JP6006545B2 - Proteoglycan production method - Google Patents
Proteoglycan production method Download PDFInfo
- Publication number
- JP6006545B2 JP6006545B2 JP2012144400A JP2012144400A JP6006545B2 JP 6006545 B2 JP6006545 B2 JP 6006545B2 JP 2012144400 A JP2012144400 A JP 2012144400A JP 2012144400 A JP2012144400 A JP 2012144400A JP 6006545 B2 JP6006545 B2 JP 6006545B2
- Authority
- JP
- Japan
- Prior art keywords
- proteoglycan
- fatty acid
- cartilage
- cartilage tissue
- acid ester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 17
- 102000016611 Proteoglycans Human genes 0.000 claims description 73
- 108010067787 Proteoglycans Proteins 0.000 claims description 73
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 33
- 229930195729 fatty acid Natural products 0.000 claims description 33
- 239000000194 fatty acid Substances 0.000 claims description 33
- 210000000845 cartilage Anatomy 0.000 claims description 31
- -1 fatty acid ester Chemical class 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 21
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 238000007654 immersion Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 241000251468 Actinopterygii Species 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 241000124008 Mammalia Species 0.000 claims description 7
- 241000237852 Mollusca Species 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 description 26
- 239000005720 sucrose Substances 0.000 description 26
- 239000007787 solid Substances 0.000 description 21
- 239000004094 surface-active agent Substances 0.000 description 19
- 238000000605 extraction Methods 0.000 description 17
- 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 16
- 239000000787 lecithin Substances 0.000 description 16
- 235000010445 lecithin Nutrition 0.000 description 16
- 229940067606 lecithin Drugs 0.000 description 16
- 229930182490 saponin Natural products 0.000 description 13
- 235000017709 saponins Nutrition 0.000 description 13
- 150000007949 saponins Chemical class 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 235000019688 fish Nutrition 0.000 description 11
- 241000271566 Aves Species 0.000 description 10
- 239000012472 biological sample Substances 0.000 description 10
- 239000007791 liquid phase Substances 0.000 description 10
- 210000002184 nasal cartilage Anatomy 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 9
- 239000007858 starting material Substances 0.000 description 9
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 8
- 229920001287 Chondroitin sulfate Polymers 0.000 description 8
- 229940059329 chondroitin sulfate Drugs 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 210000003128 head Anatomy 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 241000972773 Aulopiformes Species 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 239000002440 industrial waste Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 102000008186 Collagen Human genes 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 5
- 235000014633 carbohydrates Nutrition 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 description 4
- 101710132601 Capsid protein Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 150000004665 fatty acids Chemical class 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 235000019515 salmon Nutrition 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 229920002683 Glycosaminoglycan Polymers 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 125000004432 carbon atom Chemical group C* 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 150000005690 diesters Chemical class 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000013373 food additive Nutrition 0.000 description 3
- 239000002778 food additive Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 210000001087 myotubule Anatomy 0.000 description 3
- 235000014593 oils and fats Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 229910001220 stainless steel Inorganic materials 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- 150000005691 triesters Chemical class 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- PIGTXFOGKFOFTO-PPEDVFHSSA-N CC1(C)CC[C@@]2([C@H](O)C[C@]3(C)C(=CC[C@@H]4[C@@]5(C)CCC(O[C@@H]6O[C@@H]([C@@H](O)[C@H](O)[C@H]6O)C(O)=O)[C@@](C)(C=O)[C@@H]5CC[C@@]34C)[C@@H]2C1)C(O)=O Chemical compound CC1(C)CC[C@@]2([C@H](O)C[C@]3(C)C(=CC[C@@H]4[C@@]5(C)CCC(O[C@@H]6O[C@@H]([C@@H](O)[C@H](O)[C@H]6O)C(O)=O)[C@@](C)(C=O)[C@@H]5CC[C@@]34C)[C@@H]2C1)C(O)=O PIGTXFOGKFOFTO-PPEDVFHSSA-N 0.000 description 2
- 229920002567 Chondroitin Polymers 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004695 Polyether sulfone Substances 0.000 description 2
- 241000251125 Prionace glauca Species 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 2
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 229920006393 polyether sulfone Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 1
- PIGTXFOGKFOFTO-FVFWYJKVSA-N (2S,3S,4S,5R,6R)-6-[[(3S,4S,4aR,6aR,6bS,8R,8aR,12aS,14aR,14bR)-8a-carboxy-4-formyl-8-hydroxy-4,6a,6b,11,11,14b-hexamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound O([C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)C[C@@H](O)[C@]1(CCC(C[C@H]14)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O PIGTXFOGKFOFTO-FVFWYJKVSA-N 0.000 description 1
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000238366 Cephalopoda Species 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101800001691 Inter-alpha-trypsin inhibitor light chain Proteins 0.000 description 1
- 239000005639 Lauric acid Substances 0.000 description 1
- 229930182473 O-glycoside Natural products 0.000 description 1
- 150000008444 O-glycosides Chemical class 0.000 description 1
- 241000238413 Octopus Species 0.000 description 1
- 102100032859 Protein AMBP Human genes 0.000 description 1
- 241001423892 Somniosus microcephalus Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- VVBXXVAFSPEIJQ-CVIPOMFBSA-N [(2r)-3-[[(2r)-1-[[(2s,5r,8r,11r,12s,15s,18s,21s)-15-[3-(diaminomethylideneamino)propyl]-21-hydroxy-5-[(4-hydroxyphenyl)methyl]-4,11-dimethyl-2-(2-methylpropyl)-3,6,9,13,16,22-hexaoxo-8-propan-2-yl-10-oxa-1,4,7,14,17-pentazabicyclo[16.3.1]docosan-12-yl]am Chemical compound C([C@@H]1C(=O)N[C@@H](C(=O)O[C@H](C)[C@@H](C(N[C@@H](CCCN=C(N)N)C(=O)N[C@H]2CC[C@H](O)N(C2=O)[C@@H](CC(C)C)C(=O)N1C)=O)NC(=O)[C@H](NC(=O)[C@H](O)COS(O)(=O)=O)CC(C)C)C(C)C)C1=CC=C(O)C=C1 VVBXXVAFSPEIJQ-CVIPOMFBSA-N 0.000 description 1
- 229920001284 acidic polysaccharide Polymers 0.000 description 1
- 150000004805 acidic polysaccharides Chemical class 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 235000003084 food emulsifier Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 239000012770 industrial material Substances 0.000 description 1
- 108010093564 inter-alpha-inhibitor Proteins 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Description
本発明は、医薬品原料、医療用品材料、化粧品原料、食品材料、工業用材料等として有用なプロテオグリカンを、プロテオグリカンを含有する生物学的試料、例えば魚類、軟体動物、鳥類及び哺乳類の軟骨組織から抽出し、製造する方法に関する。 The present invention extracts proteoglycans useful as pharmaceutical raw materials, medical supplies materials, cosmetic raw materials, food materials, industrial materials, etc. from biological samples containing proteoglycans, such as fish, mollusks, birds and mammalian cartilage tissues And a manufacturing method.
