Effects of Aspergillus species inoculated and their enzymatic activities on the formation of volatile components in fermented soybean paste (Doenjang)

Abstract

Doenjang, a traditional fermented soybean paste in Korea, has been used in many dishes mainly due to its characteristic flavor properties, being closely related to volatile components produced during fermentation. The overall volatiles composition of Doenjang is affected by several factors, including microorganisms inoculated, raw materials and fermentation periods as well as fermentation conditions. Among them, the diverse biochemical reactions occurred in molds can hydrolyze macromolecules constituting raw ingredients, which can be precursors for various volatile components. In this study, the volatile components of rice-koji Doenjang produced in accordance with Aspergillus species (A. oryzae, A. sojae, and A. kawachii) during 8 weeks fermentation were compared using GC-MS analysis and multivariate statistical analyses, such as principal component analysis (PCA) and partial least square regression (PLSR) analysis. Also, the activities of diverse enzymes (α-amylase, protease, lipase and esterase) were determined to investigate their effects on the formation of volatile compounds. A total of 108 volatiles were identified, with esters being as predominant. Regardless of fungi types, carbonyls, including 4-methylheptan-2-one, heptan-2-one, (E)-hept-2-enal, and hexanal, were found to contribute mainly to early phase fermentation (0-2 weeks), whereas the contents of ethyl esters of long-chained fatty acids were considerably enhanced at latter stage of fermentation (5-8 weeks). In general, the activities of enzymes studied, except for α-amylase, increased as fermentation proceeded, although there were significant differences depending on fungi species. In particular, lipase activity showed a high correlation with the formation of long-chained fatty acid esters, which could be related to the distinctive organoleptic properties of rice-koji Doenjang.;본 연구에서는 쌀코지 된장의 생산 중 코지 제조 과정에 있어서 접종되는 Aspergillus 속 곰팡이의 종류 (A. oryzae, A. sojae, A. kawachii)에 따른 휘발성 성분의 차이를 비교하고, 이들의 생성에 영향을 미칠 것으로 사료되는 여러가지 효소 (α-amylse, protease, lipase, esterase)활성과의 상관관계를 규명하고자 하였다. 8주 간의 발효 기간 동안 쌀코지 된장의 휘발성 성분을 프로파일링하기 위해 고체상미세추출법 (Solid-phase microextraction, SPME)을 이용하여 추출하고, 가스 크로마토그래피-질량분석법 (gas chromatography-mass spectrometry, GC-MS)으로 분석하였으며, 각 효소의 활성은 2, 4, 8 주의 발효 기간 동안 측정하였다. 발효 기간이 길어짐에 따라 ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl dodecanoate, ethyl tetradecanoate, ethyl pentadecanoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl octadecanoate, ethyl octadec-9-enoate, ethyl octadeca-9,12-dienoate, ethyl octadeca-9,12,15-trienoate와 같은 장쇄지방산의 에스테르의 생성이 증가하는 경향을 보였으며, 휘발성 성분 프로파일의 균주별 차이는 점차 감소하였다. 동정 및 정량한 쌀코지 된장의 휘발성 성분의 형성에 대한 효소 활성의 영향을 알아보기 위해 부분최소제곱회귀 분석 (partial least square regression, PLSR)을 수행하였다. 그 결과, α-amylse 활성은 2-methylpyrazin, 3-hydroxy-2-methylpyran-4-one (maltol), furan-2-carbaldehyde (furfural)과 같은 당분해 산물들의 생성과 관련이 있었으며, protease는 아미노산인 phenylalanine에서 기인하는 것으로 알려진 ethyl 3-phenylpropanoate, 2-phenylethyl acetate, (E)-2-phenylbut-2-enal, 2-phenylethanol과 같은 물질의 생성에 영향을 미치는 것으로 나타났다. 특히 lipase의 활성은 된장의 향미에 중요한 역할을 하는 에스테르 화합물의 생성과 연관성을 보였는데, 이 중 sweet, fruity 한 향 특성에 기여한다고 알려진 methyl 2-methylprop-2-enoate와 pentyl propanoate가 높은 상관관계를 나타내었다.