KR20110055149A - Extraction method of chondromucoprotein for cartilage of cuttlefish and method for chondroitin sulfate using chondromucoprotein - Google Patents
Extraction method of chondromucoprotein for cartilage of cuttlefish and method for chondroitin sulfate using chondromucoprotein Download PDFInfo
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- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 229920001287 Chondroitin sulfate Polymers 0.000 title claims abstract description 36
- 229940059329 chondroitin sulfate Drugs 0.000 title claims abstract description 36
- 210000000845 cartilage Anatomy 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000000605 extraction Methods 0.000 title claims abstract description 14
- 241000238371 Sepiidae Species 0.000 title abstract 5
- 108091005804 Peptidases Proteins 0.000 claims abstract description 19
- 239000004365 Protease Substances 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims abstract description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract 3
- 241000238366 Cephalopoda Species 0.000 claims description 25
- 102000035195 Peptidases Human genes 0.000 claims description 16
- 230000007062 hydrolysis Effects 0.000 claims description 13
- 238000006460 hydrolysis reaction Methods 0.000 claims description 13
- 235000019419 proteases Nutrition 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 229920002567 Chondroitin Polymers 0.000 claims description 7
- 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 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 238000003809 water extraction Methods 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 235000019833 protease Nutrition 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims 2
- 238000001914 filtration Methods 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 235000013376 functional food Nutrition 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 239000002537 cosmetic Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 229920002683 Glycosaminoglycan Polymers 0.000 description 3
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 3
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000005868 electrolysis reaction Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000001223 reverse osmosis Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 102000018389 Exopeptidases Human genes 0.000 description 1
- 108010091443 Exopeptidases Proteins 0.000 description 1
- 241000251511 Holothuroidea Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000001621 Mucoproteins Human genes 0.000 description 1
- 108010093825 Mucoproteins Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- -1 isopropyl alcohols Chemical class 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/10—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from hair, feathers, horn, skins, leather, bones, or the like
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- 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
-
- 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
- A23V2250/00—Food ingredients
- A23V2250/54—Proteins
- A23V2250/542—Animal Protein
- A23V2250/543—Fish protein
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- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cosmetics (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
본 발명은 오징어 연골에서의 콘드로뮤코단백질(chondromucoprotein)의 추출방법 및 콘드로뮤코단백질을 이용한 콘드로이친황산(chondroitin sulfate)의 제조방법에 관한 것으로서, 보다 구체적으로는 오징어 연골에서 콘드로뮤코단백질을 추출하고 이를 이용하여 기능성 물질인 콘드로이친황산을 간편하고 효율적으로 대량 제조할 수 있는 제조방법을 제공코자 하는 것이다.The present invention relates to a method for extracting chondroicocoprotein (chondromucoprotein) from squid cartilage and to a method for preparing chondroitin sulfate (chondroitin sulfate) using chondroicoprotein, and more specifically, to extract chondroicoprotein from squid cartilage. And to use this to provide a manufacturing method that can be easily and efficiently mass-produced functional substance chondroitin sulfate.
콘드로이친황산은 포유동물을 비롯한 각종 동물의 연골조직에 다량 함유되어 있는 점질성 다당류로 관절염 예방 및 치료, 각막보호, 피부노화억제, 항종양 등 다양한 생리활성을 가지고 있다. Chondroitin sulfate is a viscous polysaccharide contained in cartilage tissues of various animals including mammals and has various physiological activities such as prevention and treatment of arthritis, corneal protection, skin aging inhibition, and anti-tumor.
이러한 생리활성으로 인해 콘드로이친황산은 부가가치가 높은 기능성 소제로 건강기능식품 및 화장품, 의약품 소재로 널리 이용되고 있다.Due to this physiological activity, chondroitin sulfate is a high value-added functional ingredient and is widely used as a health functional food, cosmetics, and pharmaceutical materials.
콘드로이친황산을 제조하기 위해서 팔쇼 등(Falshaw, R. et al., Carbohydrate polymers, 41, 357-364(2000))은 오징어 껍질 및 연골조직으로부터 음이온 교환크로마토그래피 및 크기 배재 크로마토그래피 등을 수행하여 콘드로이친황산을 분리하였으나, 이 방법은 고가의 장치가 필요하고 처리량도 제한적이라 산업에 적용하기 힘든 단점이 있다. To prepare chondroitin sulfate, Falshaw et al. (Falshaw, R. et al., Carbohydrate polymers, 41, 357-364 (2000)) performed chondroitin by anion exchange chromatography and size exclusion chromatography from squid skin and cartilage tissue. Sulfuric acid was separated, but this method requires expensive equipment and limited throughput, which makes it difficult to apply to industry.