プロテオグリカンは、１本のコアタンパク質に数本から数十本の直鎖状の糖鎖が共有結合してなる、非常に複雑かつ多種の構造を有する糖タンパク質の総称であり、軟骨組織に存在するプロテオグリカンに含まれる糖鎖の代表的なものがコンドロイチン硫酸である。 Proteoglycan is a general term for glycoproteins having very complex and various structures, in which several to several tens of linear sugar chains are covalently bonded to one core protein, and exist in cartilage tissue. A typical sugar chain contained in proteoglycan is chondroitin sulfate.
コンドロイチン硫酸は、保湿性、生体適合性あるいは潤滑性に優れる等の高い有用性から産業上注目されている成分であり、天然資源からの効果的な回収、製造法が種々開発されている。 Chondroitin sulfate is a component that has attracted industry attention due to its high usefulness such as moisture retention, biocompatibility, and lubricity, and various effective recovery and production methods from natural resources have been developed.
軟骨組織においては、コンドロイチン硫酸はそれ自体単独では存在せず、タンパク質と共有結合を介して形成された複合体すなわちプロテオグリカンの形で存在している。しかし、プロテオグリカンをそのまま抽出することは糖タンパク質複合体という複雑な構造故に困難な場合が多く、そのためプロテオグリカンのコアタンパク質部分を徹底的に分解してコンドロイチン硫酸のみを抽出しようという方法が主に採用されてきた。この方法の製造物はコンドロイチン硫酸等のムコ多糖類である。 In cartilage tissue, chondroitin sulfate does not exist by itself, but exists in the form of a complex formed through covalent bonding with a protein, ie, proteoglycan. However, it is often difficult to extract proteoglycan as it is because of the complex structure of glycoprotein complex. Therefore, the method of extracting only chondroitin sulfate by thoroughly degrading the core protein part of proteoglycan is mainly adopted. I came. The product of this method is a mucopolysaccharide such as chondroitin sulfate.
一方、コンドロイチン硫酸としてではなく、プロテオグリカンそのままを回収、製造し利用する試みもなされている。特に、魚類、鳥類及び哺乳類の軟骨組織には、コンドロイチン硫酸を主要糖鎖とするプロテオグリカンが含まれている一方、これらの軟骨組織は通常廃棄処分となっていたことから、廃棄物の有効利用を兼ねた軟骨組織からのプロテオグリカンの製造法が幾つか提唱されている。 On the other hand, attempts have been made to collect, produce and use proteoglycan as it is, not as chondroitin sulfate. In particular, the cartilage tissue of fish, birds and mammals contains proteoglycans containing chondroitin sulfate as the main sugar chain. Several methods for producing proteoglycan from cartilage tissue which have also been proposed have been proposed.
例えば、サケ鼻軟骨からグアニジン塩酸を用いてプロテオグリカンを抽出する方法（特許文献１）や酢酸を用いる方法（特許文献２）、アルカリを用いる方法（特許文献３）等の方法が報告されている。しかしこれまでの方法は、いずれも抽出、精製コストが高い、あるいはプロテオグリカンの糖鎖構造が破壊されるなど、残念ながら未だ実用化のレベルに達しているとは言えない。 For example, methods such as a method for extracting proteoglycan from salmon nasal cartilage using guanidine hydrochloride (Patent Document 1), a method using acetic acid (Patent Document 2), and a method using alkali (Patent Document 3) have been reported. However, none of the conventional methods has yet reached the level of practical use, such as high extraction and purification costs or the destruction of the sugar chain structure of proteoglycan.
本発明は、魚類、軟体動物、鳥類又は哺乳類等、特にそれらの廃棄部位から低コストで経口摂取可能なプロテオグリカンをその糖鎖構造を破壊することなく製造する方法の開発を課題とするものである。 An object of the present invention is to develop a method for producing proteoglycan that can be ingested at low cost from fish, mollusks, birds, mammals, etc., particularly from their disposal sites without destroying the sugar chain structure. .
本発明者らは、界面活性剤の水溶液、特に食品添加物として認可されているサポニン、レシチンあるいはスークロース脂肪酸エステル等の水溶液を使用することにより、糖タンパク質複合体であるプロテオグリカンを効率よく軟骨組織その他のプロテオグリカンを含有する生物学的試料から回収することができることを見出し、下記の各発明を完成した。 By using an aqueous solution of a surfactant, particularly an aqueous solution of a saponin, lecithin, sucrose fatty acid ester or the like that is approved as a food additive, the present inventors can efficiently produce a proteoglycan that is a glycoprotein complex as a cartilage tissue or the like. It was found that it can be recovered from a biological sample containing a proteoglycan, and the following inventions have been completed.
（１）プロテオグリカンを含有する生物学的試料を界面活性剤の水溶液に浸漬する工程、及び浸漬後の溶液を回収する工程を含む、プロテオグリカンの製造方法。 (1) A method for producing proteoglycan, comprising a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant and a step of recovering the solution after immersion.
（２）界面活性剤がサポニン、レシチン若しくはスークロース脂肪酸エステル、又はこれらの混合物である、（１）に記載の製造方法。 (2) The production method according to (1), wherein the surfactant is saponin, lecithin, sucrose fatty acid ester, or a mixture thereof.
（３）回収した溶液からプロテオグリカンを分離する工程をさらに含む、（１）又は（２）に記載の製造方法。 (3) The production method according to (1) or (2), further comprising a step of separating proteoglycan from the collected solution.
（４）プロテオグリカンを含有する生物学的試料が、魚類、軟体動物、鳥類若しくは哺乳類の軟骨組織、筋肉繊維又は皮である、（１）〜（３）の何れかに記載の製造方法。 (4) The production method according to any one of (1) to (3), wherein the biological sample containing proteoglycan is fish, mollusk, avian or mammalian cartilage tissue, muscle fiber, or skin.
（５）プロテオグリカンを含有する生物学的試料が、魚類、鳥類又は哺乳類の軟骨組織である、（４）に記載の製造方法。 (5) The production method according to (4), wherein the biological sample containing proteoglycan is fish, birds or mammalian cartilage tissue.
本発明は、従来の抽出方法に比べ、プロテオグリカンを未変成、未分解の状態で簡便かつ短時間で回収することができ、プロテオグリカンの製造コストを大きく減ずることができる。 According to the present invention, proteoglycan can be easily recovered in an unmodified and undegraded state in a short time as compared with conventional extraction methods, and the production cost of proteoglycan can be greatly reduced.
また、本発明の方法は、本来廃棄処分とされてきた魚類、鳥類及び哺乳類などの廃棄部位から産業上有用性の高いプロテオグリカンを回収することができ、産業廃棄物の有効利用並びに産業廃棄物自体の減量化にも貢献することができる。 In addition, the method of the present invention can recover proteoglycans having high industrial usefulness from disposal sites such as fish, birds and mammals that have been originally disposed of, and can effectively utilize industrial waste and industrial waste itself. It can also contribute to the reduction of weight.
