갈락토오스로부터 Bifidobacterium longum subsp. longum RD47 조효소를 이용한 갈락토올리고당 합성

학위논문 (석사)-- 서울대학교 대학원 : 식품영양학과, 2015. 8. 지근억.GOS는 모유 성분 중에 3 번째로 많이 존재하는 올리고당의 한 가지이 다. 이는 모유나 동물 유즙에 천연적으로 존재한다. 프리바이오틱스 (prebiotics)는 비피더스 유산균의 성장을 촉진시키는 인자이며 올리고당 과 식이 섬유가 이에 포함되며 이 중 GOS가 가장 널리 알려져 있다. 본 연구의 목적은 GOS를 합성하는 방법을 새롭게 하고 새로운 GOS 합 성하는 데에 있다. 균 선별 실험을 통해서 α-와 β-galactosidase 활성이 높은 프로바이오틱 스(probiotics)인 Bifidobacterium longum subsp. longum RD47 (RD 47) 을 선발하였다. RD 47이 modified MRS(기존 MRS 배지에 덱스트란 대 신에 라피노오스를 2% (w/v) 첨가하는 것)에 배양하였을 때 α-와 β- galactosidase의 활성이 가장 높았다. 올리고당 합성반응 조건으로는 pH 6, 37°C에서 GOS의 합성수율이 가장 높았다. RD 47의 조효소액을 이용하여 갈락토오스(galactose)를 원료로 하여 새 로운 GOS가 합성된 것을 확인할 수 있었다. TLC 결과에는 4가지 GOS 가 합성된 것을 확인할 수 있었다. 합성된 GOS는 GOSⅡ-Ⅴ로 명명하 였고, GOSⅡ는 di-galactose, GOSⅢ는 tri-galactose, GOSⅣ는 tert-galactose, GOSⅤ는 penta-galactose인 것으로 추정하였다. 그중에 GOSⅢ가 가장 많이 합성되었고 그 다음으로는 GOSⅡ인 것을 확인할 수 있었다. SPE (Solid phase extraction) tube로 합성된 GOS를 정제 및 분리하였 을 때 GOSⅢ가 정제 분리되었다. 600 MHz NMR (Nuclear Magnetic Resonance)로 GOSⅢ의 구조분석을 진행하였다. 예상 구조는 β? D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-Dgalactopyranose이다. 본 연구를 통해서 GOS를 합성하는 방법을 새롭게 제시하였고 안전성에 문제가 없는 방법으로서 비피도박테리움의 조효소액으로 갈락토오스를 이용하여 신규한 GOS를 생산할 수 있다는 것을 규명하였다. 앞으로 이 는 식품, 의약, 생물학 등 영역에서 유용하게 활용될 것으로 기대된다.1. 서론 ····················································································································1 1.1 갈락토올리고당(galactooligosaccharides,GOS)의 정의······················· 1 1.2 GOS의 기능성 ······························································································1 1.3 GOS의 합성 방법························································································3 2. 실험재료 및 방법 ···························································································5 2.1. 실험재료 및 시약························································································5 2.2. 사용균주 및 배양조건 ················································································6 2.3. 효소추출 및 활성 분석··············································································7 2.3.1. 조효소의 추출 ·····················································································7 2.3.2. 효소의 활성 측정 및 분석 ······························································7 2.4. GOS의 합성·································································································8 2.4.1. GOS의 합성 조건 ··············································································8 2.4.2. GOS의 합성 결과 ··············································································8 2.5. 합성된 GOS의 정제 및 분석···································································9 2.5.1. 합성된 GOS의 분리 및 정제 ··························································9 2.5.1.1 유리판 TLC를 이용한 GOS 분리 및 정제 ····························9 2.5.1.2 Solid phase extraction (SPE)법으로 GOS 분리 및 정제·· 9 2.5.2. 합성된 GOS의 구조 분석······························································10 3. 실험 결과 및 고찰 ·······················································································11 3.1 GOS의 합성······························································································11 3.1.1 GOS의 합성 조건 ·············································································11 3.1.2 합성된 GOS의 종류·········································································14 3.2. 합성된 GOS의 구조분석·····································································16 3.2.1 유리판 TLC로 GOS의 분리 및 정제···········································16 3.2.2 SPE로 GOS의 분리 및 정제·························································18 3.2.3 NMR로 GOS의 구조 분석 ·····························································22 4. 요약 및 결론 ·································································································29 참고문헌 ···············································································································30 Abstract in English ···························································································36Maste