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Development of High Purity Purification Method of Chondroitin Sulfate Extracted from Skate Cartilage

홍어 연골로부터 추출된 콘드로이틴 황산의 고순도 정제방법 개발

  • Jeong, Kap-Seop (Department of Food Science & Nutrition, Tongmyong University)
  • Received : 2016.03.17
  • Accepted : 2016.06.02
  • Published : 2016.06.30

Abstract

A purification method was established for high-purity chondroitin sulfate from skate cartilage. Hydrolytic extraction of skate backbone cartilage was investigated with the proteases alcalase and protamex, and the extraction contents of chondroitin sulfate were measured with several physicochemical processes. The yield of extract from skate cartilage with $40^{\circ}Brix$ concentration was 23.3% with 2% alcalase hydrolysis, which was decreased to 8.47% and 3.37% with the first and second additional ethanol purifications, respectively. The yield was 16.62% with one ethanol purification after hydrolysis with a mixture of 1% alcalase and 1% protamex. The content of chondroitin sulfate was measured as 39.88-45.08% with different ratios of ethanol solvent. The content was 42.92% at a solvent ratio of 1:1 with alcalase protease and 45.08% with a ratio of 1:2 using a protease mixture of alcalase and protamex. The molecular weight range of chondroitin sulfate was about 110-310 thousand Da, and the purity of chondroitin sulfate was 24.87-49.92% with a mixture of alcalase and protamex in GPC analysis. The maximum purity of chondroitin sulfate was 53.93% after ultrafiltration. The odor strength of chondroitin sulfate was decreased by 33% and 38% after ethanol purification and additional filtration with activated carbon, respectively. The odor concentration of ammonia and TMA from chondroitin sulfate was decreased by 52.1% and 37.89% with activated carbon filtration and two ethanol purifications, respectively, but it was necessary to eliminate the odor components efficiently using additional physicochemical processes.

Alcalase와 protamex의 두 가지 단백질 분해효소를 사용하여 홍어연골로부터 콘드로이틴 황산을 분해추출하고, 몇가지 물리화학적 공정을 적용하여 고순도의 콘드로이틴 황산을 얻기 위한 정제방법을 검토하였다. 2% alcalase로 홍어연골을 가수분해하고 $40^{\circ}Brix$로 농축한 추출물의 수율은 23.3%이었으나, 에탄올로 1회 및 2회 추가 정제한 경우 각각 8.47%, 3.37%로 얻어졌다. 1% alcalase와 1% protamex로 혼합 사용하여 추출분해하고, $40^{\circ}Brix$로 농축한 다음 1회 에탄올 정제한 경우는 추출수율이 16.62%로 측정되어 단백질 분해효소를 혼합한 경우가 더 높은 수율을 보였다. 추출된 콘드로이틴 황산의 함량은 에탄올 용매비에 따라 39.88~45.08%의 범위로 측정되었으며, alcalase만 사용시에는 용매비 1:1에서 42.92%로 가장 높게 측정되었고, alcalase와 protamex를 혼합 사용한 경우에는 용매비 1:2에서 45.08%로 가장 높았으며, 에탄올에 의한 농축물의 정제시 교반 시간은 2시간이 효과적이었다. 콘드로이틴 황산의 GPC (Gel Permeation Chromatography) 측정 결과 alcalase와 protamex를 혼합 사용한 경우 에탄올 정제 횟수에 따라 콘드로이틴 황산의 분자량과 순도는 각각 11만~31만 Da.과 24.87%~49.92%의 범위로 측정되었으며, 한외여과를 통하여 분자량 약 11만 Da., 최고 순도 53.93%의 콘드로이틴 황산을 얻을 수 있었다. 콘드로이틴 황산의 냄새 강도는 에탄올 정제만으로는 33%, 활성탄 처리와 에탄올 정제를 병행한 경우 38%의 감소효과가 얻어졌으며, 활성탄 처리와 2회의 에탄올 정제시 암모니아는 52.1%, TMA (trimethylamine)는 37.89%의 탈취효과가 있었으나, 냄새성분의 충분한 제거를 위해서는 추가적인 물리화학적 처리가 요구되었다.

Keywords

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