また、好ましい態様の本発明では高温で抽出するため、組織内に内包しているタンパク質分解酵素を失活させるためのタンパク質分解酵素阻害剤（インヒビター）の添加を必ずしも必要としない。かかるインヒビターの効果は全てのタンパク質分解酵素に有効ではない上に、インヒビター自身が人体に有害なものが多く、食品材料としてのプロテオグリカンを製造するに当たり、インヒビターを使用することは好ましくないが、本発明はインヒビターを必要とはせず、従って上記の問題を回避することが可能である。 Further, in the preferred embodiment of the present invention, since extraction is performed at a high temperature, it is not always necessary to add a proteolytic enzyme inhibitor (inhibitor) for inactivating the proteolytic enzyme contained in the tissue. The effect of such an inhibitor is not effective for all proteolytic enzymes, and the inhibitor itself is often harmful to the human body, and it is not preferable to use an inhibitor in producing proteoglycan as a food material. Does not require an inhibitor and therefore it is possible to avoid the above problems.
本発明は、プロテオグリカンを含有する生物学的試料を界面活性剤の水溶液に浸漬する工程、及び浸漬後の溶液を回収する工程を含む、プロテオグリカンの製造方法である。 The present invention is a method for producing proteoglycan, comprising a step of immersing a biological sample containing proteoglycan in an aqueous solution of a surfactant and a step of recovering the solution after immersion.
本発明は、一般に熱やアルカリに不安定なタンパク質を含むプロテオグリカンを、界面活性剤、好ましくは食品添加物である界面活性剤、特に好ましくはサポニン、レシチン若しくはスークロース脂肪酸エステル単体、あるいはこれらの混合の水溶液を用いるという新しい発想の下に抽出、製造する方法である。 The present invention generally comprises a proteoglycan containing a protein unstable to heat and alkali, a surfactant, preferably a surfactant as a food additive, particularly preferably a saponin, lecithin or sucrose fatty acid ester alone, or a mixture thereof. This is a method of extraction and production under the new idea of using an aqueous solution.
本発明においては、プロテオグリカンとして任意のプロテオグリカンが使用できる。プロテオグリカンは酸性多糖であるグリコサミノグリカンとタンパク質の複合体であるが、コアタンパク質とそのタンパク質に結合している糖鎖の結合力がタンパク質のセリン残基あるいはスレオニン残基を介したＯ−グリコシド結合であるためアルカリに対して弱く、簡単に分離してしまう性質を有している。そのため、その抽出や精製は一般に極めて困難であり、タンパク質だけからなるコラーゲンや糖質だけからなるコンドロイチン硫酸などの抽出とは異なる注意が必要である。このため、従来技術は、工程が複雑になる、あるいは手作業を伴う部分が多いなど、大量生産に向かないものであった。 In the present invention, any proteoglycan can be used as the proteoglycan. Proteoglycan is a complex of glycosaminoglycan, which is an acidic polysaccharide, and protein, but the O-glycoside has a binding force between the core protein and the sugar chain bound to the protein via the serine residue or threonine residue of the protein. Since it is a bond, it is weak against alkalis and easily separated. Therefore, the extraction and purification are generally very difficult, and different cautions are required from the extraction of collagen consisting only of protein and chondroitin sulfate consisting only of carbohydrates. For this reason, the conventional technology is not suitable for mass production because the process is complicated or there are many parts that involve manual work.
本発明においては、界面活性剤は、陰イオン界面活性剤、陽イオン界面活性剤、両面界面活性剤及び非イオン界面活性剤の任意の界面活性剤が使用できる。界面活性剤として、食品添加物又は食品用乳化剤として承認されている、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル、プロピレングリコール脂肪酸エステル、スークロース脂肪酸エステル、ソルビタン脂肪酸エステル、大豆リン脂質（レシチン）、サポニン等が好ましく、特に好ましくは、サポニン、レシチンあるいはスークロース脂肪酸エステル単体、あるいはこれらの混合物である。
スークロース脂肪酸エステルには、スークロース１分子に脂肪酸１分子が付加してできるショ糖脂肪酸モノエステル、脂肪酸２分子が付加してできるジエステル、脂肪酸３分子が付価してできるトリエステルが含まれ、理論上は、脂肪酸８分子の付加したオクタエステルまで存在し得るが、食品用に適するのは、モノエステル、ジエステル、トリエステル及びこれらの混合物である。スークロース脂肪酸エステルとしては、エマルジョンの乳化安定性の観点から、好ましくはＨＬＢ１３〜１９、特に１４〜１９、最適にはＨＬＢ値１９のスークロース脂肪酸エステルである。エマルジョンコストの低減の観点も加えれば、モノエステルとジエステルとトリエステルの混合物（以下、スークロース脂肪酸混合エステルという）が望ましく、特にＨＬＢ値１４〜１６のスークロース脂肪酸混合エステルとＨＬＢ１９のスークロース脂肪酸モノエステルとの混合物が望ましい。脂肪酸としては、好ましくは炭素数１０〜２２、さらに好ましくは炭素数１２〜１８の飽和又は不飽和脂肪酸である。なかでも、炭素数１２の飽和脂肪酸であるラウリン酸が好ましい。
レシチンには、植物レシチン、分別レシチン、卵黄レシチン、酵素処理レシチン、酵素分解レシチン等が含まれる。レシチンとしては、ＨＬＢ値３〜４の植物性レシチンが好ましい。
サポニンには、キラヤサポニン、大豆サポニン、茶サポニンなどが含まれ、キラヤサポニンが好ましい。
In the present invention, as the surfactant, any surfactant such as an anionic surfactant, a cationic surfactant, a double-sided surfactant and a nonionic surfactant can be used. As surfactants, approved as food additives or food emulsifiers, glycerin fatty acid ester, polyglycerin fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, soybean phospholipid (lecithin), saponin, etc. Saponin, lecithin, sucrose fatty acid ester alone, or a mixture thereof is preferable.
Sucrose fatty acid esters include sucrose fatty acid monoester formed by adding one molecule of sucrose to one molecule of sucrose, diester formed by adding two molecules of fatty acid, and triester formed by adding three molecules of fatty acid. The above may be present up to octaesters with 8 molecules of fatty acid, but suitable for food are monoesters, diesters, triesters and mixtures thereof. The sucrose fatty acid ester is preferably a sucrose fatty acid ester having an HLB value of 13 to 19, particularly 14 to 19, and most preferably having an HLB value of 19, from the viewpoint of emulsion stability of the emulsion. From the viewpoint of reducing the emulsion cost, a mixture of monoester, diester and triester (hereinafter referred to as sucrose fatty acid mixed ester) is desirable. In particular, a sucrose fatty acid mixed ester having an HLB value of 14 to 16 and a sucrose fatty acid monoester having HLB 19 A mixture of The fatty acid is preferably a saturated or unsaturated fatty acid having 10 to 22 carbon atoms, more preferably 12 to 18 carbon atoms. Of these, lauric acid, which is a saturated fatty acid having 12 carbon atoms, is preferable.
