiflorum using Aspergillus shirousamii | |
Gao et al. | ACE inhibitory, antitumor and antioxidant activities of submerged culture materials of three medicinal mushrooms | |
Jung et al. | Osteoblastic differentiation of mesenchymal stem cells by mumie extract | |
KR102437929B1 (en) | Food composition for relieving menopausal symptom comprising extracts of Fabaton soybean leaves and active wild-cultivated ginseng and preparation method thereof | |
KR102071131B1 (en) | A functional soybean paste for improving vascular disorder | |
KR20140081013A (en) | (Composition comprising the extract of crude drug complex for promoting bone growth) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
E902 | Notification of reason for refusal | ||
E601 | Decision to refuse application |