미국특허 제4,302,577호에는 소의 기관 연골을 산과 알칼리 용액 중에서 효소 분해한 후 세틸 피리디니움 클로라이드(CPC) 또는 n-알킬 디메틸 벤질 암모늄 클로라이드를 가하여 콘드로이친황산과의 복합체를 형성시킨 다음 알칼리 조건하에서 복합체를 재 분리하고 중화시키는 일련의 과정을 통해 콘드로이친황산을 분리하는 방법이 등록되어 있으나, 이 방법은 과정이 복잡하며 CPC와 n-알킬 디메틸 벤질 암모늄 클로라이드와 같은 유독물을 사용함으로 식품산업에 적용하기 어렵다. U.S. Patent No. 4,302,577 discloses a complex of bovine organ cartilage in acid and alkaline solution followed by the addition of cetyl pyridinium chloride (CPC) or n-alkyl dimethyl benzyl ammonium chloride to form a complex with chondroitin sulfate. Although a method of separating chondroitin sulfate is registered through a series of re-separation and neutralization processes, the process is complicated and difficult to apply to the food industry by using toxic substances such as CPC and n-alkyl dimethyl benzyl ammonium chloride.
대한민국 특허공고 제167831호에는 해삼을 단백분해효소로 가수분해한 후 이가수분해물을 알코올로 분별 침전시켜 콘드로이친황산을 분리하는 방법이 기재되어 있는데, 이 방법은 비교적 간단한 공정으로 이루어져 있으나 대상 원료인 해삼이 고가여서 산업화가 힘들고, 또한 질소의 함량이 높은 문제를 안고 있다. Korean Patent Publication No. 167831 describes a method for separating chondroitin sulfate by hydrolyzing sea cucumber with proteolytic enzymes and then precipitating dihydrolyzate with alcohol, which is a relatively simple process. Due to this high price, industrialization is difficult and the nitrogen content is high.
한편 대한민국 공개특허 제99-33739호에는 멍게껍질을 단백분해효소로 가수분해한 후 트리클로로아세트산(TCA)으로 침전시키고 상등액을 다시 에탄올에 침전시켜 조당류를 얻고, 이 조당류를 NaCl이 함유된 인산완충용액으로 유리칼럼 크로마토그래피를 수행한 후 에탄올로 침전시키고, 이 침전물을 다시 세파덱스G-52칼럼 크로마토그래피를 수행한 후 에탄올에 침전시킨 다음 투석 및 동결 건조하는 일련의 방법으로 정제된 황산 다당류를 얻는 방법이 기재되어 있다.On the other hand, Korean Patent Publication No. 99-33739 discloses that the sea shell is hydrolyzed by protease and then precipitated with trichloroacetic acid (TCA), and the supernatant is precipitated again with ethanol to obtain crude sugars. Purified sulfuric acid by phosphate buffer solution, precipitated with ethanol, and precipitated again with Sephadex G-52 column chromatography, precipitated in ethanol, and purified by dialysis and lyophilization. A method of obtaining polysaccharides is described.
그러나 이 역시 유독물인 TCA를 사용하고 공정이 복잡하여 전공정이 1주일 이상의 시간이 소요되어 산업적인 효용이 없다고 할 수 있다. However, this also uses toxic TCA and the process is complicated, so the entire process takes more than a week, so there is no industrial utility.
대한민국 특허 제416884호에는 동물의 연골조직을 엔도프로타제(endoprotase)로 가수분해하고 이 가수분해물에 엔도프로타제와 액소펩티다제(exopeptidase)의 혼합물로 가수분해하고 여기에 염을 가하고 알코올로 분별침전 시키고 이 침전물을 탈염하여 콘드로이친황산을 정제하는 방법이 게시되어 있으나, 이 방법 또한 공정이 복잡하고 염을 가하고 나중에 가한 염을 다시 탈염하는 등의 공정으로 시간의 소요가 많아 대량생산하기에는 부족한 면이 많고 분별 침전시 사용되는 이소프로필알콜은 식품류에 사용이 엄격히 규제되어 있다.Korean Patent No. 416884 describes hydrolysis of animal cartilage tissue with endoprotase, hydrolysis with a mixture of endoprotase and exopeptidase, adding salt to it and fractionating with alcohol. There is a method of refining chondroitin sulfate by precipitating and desalting the precipitate, but this method is also complicated to process and adds salt and desalting salt added later. Many isopropyl alcohols used for fractional precipitation are strictly regulated for use in foods.