Lecithin includes plant lecithin, fractionated lecithin, egg yolk lecithin, enzyme-treated lecithin, enzyme-decomposed lecithin and the like. As lecithin, vegetable lecithin having an HLB value of 3 to 4 is preferable.
Saponins include quillaja saponins, soybean saponins, tea saponins and the like, with quillaja saponins being preferred.
本発明は、プロテオグリカンを含む生物学的試料、例えば魚類、軟体動物、鳥類若しくは哺乳類の軟骨組織、筋肉繊維又は皮に対して適用することができるが、軟骨組織への適用が好ましい。本発明において使用される軟骨組織は、魚類、鳥類及び哺乳類、特にそれらの廃棄部位のいずれも利用することができる。本発明において軟骨組織とは、軟骨単独あるいは軟骨の周辺部位、例えば骨、筋肉繊維、皮等を含む組織のいずれも意味する。 The present invention can be applied to biological samples containing proteoglycans, such as fish, mollusk, avian or mammalian cartilage tissue, muscle fibers or skin, but application to cartilage tissue is preferred. As the cartilage tissue used in the present invention, any of fish, birds and mammals, particularly those waste sites can be used. In the present invention, the cartilage tissue means either cartilage alone or a tissue including a peripheral part of the cartilage, for example, bone, muscle fiber, skin and the like.
本発明においては、一般に流通しているヨシキリザメ、アオザメなどの軟骨魚類のヒレ、軟骨部分に対する利用はもちろん、特に一般に氷頭とよばれている、サケの頭部にその平均重量で約６％含まれている鼻軟骨組織の利用が好ましい。北海道沿岸部で漁獲されたサケ（大半はシロサケである。）が、様々な加工品として処理される際、その頭部は不要とされることが多く、そのため切断された頭部は、一部魚粉に加工され利用されてはいるものの、その大半は産業廃棄物として廃棄処理されている。氷頭はその様な廃棄物から簡便、安価かつ安定的に入手することができる。 In the present invention, it is used not only for fins and cartilage parts of cartilaginous fish such as blue sharks and blue sharks that are generally available, but also in general, generally called ice heads, and contains about 6% in average weight in the salmon head. Use of nasal cartilage tissue is preferred. When salmon caught on the coast of Hokkaido (mostly salmon) are processed as various processed products, their heads are often unnecessary, so some of the cut heads Although processed into fish meal and used, most of it is disposed of as industrial waste. Ice heads can be obtained simply, inexpensively and stably from such waste.
本発明では、氷頭の他、エイの軟骨組織、サメの軟骨組織等の魚類由来の軟骨組織、ニワトリの軟骨組織等の鳥類の軟骨組織、さらにはウシの喉軟骨や気管支軟骨、クジラの軟骨等の哺乳動物由来の軟骨組織も利用することができる。さらに、軟体動物であるイカやタコの表皮・軟骨組織にもプロテオグリカンが存在することが知られており、これら軟体動物の表皮・軟骨等も本発明で利用することができる。また哺乳動物血液中には硫酸を殆ど含まないコンドロイチン・タンパク質複合体（ビクニン）が存在しており、またこのコンドロイチンは哺乳動物血液中のムコ多糖のほぼ１００％を占めることが報告されている（Salier J.P., Rouet P., Raguenez G., Daveau M.: The inter-alpha-inhibitor family: from structure to regulation Biochem. J., 315, 1-9, 1996）ことから、哺乳動物の血液由来コンドロイチンは本発明におけるプロテオグリカンを含む生物学的試料の一例として有用である。上記のプロテオグリカンを含む生物学的試料の多くも産業廃棄物であり、その入手は容易である。これらの原料は、次に説明する界面活性剤水溶液への浸漬に先だって、表面積を増加させてプロテオグリカンの抽出量を上げるために、可能な限り細かく破砕すること、脱脂することなどの前処理を行なうことが好ましい。 In the present invention, in addition to ice head, fish cartilage tissue such as ray cartilage tissue, shark cartilage tissue, avian cartilage tissue such as chicken cartilage tissue, bovine throat cartilage, bronchial cartilage, whale cartilage A cartilage tissue derived from mammals such as can also be used. Furthermore, it is known that proteoglycan is also present in the epidermis / cartilage tissue of squid and octopus which are molluscs, and the epidermis / cartilage of these molluscs can also be used in the present invention. In addition, chondroitin protein complex (bikunin) containing almost no sulfate is present in mammalian blood, and this chondroitin is reported to occupy almost 100% of mucopolysaccharide in mammalian blood ( Salier JP, Rouet P., Raguenez G., Daveau M .: The inter-alpha-inhibitor family: from structure to regulation Biochem. J., 315, 1-9, 1996). The present invention is useful as an example of a biological sample containing proteoglycan. Many of the biological samples containing the above proteoglycans are also industrial waste and are readily available. Prior to immersion in the surfactant aqueous solution described below, these raw materials are subjected to pretreatment such as crushing and degreasing as finely as possible in order to increase the surface area and increase the amount of proteoglycan extracted. It is preferable.
本発明で使用する界面活性剤の中でも、プロテオグリカンの抽出効率、後処理の簡便さ等から、スークロース脂肪酸エステル類の使用が好ましく、特にスークロース脂肪酸エステル（Ｃ１２）の使用が好ましい。 Among the surfactants used in the present invention, the use of sucrose fatty acid esters is preferred, and the use of sucrose fatty acid ester (C12) is particularly preferred from the standpoint of proteoglycan extraction efficiency and ease of post-treatment.
界面活性剤、例えばサポニン、レシチン若しくはスークロース脂肪酸エステル又はこれらの混合物の水溶液の界面活性剤濃度は、好ましくは０．０１〜２０重量％、特に好ましくは０．１〜１０重量％、さらに好ましくは０．５〜５重量％である。浸漬時間は、適宜設定できるが、例えば０．１〜２重量％の界面活性剤、例えばサポニン、レシチン若しくはスークロース脂肪酸エステル、又はこれらの混合物の水溶液を用いた場合の浸漬時間は、１０時間以上とすることが好ましい。また２〜５重量％の界面活性剤、例えばサポニン、レシチン若しくはスークロース脂肪酸エステル又はこれらの混合物の溶液を用いる場合の浸漬時間は、１２時間程度とすることが好ましい。これらの条件により、より高分子のプロテオグリカンを回収、製造することができる。 The surfactant concentration of an aqueous solution of a surfactant, such as saponin, lecithin or sucrose fatty acid ester or a mixture thereof, is preferably 0.01 to 20% by weight, particularly preferably 0.1 to 10% by weight, and more preferably 0. 0.5 to 5% by weight. The immersion time can be set as appropriate. For example, the immersion time when using an aqueous solution of 0.1 to 2% by weight of a surfactant such as saponin, lecithin or sucrose fatty acid ester, or a mixture thereof is 10 hours or more. It is preferable to do. The immersion time when using a solution of 2 to 5% by weight of a surfactant such as saponin, lecithin or sucrose fatty acid ester or a mixture thereof is preferably about 12 hours. Under these conditions, higher-molecular proteoglycans can be recovered and produced.
