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Comparision of volatile and non-volatile metabolites produced by Saccharomycopsis fibuligera KJJ81 based on cultivation media and cultivation time

Title
Comparision of volatile and non-volatile metabolites produced by Saccharomycopsis fibuligera KJJ81 based on cultivation media and cultivation time
Authors
정지혜
Issue Date
2016
Department/Major
대학원 식품공학과
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
김영석
Abstract
본 연구에서는 누룩에 존재하는 Saccharomycopsis fibuligera KJJ81의 대사산물분석을 수행하였다. 먼저, 다섯 종류의 탄소원에서 배양시켰을 때 나오는 휘발성 대사산물을 비교 분석 하였다. 그 다음으로, 배양 시간에 따른 휘발성, 비휘발성 대사산물을 분석하였다. S. fibuligera KJJ81는 YP 액체배지에 다섯 종류의 탄소원 (glucose, galactose, fructose, maltose, xylose)을 각각 첨가하여 18시간 배양하였다. 휘발성 대사산물 추출 방법으로는 stir bar sorptive extraction (SBSE)방법을 사용하였으며, GC-MS를 이용해 분석하였다. 비휘발성 대사산물은 fast filtration 방법으로 추출한 뒤 유도체화 하여 GC-TOF/MS를 이용하여 분석하였다. AMDIS를 이용하여 데이터를 처리한 뒤, PLS-DA를 이용해 S. fibuligera KJJ81 시료 군집에 영향을 미치는 주요 대사산물을 선정하였다. 먼저, 탄소원에 따른 휘발성 대사산물 분석 결과 1-Octanol, (2E,6E)-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl acetate, 4-(2-methyl-2-propanyl)-cyclohexyl acetate and 5-[(2E)-2-octen-1-yl]dihydro-2(3H)-furanone 가 xylose와 galactose 그룹에 영향을 미치는 주요 휘발성 대사산물로 선정되었다. Fructose와 maltose 그룹에 영향을 미치는 주요 휘발성 대사산물로는 1-Octen-3-ol, 2-furylmethanol, 3-methyl-1-butanol, phenol, 3-methylbutyl acetate, 5-hexyldihydro-2(3H)-furanone, 7,9-bis(2-methyl-2-propanyl)-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione and (methyldisulfanyl)- methane가 선정되었다. 특이하게도 glucose 배지에서는 2-phenylethanol, 2-phenylethyl acetate 그리고 ethyl phenylacetate와 같은 phenyl 그룹이 붙거나 phenylalanine으로부터 유래한 휘발성 대사산물들이 많이 검출되었다. Glucose 배지에서 배양한 S. fibuligera KJJ81의 휘발성 및 비휘발성 대사산물을 배양 시간별로 분석 한 결과, 2-phenylethanol의 양은 계속해서 증가하는 경향을 보였다. 반면 2-phenylethyl acetate는 배양 14시간까지만 증가하는 경향을 보였다. 상대적으로 높은 수준으로 2-phenylethanol과 2-phenylethyl acetate가 검출된 반면 phenylalanine은 낮은 농도로 검출되었다. Arabitol은 배양 4시간까지는 증가하다 감소하는 경향을 보였으며, mannose는 배양 8시간까지 감소하다 이후 증가하는 경향을 보였다. Mannitol의 경우, 배양초기에는 큰 변화를 보이지 않다가 배양 24시간부터 증가하는 경향을 나타냈다. S. fibuligera KJJ81에서 palmitic acid, stearic acid 그리고 oxalic acid가 높은 농도로 검출되었다. Palmitic acid, stearic acid의 경우 배양 시간에 따라 지속적으로 그 양이 변하였으며, oxalic acid는 14시간때에 가장 높은 농도로 검출되었다. 마지막으로 이 논문은 S. fibuligera KJJ81의 대사산물에 대해 연구한 첫 번째 연구라는 의의를 갖는다.;Saccharomycopsis fibuligera KJJ81 is an amylolytic yeast which can be obtained in nuruk, a microbial mixed-strain starter. Volatile and non-volatile metabolites produced by S. fibuligera KJJ81 were analyzed depending on different carbon sources and cultivation times. S. fibuligera KJJ81 was cultivated in YP broth with different carbon sources (glucose, fructose, galactose, xylose and maltose) for 18 hours. Volatile metabolites were extracted using stir bar sorptive extraction (SBSE) method and analyzed using GC-MS. Non-volatile metabolites were extracted with fast filtration method and derivatized for GC-TOF/MS analysis. Partial least squares discriminant analysis (PLS-DA) was applied to estimate the significant differences and detect major metabolites which cluster between S. fibuligera KJJ81 samples. 1-Octanol, (2E,6E)-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl acetate, 4-(2-methyl-2-propanyl)-cyclohexyl acetate and 5-[(2E)-2-octen-1-yl]dihydro-2(3H)-furanone were detected as main contributors in xylose and galactose group. On the other hand, 1-Octen-3-ol, 2-furylmethanol, 3-methyl-1-butanol, phenol, 3-methylbutyl acetate, 5-hexyldihydro-2(3H)-furanone, 7,9-bis(2-methyl-2-propanyl)-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione and (methyldisulfanyl)- methane were grouped in fructose and maltose cluster. Volatile metabolites with phenyl group and derived from phenylalanine, such as 2-phenylethanol, 2-phenylethyl acetate and ethyl phenylacetate, were predominantly found in glucose media. According to cultivation times, the amount of 2-phenylethanol continuously increased. On the other hand, the level of 2-phenylethyl acetate increased until 14 hours. Although the levels of 2-phenylethanol and 2-phenylethyl acetate were high, a relatively small amount of phenylalanine was detected during S. fibuligera KJJ81 growth. Considerable phenylalanine degradation can be possible explanation for this tendency. The level of mannitol were retained during cultivation and increased from 24 hours of cultivation. As far as we know, this tendency could be explained by synthesis of mannitol from glucose in YPD media. Palmitic acid is a predominant fatty acid in eukaryotes and a large amount of it was detected in S. fibuligera KJJ81. In addition, oxalic acid was the dominant organic acids. Oxalic acid exists in most living organisms and has negative influence on beer industry.
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