이에 본 발명자는 오징어 연골에서 콘드로뮤코단백질을 추출하고 이를 이용하여 건강기능성식품의 소재 및 화장품소재와 의약품 소재로 사용이 가능한 콘드로이친황산을 간단한 공정과 효율적 정제방법으로 생산원가를 절감하고 대량생산하는 방법을 제공함에 발명의 기술적 과제를 두고 본 발명을 완성한 것이다.Therefore, the present inventors extract chondroicoprotein from squid cartilage and reduce the production cost and mass-produce the chondroitin sulfate, which can be used as a health functional food material, cosmetic material and pharmaceutical material, by simple process and efficient purification method. The present invention has been made in view of the technical problem of the present invention.
과제 해결수단으로 본 발명에서는 염분을 제거한 분쇄된 오징어 연골을 물에 대한 중량비율이 20~30%(W/V), 온도 120~135℃, 추출시간 90~150분, 압력 0.3~1.0MPa으로 열수추출하고 필터하여 콘드로뮤코단백질을 추출하였다.In the present invention as a means for solving the problem, the weight ratio of the squid cartilage to remove the salt is 20 to 30% (W / V), temperature 120 ~ 135 ℃, extraction time 90 ~ 150 minutes, pressure 0.3 ~ 1.0MPa Hot water was extracted and filtered to extract chondromocoprotein.
또한 열수추출한 후 남은 오징어 연골을 단백분해효소로 가수분해하여 콘드로뮤코단백질을 추출하였다.In addition, chondroitin protein was extracted by hydrolyzing the remaining squid cartilage with hydrolase after hydrothermal extraction.
그리고 본 발명에서는 상기 열수추출 또는 단백분해효소로 가수분해 추출한 콘드로뮤코단백질 또는 이들을 혼합한 것을 단백분해효소로 다시 가수분해하여 콘드로이친황산을 제조하였으며, 이렇게 제조된 콘드로이친황산을 다단계 분리막 공정을 이용하여 여러 단계로 분획하여 정제하였다.In the present invention, chondroicosulfuric acid hydrolyzed by hydrothermal extraction or protease, or a mixture thereof, was hydrolyzed again using protease to prepare chondroitin sulfate, and thus prepared chondroitin sulfate using a multistage membrane process. Purification by fractionation in several steps.
본 발명에서는 오징어 연골을 고온, 가압 하에서 열수추출과 단백분해효소로 가수분해하는 두가지 방법을 혼용하여 오징어 연골에 함유된 콘드로뮤코단백질(뮤코다당단백질)을 추출하고 이 추출물을 다시 단백분해효소로 가수분해하여 다단계 막분리공정을 사용하여 간략하게 정제된 콘드로이친황산을 생산하고, 이렇게 얻어진 콘드로이친황산은 건강기능식품의 소재 및 의약품원료, 화장품의 원료로 이용할 수 있다. In the present invention, a mixture of two methods of hydrothermal extraction and proteolytic enzyme hydrolysis of squid cartilage under high temperature and pressure is used to extract chondrogen mucoprotein (mucopolysaccharide protein) contained in squid cartilage, and the extract is re-protease. Hydrolyzed to produce purified Chondroitin Sulfate simply by using a multi-stage membrane separation process. Chondroitin Sulfate thus obtained can be used as a raw material for health functional foods, as a raw material for pharmaceuticals and cosmetics.
이하 본 발명에서 제공하는 오징어 연골에서의 콘드로뮤코단백질의 추출방법 과 추출된 콘드로뮤코단백질을 이용한 콘드로이친황산의 제조방법을 상세히 설명한다.Hereinafter, a method for extracting chondroicoprotein from squid cartilage provided by the present invention and a method for preparing chondroitin sulfate using the extracted chondroicoprotein are described in detail.