界面活性剤の水溶液への生物学的試料の浸漬は、好ましくは３０℃〜１００℃、より好ましくは４０℃〜８０℃、特に好ましくは５０℃〜７０℃で行う。特に、浸漬温度を５０℃〜６０℃とすることにより、プロテオグリカンは、殆ど分解されず、高分子の糖タンパク質複合体として抽出することができる。 The immersion of the biological sample in the aqueous solution of the surfactant is preferably performed at 30 ° C to 100 ° C, more preferably 40 ° C to 80 ° C, and particularly preferably 50 ° C to 70 ° C. In particular, by setting the immersion temperature to 50 ° C. to 60 ° C., proteoglycan is hardly decomposed and can be extracted as a polymer glycoprotein complex.
浸漬は、軟骨１重量部に対して界面活性剤の水溶液、例えばサポニン、レシチン又はスークロース脂肪酸エステル又はこれらの混合物の水溶液を好ましくは１〜５０重量部、より好ましくは２〜３０重量部、さらに４〜２０重量部、特に好ましくは６〜１５重量部を用いて行う。好ましくは、ミキサーあるいはスターラーなどを用いて攪拌しながら浸漬する。 The immersion is preferably 1 to 50 parts by weight, more preferably 2 to 30 parts by weight, and more preferably 2 to 30 parts by weight of an aqueous solution of a surfactant, for example, an aqueous solution of saponin, lecithin or sucrose fatty acid ester or a mixture thereof. -20 parts by weight, particularly preferably 6-15 parts by weight. Preferably, it is immersed while stirring using a mixer or a stirrer.
軟骨組織からのプロテオグリカンの抽出は、例えばカルバゾール法（Johnら、ANALYTICAL BIOCHEMISTRY、1967年、第19巻、第119-132頁）によってウロン酸量を検出ないし定量することでモニターすることができるが、その他公知の方法によってウロン酸を検出し、モニターしてもよい。 Extraction of proteoglycan from cartilage tissue can be monitored by detecting or quantifying the amount of uronic acid by, for example, the carbazole method (John et al., ANALYTICAL BIOCHEMISTRY, 1967, Vol. 19, pp. 119-132). In addition, uronic acid may be detected and monitored by a known method.
浸漬が終了した界面活性剤の水溶液は、プロテオグリカンを抽出した後の残渣を多く含むので、これらを濾過、遠心分離その他の方法で取り除くことが好ましい。プロテオグリカンを含む抽出液はそのまま製品として利用してもよいが、プロテオグリカンの各種用途に対して求められる純度にまで適当な方法でプロテオグリカンを分離ないし精製することが好ましい。 The aqueous solution of the surfactant that has been soaked contains a large amount of residue after extraction of the proteoglycan, and it is preferable to remove these by filtration, centrifugation, or other methods. Although the extract containing proteoglycan may be used as a product as it is, it is preferable to separate or purify proteoglycan by an appropriate method up to the purity required for various uses of proteoglycan.
本発明において、プロテオグリカンの精製方法としては公知の方法を使用でき、格別の方法を要するものではないが、好ましい方法として遠心分離法を挙げることができる。遠心分離操作によって、細かい固形物を沈殿残渣として簡便に除去することができる。 In the present invention, a known method can be used as a method for purifying proteoglycan, and a special method is not required, but a preferable method is a centrifugation method. A fine solid can be easily removed as a precipitation residue by centrifugation.
また、遠心分離法によって回収されるプロテオグリカンを含む液相を、さらにフィルターペーパーあるいは適当な分画分子量を有する限外濾過膜分離装置などを用いて濾過してもよい。排除分子量としては概ね５万〜１００万の範囲であればよい。この操作において、分画分子量５０万以上のものを使用すれば、液相からコラーゲンも除去することができ、プロテオグリカンの純度を上げることが可能である。また、プロテオグリカンを含む液相に水を加えて液相の粘度を下げることで、膜の通過を容易にするとともに、これを繰り返すことで、わずかに生じる原料由来の特異臭を除去することも可能である。 Further, the liquid phase containing proteoglycan recovered by the centrifugal separation method may be further filtered using a filter paper or an ultrafiltration membrane separation device having an appropriate fractional molecular weight. The excluded molecular weight may be in the range of about 50,000 to 1,000,000. In this operation, if a fractional molecular weight of 500,000 or more is used, collagen can also be removed from the liquid phase, and the purity of proteoglycan can be increased. In addition, by adding water to the liquid phase containing proteoglycan to reduce the viscosity of the liquid phase, it is possible to facilitate passage through the membrane, and by repeating this, it is also possible to remove the slightly specific odor originating from the raw material It is.
さらに、得られた濃縮液を食塩飽和エタノールに加えることで、ゲル状のプロテオグリカンを回収することもできる。このゲル状プロテオグリカンは真空凍結乾燥機を用いて固形物にしてもよい。あるいは、スプレードライヤーで乾燥させ、粉末状固形物とすることもできる。 Furthermore, gel-like proteoglycan can also be collect | recovered by adding the obtained concentrate to salt saturated ethanol. This gel proteoglycan may be made into a solid using a vacuum freeze dryer. Alternatively, it can be dried with a spray dryer to form a powdery solid.
以下に実施例を挙げて本発明をさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.
＜実施例１＞
−４０℃で冷凍保管したアオザメから摘出した軟骨を電動のミートチョッパーで細かく破砕しミンチ状にしたものを２００．００ｇ用意し、出発原料とした。５リットルの抽出用容器にあらかじめ０℃に冷却しておいた蒸留水２３７６．０ｇを入れ、さらに固形のスークロース脂肪酸エステル（Ｃ１２；ＨＬＢ１４〜１８）２４．０ｇを投入し、総量２４００．００ｇ（１．０重量％）のスークロース脂肪酸エステル水溶液を準備した。この抽出用容器に出発原料２００．００ｇを投入し、スターラーを用いて攪拌しながら、１２時間浸漬した。
<Example 1>
200.00 g of cartilage extracted from a gray shark frozen at −40 ° C. and minced with an electric meat chopper was prepared as a starting material. 2376.0 g of distilled water cooled to 0 ° C. in advance in a 5 liter extraction container, and further charged with 24.0 g of solid sucrose fatty acid ester (C12; HLB14-18) for a total amount of 2400.00 g (1 0.0 wt%) sucrose fatty acid ester aqueous solution. 200.00 g of starting material was put into this extraction container and immersed for 12 hours while stirring with a stirrer.
