Up to 35% of (R)-methyl 2-methylbutanoate (M2MB) was observed in a beverage fermented with shiitake. As M2MB naturally occurs typically in high excesses of the (S)-enantiomer, the origin of the (R)-ester was elucidated by stable isotope labeled precursor-feeding studies. (R)-2-Methylbutanoic acid was identified as the main precursor in the substrate wort. Trace amounts of (R)-M2MB were produced by transformation of unsaturated secondary metabolites (tiglic aldehyde and tiglic acid) derived from l-isoleucine. Surprisingly, shiitake esterified (R)-2-methylbutanoic acid faster to (R)-M2MB than the corresponding (S)-enantiomer. Concurrently, spontaneous non-enantioselective degradation of M2MB occurred in shiitake. This explains diverse enantiomeric ratios of M2MB and different enantiomeric ratios of 2-methylbutanoic acid and M2MB in the beverage. As the odor threshold values of (R)-and (S)-M2MB differ significantly, these findings are of high relevance for the overall flavor of the fermented beverage and elucidate the discrepancy of enantiomeric ratios of 2-alkyl-branched acids and esters reported in nature.

在用香菇发酵的饮料中观察到高达35％的（R）-2-甲基丁酸甲酯（M2MB）。由于M2MB通常自然地以高过量的（S）-对映异构体天然存在，因此（R）-酯的来源已通过稳定的同位素标记的前体摄食研究得以阐明。（R）-2-甲基丁酸被鉴定为底物麦芽汁中的主要前体。痕量的（R）-M2MB通过转化衍生自1-异亮氨酸的不饱和次级代谢产物（三聚醛和三聚酸）而产生。令人惊讶地，香菇酯化（[R）-2-甲基丁酸更快（[R）-M2MB比相应的（小号）-对映体。同时，香菇中发生了M2MB的自发非对映选择性降解。这解释了饮料中M2MB的不同对映体比率以及2-甲基丁酸和M2MB的不同对映体比率。由于（R）-和（S）-M2MB的气味阈值存在显着差异，因此这些发现与发酵饮料的整体风味高度相关，并阐明了所报告的2-烷基支链酸和酯的对映体比例的差异在自然界。