1. 오징어 연골에서의 콘드로뮤코단백질의 추출방법1. Extraction of Chondromocoproteins from Squid Cartilage
본 발명에서 제공하는 콘드로뮤코단백질의 추출방법으로는 열수추출방법과, 열수추출 후 남은 오징어 연골을 단백분해효소로 가수분해하는 두가지 방법으로 이루어지는 것으로, Chondroitin protein extraction method provided by the present invention comprises a hot water extraction method and two methods of hydrolyzing the squid cartilage remaining after the hot water extraction with protease,
먼저 오징어 연골을 적당한 크기(1~3㎜정도)로 분쇄한 다음 정제된 물(상수도, 증류수 등)에 침지시켜 3~6시간 팽윤시킨 다음 수세하여 오징어 연골이 갖고 있는 염분을 제거한다.First, squid cartilage is crushed to a suitable size (about 1-3mm), immersed in purified water (water, distilled water, etc.) and swollen for 3 to 6 hours, and then washed with water to remove salts of squid cartilage.
1)열수추출방법 1) Hot water extraction method
상기와 같이 오징어 연골에서 염분을 제거하고 적당한 크기로 분쇄한 상태에서 반응기에 넣고 고온.가압하여 열수추출 한다. Remove salt from squid cartilage as above and put it into the reactor in a state of crushing to a suitable size and extract hot water by high temperature and pressure.
상기 열수추출 조건으로는 오징어 연골과 물에 대한 비율은 20~30%(W/V)가 적당하며, 온도는 120~135℃, 추출시간은 90~150분 정도가 좋으며, 이때 압력은 0.3~1.0MPa 정도가 바람직하다. As the hot water extraction conditions, the ratio of squid cartilage and water is 20 to 30% (W / V), and the temperature is 120 to 135 ° C and the extraction time is about 90 to 150 minutes, and the pressure is 0.3 to About 1.0 MPa is preferable.
상기와 같이 열수추출 하는 매우 간단한 방법으로 오징어 연골에 포함되어 있는 콘드로뮤코단백질을 회수할 수 있으며 회수율은 오징어 연골에 포함되어 있는 전체 콘드로뮤코단백질 대비 대략 10~20%이며, 열수추출된 콘드로뮤코단백질은 필터하여 보관한다.As described above, it is possible to recover the chondromocoproteins contained in squid cartilage by a very simple method of hot water extraction, and the recovery rate is about 10-20% of the total chondromocoproteins contained in the squid cartilage. Romucoproteins are filtered and stored.
2)단백분해효소로 가수분해하는 방법2) Hydrolysis with Protease
상기 열수추출하고 필터하여 남은 오징어 연골을 단백분해효소로 가수분해하여 나머지 콘드로뮤코단백질을 추출하고 필터하여 모은다. The hot water extracted and filtered to hydrolyze the remaining squid cartilage with protease to extract and filter the remaining chondromocoproteins.
이때 사용하는 효소로는 알카라제와 프로티나아제 등이 있으며, 다른 알려진 효소들을 사용하여도 무방하다.At this time, the enzymes used include alkalase and proteinase, and other known enzymes may be used.
효소반응 조건은 효소의 양은 오징어 연골 건조물 중량에 대한 비율이 0.5~2.5%(W/V)가 적당하며, pH는 5.0~10.0이 적당하고 이때 pH조절은 완충용액이 아닌 전기분해수를 이용하여 pH를 맞춘다. Enzyme reaction condition is suitable for the amount of enzyme is 0.5 ~ 2.5% (W / V) to the weight of dried squid cartilage, pH is 5.0 ~ 10.0, and the pH is adjusted by using electrolysis water, not buffer solution. Adjust the pH.
온도는 40~55℃가 적당하며, 시간은 4~6시간이 적당하다. Temperature is suitable 40 ~ 55 ℃, time is 4 ~ 6 hours is appropriate.
상기와 같이 가수분해하여 추출한 콘드로뮤코단백질 내에는 단백분해효소가 활동하고 있으므로 90 ~ 95℃에서 약 15분 가열하여 효소를 실활시킨다.The protease is active in the hydrolyzed chondroitin protein extracted as described above, so that the enzyme is inactivated by heating at 90-95 ° C. for about 15 minutes.
2.콘드로뮤코단백질을 이용한 콘드로이친황산의 제조방법2. Preparation of Chondroitin Sulfate Using Chondroitin Protein
그리고 본 발명에서는 상기와 같이 열수추출방법과, 열수추출 후 남은 오징어 연골을 단백분해효소로 가수분해하는 방법으로 추출한 콘드로뮤코단백질을 이용하여 콘드로이친황산을 제조한다.In the present invention, chondroitin sulfate is prepared by using chondroitin sulfate extracted by hydrothermal extraction method and hydrolysis of squid cartilage remaining after hydrothermal extraction with protease.
