Comparison of non-volatile and volatile metabolites produced by Lichtheimia ramosa according to cultivation media and growth

Abstract

본 연구에서는 전통 누룩에 존재하는 Lichtheimia ramosa 균의 비휘발성 성분 (당, 당알코올, 아미노산, 유기산, 지방산)과 휘발성 성분 (향기성분)을 비교 분석하여 균의 특징을 알고자 하였다. 총 세 가지 배지 (complete media (CM), potato dextrose broth (PDB), sabouraud dextrose broth (SDB))에서 총 48시간 동안 발효시키며 12시간마다 비휘발성 및 휘발성 성분들을 분석하였다. 비휘발성 대사산물을 분석하기 위하여 fast filtration이라는 방법을 이용하여 추출 및 quenching하고, methoxyamine hydrochloride와 N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)를 이용하여 유도체화시켰다. 유도체화시킨 시료는 gas chromatography time-of-flight mass spectrometry (GC-TOF/MS)를 이용하여 분석하였다. 휘발성 성분을 흡착하기 위해 Stir bar sorptive extraction (SBSE)방법을 이용하였으며 GC-MS를 이용하여 분석하였다. 균의 초기 증식단계에서는 2-phenylethanol과 같은 성분이 검출되었고, 증식하는 동안은 당으로부터 유래한 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), 2-hydroxy-3-methyl-2-cyclopenten-1-one (cyclotene)과 같은 성분들이 많이 생성되었다. 균의 비증식 단계에서는 3-methyl-2-buten-1-ol과 α-humlene과 같은 terpene류가 많이 검출되었다. CM 배지의 경우 sucrose와 fructose같은 당류가 다른 배지에 비해 많이 함유되어있었고, 당으로부터 유래한다고 알려진 5-(hydroxymethyl)-2-furaldehyde와 2-furaldehyde 성분이 다른 배지에서보다 많이 검출되었다. SDB 배지의 경우 다른 배지들보다 아미노산 함량이 높았고phenylalanine으로부터 유래한 2-phenylethanol이 많이 검출되었다.;In this study, non-volatile and volatile metabolites of Lichtheimia ramosa were analyzed to identify characteristic of L. ramosa which is one of the predominant filamentous fungi in Korean traditional nuruk. Non-volatile metabolites found in cell mass of Lichtheimia ramosa were analyzed using GC-TOF/MS and volatile metabolites found in fermentation broth and cell mass of Lichtheimia ramosa were analyzed using GC-MS. During cultivation, non-volatile and volatile metabolites were compared by using multivariate statistical analysis. Detected non-volatile metabolites includes amino acids, carbohydrates, fatty acids, and organic acids. Analyzed volatile compounds contains acids, alcohols, aldehydes, benzene and benzene derivatives, esters, furan and furan derivatives, ketones, sulfur compound, terpenes, hydrocarbon, and miscellaneous. Regardless of types of media, 2-phenylethanol, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), 2-hydroxy-3-methyl-2-cyclopenten-1-one (cyclotene), 3-methyl-2-buten-1-ol, and α-humulene were detected as important metabolites which contribute to separating samples according to cultivation times. In early stage of proliferation, 2-phenylethanol was produced. Compounds derived from carbohydrates such as 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), and 2-hydroxy-3-methyl-2-cyclopentne-1-one (cyclotene) were increased as cultivation time imcreased. Contents of terpenes such as 3-methl-2-buten-1-ol and α-humulene increased as cultivation proceeded. In CM, contents of 5-(hydroxymethyl)-2-furaldehyde and 2-furaldehyde were high compared to the other media. These compounds is known as derived from carbohydrates. Actually, CM contains lots of carbohydrates against PDB and SDB. Amount of 2-phenylethanol derived from phenylalanine, was detected in volatile compounds of L. ramosa cultivated in SDB which have a lot of phenylalanine.