浸漬終了後、内容物を１mm角のステンレススチール製こし器をセットした別の容器に移し、プロテオグリカンを含む抽出液を回収した。 After the immersion, the contents were transferred to another container in which a 1 mm square stainless steel strainer was set, and the extract containing proteoglycan was collected.
抽出液をＩＷＡＫＩ ＣＦＳ−４００型の遠心分離機を用いて１５００×ｇ、３０分の遠心分離を行い、固形分ならびに油脂分を除去して、プロテオグリカンを含む液相を回収した。 The extract was centrifuged at 1500 × g for 30 minutes using an IWAKI CFS-400 type centrifuge to remove solids and oils and fats, and a liquid phase containing proteoglycans was recovered.
さらにこの液相を、フィルターペーパー（アドバンテック社製）を用いて濾過し、濾液の６倍量の蒸留水を加えた後、日本ミリポア製 ＰＲＥＰ／ＳＣＡＬＥ ＴＦＦ膜（分画分子量１０万）を用いて分画と濃縮を同時に行った。 Further, this liquid phase is filtered using a filter paper (manufactured by Advantech), 6 times the amount of distilled water is added to the filtrate, and then a PREP / SCALE TFF membrane (manufactured by Nippon Millipore) (molecular weight cut off 100,000) is used. Fractionation and concentration were performed simultaneously.
得られた濃縮液の一部を採取し、液中の固形分重量を測定した。測定は、乾燥炉（ＹＡＭＡＴＯ ＤＸ４０１）により、１０５℃、１６時間乾燥させ、完全に水分を蒸発させた後、残った固形分をデジタル計量器（Ａ＆Ｄ社 ＧＦ−４００）で精密に測定した。その結果、２００．００ｇの出発原料から、換算値でその２．７５％に相当する５．５ｇの乾燥固形分を得ることができた。 A part of the concentrated liquid obtained was collected and the solid content weight in the liquid was measured. The measurement was performed by drying at 105 ° C. for 16 hours with a drying furnace (YAMATO DX401), and after completely evaporating water, the remaining solid content was precisely measured with a digital meter (A & D GF-400). As a result, 5.5 g of dry solid content corresponding to 2.75% of the converted value was obtained from 200.00 g of the starting material.
また、濃縮液をアミノ酸自動分析装置（日立製作所社製、Ｌ−８５００ Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いてアミノ酸量を測定することで濃縮液中のコラーゲン量を定量するとともに、カルバゾール法によりウロン酸量を定量してプロテオグリカン量を計算した。 In addition, the amount of amino acid in the concentrated solution is measured by using an amino acid automatic analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) to quantify the amount of collagen in the concentrated solution, and the amount of uronic acid is determined by the carbazole method. The amount of proteoglycan was calculated by quantification.
さらに高速液体クロマトグラフィ装置（島津製作所、カラムＡｓａｈｉｐａｋ）を用いて、プロテオグリカンの分子量を測定した。 Further, the molecular weight of proteoglycan was measured using a high performance liquid chromatography apparatus (Shimadzu Corporation, column Asahipak).
これらの分析の結果、固形分中にタンパク質３５．０％、灰分２１．５％、炭水化物４２．９％、脂質０．６％が存在することが判った。特許文献１によれば、プロテオグリカンのコアタンパク質の重量比は約７．０％と記載されており、従って本発明におけるプロテオグリカンは、炭水化物が約４２．９％であることから計算して、純度は約４６％と推定される。またプロテオグリカンの分子量は約２００万であった。 As a result of these analyses, it was found that 35.0% protein, 21.5% ash, 42.9% carbohydrate, and 0.6% lipid were present in the solid content. According to Patent Document 1, the weight ratio of the core protein of proteoglycan is described as about 7.0%. Therefore, the proteoglycan in the present invention has a purity of about 42.9% as calculated from the carbohydrate. It is estimated at about 46%. The molecular weight of proteoglycan was about 2 million.
上記の実施例１に示す操作において、スークロース脂肪酸エステル溶液の濃度を０．１％、５．０％、１０％に変えて２４時間浸漬、抽出を行ったときのプロテオグリカンの回収量（ウロン酸量）の経時変化を調べた結果、ほぼ同様な結果が得られた。また、上記の実施例１に示す操作において、スークロース脂肪酸エステルを０．６６６Ｎの酢酸に変えて２４時間浸漬、抽出を行ったときのプロテオグリカンの回収量（ウロン酸量）の経時変化を調べた結果、実施例１に比べて劣った。 In the operation shown in Example 1 above, the recovery amount of proteoglycan (the amount of uronic acid) when the concentration of the sucrose fatty acid ester solution was changed to 0.1%, 5.0%, and 10% for 24 hours soaking and extraction As a result of examining the change over time of), almost the same result was obtained. In addition, in the operation shown in Example 1 above, the results of examining the change over time in the recovered amount of proteoglycan (uronic acid amount) when sucrose fatty acid ester was changed to 0.666N acetic acid and immersed and extracted for 24 hours Compared with Example 1, it was inferior.
＜実施例２＞
−４０℃で冷凍保管したシロサケの頭部から摘出した鼻軟骨を電動のミートチョッパーで細かく破砕しミンチ状にしたものをアセトンに浸漬し、鼻軟骨から脱脂及び脱水を行った。処理後の鼻軟骨を通風又は減圧乾燥したもの２４．００ｇを出発原料とした。５リットルの抽出用容器にあらかじめ５℃に冷却しておいた蒸留水２９７０．０ｇを入れ、さらに実施例１で使用した固形のスークロース脂肪酸エステル３０．０ｇを投入し、総量３０００．００ｇ（１．０重量％）のスークロース脂肪酸エステル水溶液を準備した。この抽出容器に出発原料２４．００ｇを投入し、スターラーを用いて攪拌しながら、１２時間浸漬した。
<Example 2>
The nasal cartilage extracted from the head of a salmon salmon frozen at −40 ° C. was finely crushed with an electric meat chopper and minced, and immersed in acetone to degrease and dehydrate the nasal cartilage. 24.00 g of the nasal cartilage that had been treated and ventilated or dried under reduced pressure was used as a starting material. Into a 5 liter extraction vessel, 2970.0 g of distilled water cooled to 5 ° C. in advance was added, and 30.0 g of the solid sucrose fatty acid ester used in Example 1 was added, for a total amount of 3000.00 g (1. 0% by weight) of a sucrose fatty acid ester aqueous solution was prepared. 24.00 g of starting material was put into this extraction container and immersed for 12 hours while stirring with a stirrer.
浸漬終了後、内容物を１mm角のステンレススチール製こし器をセットした別の容器に移し、鼻軟骨を除去して、プロテオグリカンを含む抽出液を回収した。 After the immersion, the contents were transferred to another container set with a 1 mm square stainless steel strainer, the nasal cartilage was removed, and the extract containing proteoglycan was collected.