1)콘드로이친황산의 제조방법1) Method of producing chondroitin sulfate
본 발명에서의 콘드로이친황산의 제조방법으로는 상기 열수추출 및 가수분해에 의해 추출된 콘드로뮤코단백질(뮤코다당단백질)을 단독 또는 혼합하여 다시 단백분해효소로 가수분해하면 된다.In the method for preparing chondroitin sulfate in the present invention, chondroimucoprotein (mucopolysaccharide protein) extracted by hot water extraction and hydrolysis may be hydrolyzed by protease alone or in combination.
이때 사용하는 효소로는 알카라제와 프로티나아제 등이 있으며, 다른 알려진 효소들을 사용하여도 무방하다.At this time, the enzymes used include alkalase and proteinase, and other known enzymes may be used.
효소반응 조건은 효소의 양은 오징어 연골 건조물 중량에 대한 비율이 0.25~1.25%(W/V)가 적당하며, pH는 5.0~10.0이 적당하고 이때 조절은 완충용액이 아닌 전기분해수를 이용하여 pH를 맞춘다. Enzyme reaction condition is suitable for the amount of enzyme is 0.25 ~ 1.25% (W / V) to the weight of dried squid cartilage, pH is 5.0 ~ 10.0, and the pH is adjusted by using electrolysis water, not buffer solution. To match.
온도는 40~55℃가 적당하며, 시간은 4~6시간이 적당하다. Temperature is suitable 40 ~ 55 ℃, time is 4 ~ 6 hours is appropriate.
상기와 같이 가수분해하여 추출한 콘드로뮤코단백질 내에는 단백분해효소가 활동하고 있으므로 90 ~ 95℃에서 약 15분 가열하여 효소를 실활시킨다.The protease is active in the hydrolyzed chondroitin protein extracted as described above, so that the enzyme is inactivated by heating at 90-95 ° C. for about 15 minutes.
2)다단계 막분리 공정을 이용하여 분자량대별로 분획2) Fractionation by molecular weight band using multi-step membrane separation process
상기와 같은 단백분해효소로 가수분해 하여 얻어지는 콘드로이친황산은 다양한 크기의 입자가 혼합 구성되어 있으며, 이를 그대로 사용할 수 있으나, 본 발명에서는 다단계 막분리 공정을 이용하여 분자량대별로 분획한다. Chondroitin sulfate obtained by hydrolysis with the protease as described above is composed of particles of various sizes are mixed, it can be used as it is, in the present invention is fractionated by molecular weight band using a multi-stage membrane separation process.
즉, 분획분자량(MWCO)10kDalton이하, 10kDalton~30kDalton, 30kDalton~50kDalton, 50kDalton~100kDalton, 100kDalton이상의 여러 단계로 분획한다. That is, fractional molecular weight (MWCO) is fractionated in several stages of 10kDalton or less, 10kDalton ~ 30kDalton, 30kDalton ~ 50kDalton, 50kDalton ~ 100kDalton, 100kDalton or more.
상기 각각의 분획물을 역삼투압막(Reverse Osmosis)을 이용 농축하여 건조한다. Each fraction is concentrated to dryness using reverse osmosis membrane (Reverse Osmosis).
표 1은 분자량대별 분획 건조물의 콘드로이친황산의 함량을 분석한 결과를 나타내었다.(시험방법은 건강기능식품 기준 및 규격의 뮤코다당단백식품 콘드로이친황산 시험법)Table 1 shows the results of analyzing the content of chondroitin sulfate in the fraction dried fractions by molecular weight. (The test method is the mucopolysaccharide protein food chondroitin sulfate test method of health functional food standards and standards)
시험항목Fraction
Test Items
10,000이하MWCO
Less than 10,000
10,000~30,000MWCO
10,000-30,000
30,000~50,000MWCO
30,000 ~ 50,000
50,000~100,000MWCO
50,000-100,000
100,000이상MWCO
More than 100,000
(%)Chondroitin Sulfate
(%)
각 분획물별 콘드로이친황산의 함량이 달라 건강 기능성식품 소재와, 의약품 소재, 화장품 소재 등으로 구분하여 이용할 수 있다.The content of chondroitin sulfate in each fraction is different and can be used by dividing it into health functional food material, pharmaceutical material, and cosmetic material.
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