본 연구에서는 두 단계 효소적 방법을 이용하여 trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA)-enriched triacylglycerols (TAG)를 합성하고자 하였다. 우선 홍화씨유 추출 공액리놀레산의 초기 t10,c12-CLA와 cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA)의 함량은 각각 33.3%와 32.0%이었다. C. rugosa lipase를 반응촉매로 이용하여 홍화씨유 추출 공액리놀레산 내 c9,t11-CLA와 dodecan-1-ol을 에스터화시켜 dodecyl ester 형태로 c9,t11-CLA를 분리시켜 제거한 후t10,c12-CLA를 농축하였다. 이 때 반응은 재순환 충진형 반응기 (recirculating packed bed reactor)에서 수행하였다. 그 결과 t10,c12-CLA의 함량은 54.7%, 수율은 36.4%이었으며 이때의 최적반응 조건은 온도 20 ?C, 기질몰비율 (홍화씨유 추출 CLA: dodecan-1-ol) 1:1, 수분함량 0%, 반응시간 36시간이었다. 다음, C. antartica lipase를 반응촉매로 이용하여 t10,c12-CLA 농축물을 글리세롤 반응시켜 t10,c12-CLA enriched TAG를 합성하였다. 이 때 반응은 진공 회분식 반응기 (vacuum batch type reactor)에서 수행하였다. 그 결과 t10,c12-CLA-enriched TAG의 함량이 90% 이상이 되는 반응물을 얻었으며 이때의 반응조건은 온도 60 ?C, 기질몰비율 (글리세롤:t10,c12-CLA 농축물) 1:3, 기질량 대비 효소량 10%, 진공도 0.4 kPa, 반응시간 12시간이었다. 본 연구에서 합성한 TAG는 항비만 효능을 나타낼 것으로 기대된다.
The aim of this study was to synthesize trans-10,cis-12 conjugated linoleic acid (t10,c12-CLA)-enriched triacylglycerols (TAG) with potential anti-obesity effects via two-step enzymatic reactions. Commercial CLA mixtures, containing 33.3% t10, c12-CLA and 32.0% cis-9,trans-11 CLA (c9,t11-CLA) was esterifed with dodecan-1-ol for selectively removing the c9,t11-CLA in the form of dodecyl esters. The reaction was performed in a recirculating packed bed reactor, using an immobilized lipase from Candida rugosa as a biocatalyst. A free fatty acid fraction containing 54.7% t10,c12-CLA was produced with the yield of 36.4% of initial t10, c12-CLA under the best conditions (temperature, 20 ºC; substrate molar ratio (CLA mixtures to dodecan-1-ol), 1:1; water content, 0%; reaction time, 36 h) established in this study. The free fatty acid fraction enriched in t10,c12-CLA was esterified with glycerol for preparing t10,c12-CLA-enriched TAG. The reaction was performed in a vacuum batch type reactor, using an immobilized lipase from Candida antarctica as a biocatalyst. The best combination of temperature, enzyme loading and vacuum level was 60 ºC, 10% of the total weight of the substrates, and 0.4 kPa, respectively. The t10,c12-CLA-enriched TAG of >90% was successfully synthesized in 12 h.
목차
I. INTRODUCTION 1II. MATERIALS AND METHODS 42.1. Materials 42.2. Lipase-catalyzed selective esterification 42.3. Fractionation of free fatty acids 72.4. Purification of free fatty acids 92.5. Lipase-catalyzed glycerolysis 112.6. Fatty acid composition 132.7. Analysis of triacylglycerol composition 132.8. Analysis of fatty acid residues at the sn-2 position 14III. RESULTS AND DISCUSSION 163.1. Effect of temperature 163.2. Effect of water content 203.3. Effect of substrate molar ratio 243.4. Optimization of reaction parameters 283.5. Enzymatic synthesis of structured lipids containing trans-10,cis-12 CLA 323.6. Change in composition of reaction products 343.7. Fatty acid composition of TAG in reaction products 36IV. CONCLUSION 39V. REFERENCES 40국문초록 45