抽出液を日立ｈｉｍａｃＣＦ７Ｄ２型の遠心分離機を用いて３０００rpm、２０分の遠心分離を行い、固形分ならびに油脂分を除去して、プロテオグリカンを含む液相を回収した。 The extract was centrifuged at 3000 rpm for 20 minutes using a Hitachi himacCF7D2 type centrifuge to remove solids and oils and fats, and a liquid phase containing proteoglycan was recovered.
さらに、この液相をフィルターペーパー（アドバンテック社製）を用いて濾過し、濾液の６倍量の蒸留水を加えた後、日本ミリポア製 ＢＩＯＭＡＸ １００Ｋ ＰＯＬＹＥＴＨＥＲＳＵＬＦＯＮＥ（分画分子量１０万）を用いて分画と濃縮を同時に行った。 Furthermore, this liquid phase is filtered using a filter paper (manufactured by Advantech), 6 times the amount of distilled water is added to the filtrate, and then fractionated using BIOMAX 100K POLYETHERSULFONE (fraction molecular weight 100,000) manufactured by Nihon Millipore. And concentrating at the same time.
得られた濃縮液の一部を採取し、液中の固形分重量を測定した。測定は、乾燥炉（ＹＡＭＡＴＯ ＤＸ４０１）により、１０５℃、１６時間乾燥させ、完全に水分を蒸発させた後、残った固形分をデジタル計量器（Ａ＆Ｄ社 ＧＦ−４００）で精密に測定した。その結果、２４．００ｇの出発原料から、換算値でその２２．２１％に相当する５．５０ｇの乾燥固形分を得ることができた。 A part of the concentrated liquid obtained was collected and the solid content weight in the liquid was measured. The measurement was performed by drying at 105 ° C. for 16 hours with a drying furnace (YAMATO DX401), and after completely evaporating water, the remaining solid content was precisely measured with a digital meter (A & D GF-400). As a result, it was possible to obtain 5.50 g of dry solid content corresponding to 22.21% in terms of conversion value from 24.00 g of the starting material.
また、濃縮液をアミノ酸自動分析装置（日立製作所社製、Ｌ−８５００ Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いてアミノ酸量を測定することで濃縮液中のコラーゲン量を定量するとともに、ガランボス法によりウロン酸量を定量してプロテオグリカン量を計算した。さらに高速液体クロマトグラフィ装置（島津製作所、カラムＴＳＫ−ＧＥＬ Ｇ４０００ＰＷＸＬ）を用いて、プロテオグリカンの分子量を測定した。 In addition, the amount of amino acid in the concentrated solution is measured using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) to quantify the amount of collagen in the concentrated solution, and the amount of uronic acid is determined by the galambos method. The amount of proteoglycan was calculated by quantification. Furthermore, the molecular weight of proteoglycan was measured using a high performance liquid chromatography apparatus (Shimadzu Corporation, column TSK-GEL G4000PWXL).
これらの分析の結果、固形分中にタンパク質１４．８％、灰分１８．４％、炭水化物６４．８％、脂質０．０％が存在することが判った。また、プロテオグリカンの分子量は約１３０万であった。 As a result of these analyses, it was found that 14.8% protein, 18.4% ash, 64.8% carbohydrate, and 0.0% lipid were present in the solid content. The molecular weight of proteoglycan was about 1.3 million.
＜実施例３＞
−４０℃で冷凍保管したシロサケの頭部から摘出した鼻軟骨を電動のミートチョッパーで細かく破砕しミンチ状にしたものをアセトンに浸漬し、鼻軟骨から脱脂及び脱水を行った。処理後の鼻軟骨を通風又は減圧乾燥したもの１７．９０ｇを出発原料とした。５リットルの抽出用容器にあらかじめ５℃に冷却しておいた蒸留水２４９５．０ｇを入れ、さらに固形のキラヤサポニン５．０ｇを投入し、総量２５００ｇ（０．２重量％）のサポニン水溶液を準備した。この抽出容器に出発原料１７．９０ｇを投入し、スターラーを用いて攪拌しながら、１２時間浸漬した。
<Example 3>
The nasal cartilage extracted from the head of a salmon salmon frozen at −40 ° C. was finely crushed with an electric meat chopper and minced, and immersed in acetone to degrease and dehydrate the nasal cartilage. 17.90 g of the processed nasal cartilage that was ventilated or dried under reduced pressure was used as a starting material. Add 2495.0 g of distilled water that has been cooled to 5 ° C. in a 5 liter extraction container, and then add 5.0 g of solid Quillaja saponin to prepare a total 2,500 g (0.2 wt%) saponin aqueous solution. did. 17.90 g of starting material was put into this extraction container and immersed for 12 hours while stirring with a stirrer.
浸漬終了後、内容物を１mm角のステンレススチール製こし器をセットした別の容器に移し、鼻軟骨を除去して、プロテオグリカンを含む抽出液を回収した。 After the immersion, the contents were transferred to another container set with a 1 mm square stainless steel strainer, the nasal cartilage was removed, and the extract containing proteoglycan was collected.
抽出液を日立ｈｉｍａｃＣＦ７Ｄ２型の遠心分離機を用いて３０００rpm、２０分の遠心分離を行い、固形分ならびに油脂分を除去して、プロテオグリカンを含む液相を回収した。 The extract was centrifuged at 3000 rpm for 20 minutes using a Hitachi himacCF7D2 type centrifuge to remove solids and oils and fats, and a liquid phase containing proteoglycan was recovered.
さらに、この液相をフィルターペーパー（アドバンテック社製）を用いて濾過し、濾液の６倍量の蒸留水を加えた後、日本ミリポア製 ＢＩＯＭＡＸ １００Ｋ ＰＯＬＹＥＴＨＥＲＳＵＬＦＯＮＥ（分画分子量１０万）を用いて分画と濃縮を同時に行った。 Furthermore, this liquid phase is filtered using a filter paper (manufactured by Advantech), 6 times the amount of distilled water is added to the filtrate, and then fractionated using BIOMAX 100K POLYETHERSULFONE (fraction molecular weight 100,000) manufactured by Nihon Millipore. And concentrating at the same time.
得られた濃縮液の一部を採取し、液中の固形分重量を測定した。測定は、乾燥炉（ＹＡＭＡＴＯ ＤＸ４０１）により、１０５℃、１６時間乾燥させ、完全に水分を蒸発させた後、残った固形分をデジタル計量器（Ａ＆Ｄ社 ＧＦ−４００）で精密に測定した。その結果、１７．９０ｇの出発原料から換算値でその１８．４１％に相当する３．２９ｇの乾燥固形分を得ることができた。 A part of the concentrated liquid obtained was collected and the solid content weight in the liquid was measured. The measurement was performed by drying at 105 ° C. for 16 hours with a drying furnace (YAMATO DX401), and after completely evaporating water, the remaining solid content was precisely measured with a digital meter (A & D GF-400). As a result, 3.29 g of dry solid content corresponding to 18.41% of the converted value was obtained from 17.90 g of the starting material.
また、濃縮液をアミノ酸自動分析装置（日立製作所社製、Ｌ−８５００ Ａｍｉｎｏ Ａｃｉｄ Ａｎａｌｙｚｅｒ）を用いてアミノ酸量を測定することで濃縮液中のコラーゲン量を定量するとともに、ガランボス法によりウロン酸量を定量してプロテオグリカン量を計算した。さらに高速液体クロマトグラフィ装置（島津製作所、カラムＴＳＫ−ＧＥＬ Ｇ４０００ＰＷＸＬ）を用いて、プロテオグリカンの分子量を測定した。 In addition, the amount of amino acid in the concentrated solution is measured using an automatic amino acid analyzer (manufactured by Hitachi, Ltd., L-8500 Amino Acid Analyzer) to quantify the amount of collagen in the concentrated solution, and the amount of uronic acid is determined by the galambos method. The amount of proteoglycan was calculated by quantification. Furthermore, the molecular weight of proteoglycan was measured using a high performance liquid chromatography apparatus (Shimadzu Corporation, column TSK-GEL G4000PWXL).
これらの分析の結果、固形分中にタンパク質１７．０％、灰分２２．４％、炭水化物６０．６％、脂質０．０％が存在することが判った。また、プロテオグリカンの分子量は約１２０万であった。 As a result of these analyses, it was found that 17.0% protein, 22.4% ash, 60.6% carbohydrate, and 0.0% lipid were present in the solid content. The molecular weight of proteoglycan was about 1.2 million.
本発明は、従来の抽出方法に比べ、プロテオグリカンを未変成、未分解の状態で簡便かつ短時間で回収することができ、プロテオグリカンの製造コストを大きく減ずることができ、また、本来廃棄処分とされてきた魚類、鳥類及び哺乳類などの廃棄部位から産業上有用性の高いプロテオグリカンを回収することができ、産業廃棄物の有効利用並びに産業廃棄物自体の減量化にも貢献することができる。 Compared with conventional extraction methods, the present invention can recover proteoglycan in an unmodified and undegraded state easily and in a short period of time, greatly reducing the production cost of proteoglycan, and is essentially discarded. Proteoglycans with high industrial utility can be recovered from waste sites such as fish, birds and mammals, which can contribute to effective utilization of industrial waste and reduction of industrial waste itself.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2012144400A JP6006545B2 (en) | 2012-06-27 | 2012-06-27 | Proteoglycan production method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2012144400A JP6006545B2 (en) | 2012-06-27 | 2012-06-27 | Proteoglycan production method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2014009164A JP2014009164A (en) | 2014-01-20 |
| JP6006545B2 true JP6006545B2 (en) | 2016-10-12 |
Family
ID=50106165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2012144400A Active JP6006545B2 (en) | 2012-06-27 | 2012-06-27 | Proteoglycan production method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6006545B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6855042B2 (en) * | 2016-10-04 | 2021-04-07 | 日本薬品株式会社 | Food compositions and oral pharmaceutical compositions for the prevention, alleviation or treatment of arthropathy symptoms |
| JP2018090552A (en) * | 2016-12-07 | 2018-06-14 | 株式会社ウェルヴィーナス | Arthralgia improver |
| JP2018151206A (en) * | 2017-03-10 | 2018-09-27 | 一丸ファルコス株式会社 | Method for analyzing proteoglycan |
| WO2024029598A1 (en) * | 2022-08-04 | 2024-02-08 | 日本薬品株式会社 | Composition for improving or maintaining muscle amount or muscle strength |
| JP7402460B1 (en) * | 2023-02-16 | 2023-12-21 | 国立大学法人鳥取大学 | Proteoglycan production method and composition |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3756838B2 (en) * | 2002-04-09 | 2006-03-15 | 日本メナード化粧品株式会社 | Topical skin preparation |
| EP2455097B1 (en) * | 2009-07-16 | 2016-02-17 | Sunstar Inc. | Proteoglycan-containing material |
| JP2013039087A (en) * | 2011-08-18 | 2013-02-28 | Fuji Agri Arsh:Kk | Drinking water containing proteoglycan and method for producing the same |
-
2012
- 2012-06-27 JP JP2012144400A patent/JP6006545B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2014009164A (en) | 2014-01-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4219974B2 (en) | Proteoglycan production method | |
| JP6006545B2 (en) | Proteoglycan production method | |
| EP2024473B1 (en) | Extraction of highly unsaturated lipids with liquid dimethyl ether | |
| RU2498620C2 (en) | Method for reduction of fluoride concentration during production of krill protein concentrate | |
| JP4181305B2 (en) | Methods for extracting lipids from marine and freshwater animal tissues | |
| US8568819B2 (en) | Solid composition containing lipids from crustaceans | |
| US6346276B1 (en) | Composition containing useful substances originating in fishes and shellfishes and process for the preparation of the substances | |
| CN107105692B (en) | Solvent-free method for obtaining phospholipid-rich krill oil and neutral lipid-rich krill oil by melting and evaporation | |
| CN107586320A (en) | A kind of brown croaker air bladder reducing blood lipid oligopeptides and its application | |
| JP2012201614A (en) | Method for producing proteoglycan | |
| RU2287959C2 (en) | Method for producing of natural structurizers from fish wastes | |
| JP2009227765A (en) | Manufacturing method of plasmalogen containing lipid | |
| KR102085775B1 (en) | Method for separating and purifying DHA from tuna bark | |
| JP2012017312A (en) | Method for producing glycoprotein complex | |
| NO20100359A1 (en) | Peptide material and preparations and uses thereof. | |
| JP6855042B2 (en) | Food compositions and oral pharmaceutical compositions for the prevention, alleviation or treatment of arthropathy symptoms | |
| US20170058233A1 (en) | Method for fractionation of a protein and lipid containing material | |
| NO20100370A1 (en) | Peptide material, feed compositions and preparations, and uses thereof. | |
| KR102180261B1 (en) | Method for extraction of heparin derived from by-product of pork | |
| TR2021012454A2 (en) | PRODUCTION METHOD OF PROTEIN HYDROLysate ISOLATED FROM MECHANICALLY SEPARATED TURKEY MEAT AND PROTEIN HYDROLysate OBTAINED BY THIS METHOD | |
| TH2003002653C3 (en) | Method for extracting bamboo pulp mushrooms that can stimulate the growth of fibroblast cell cultures at high levels. | |
| CN113603768A (en) | Preparation method of fish-derived collagen | |
| RU2494642C2 (en) | Method for manufacture of natural structure forming agent | |
| Yeo et al. | Characterization of Freeze-and Spray-Dried Edible Bird’s Nest Hydrolysates | |
| JP2003289811A (en) | Water-soluble fraction of residue of dissected edible bird |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20141007 |
|
| RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20150316 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20151027 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20151224 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20160419 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20160715 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20160727 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20160906 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20160909 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6006545 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |