Involvement of methionine salvage pathway genes of Saccharomyces cerevisiae in the production of precursor compounds of dimethyl trisulfide (DMTS).

Dimethyl trisulfide (DMTS) is one of the components responsible for the unpalatable aroma of stale Japanese sake, called "hineka". Recently, a precursor compound of DMTS, 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), was identified. It was speculated that the yeast methionine salvage pathway (MTA cycle) might participate in the formation of DMTS-P1, because the chemical structure of DMTS-P1 was similar to one of the intermediate compounds of that pathway. Here, we carried out sake brewing tests using laboratory yeast strains with disrupted MTA cycle genes and found that DMTS-P1 was hardly produced by Δmeu1, Δmri1, and Δmde1 strains. Furthermore, the DMTS producing potential (production of DMTS during storage of sake) decreased in sake made with Δmri1 and Δmde1. We constructed sake yeast strains with a disrupted MRI1 or MDE1 gene and confirmed a decline in the DMTS-P1 content and DMTS producing potential of sake made with these disruptants. The results of sake brewing tests using MTA cycle disruptants suggested that SPE2 is responsible for the production of DMTS precursors other than DMTS-P1: although the DMTS-P1 content was higher in Δspe2 sake than in Δmri1 or Δmde1 sake, the DMTS producing potential of Δspe2 sake was as low as that of Δmri1 or Δmde1 sake. Sake brewing tests using BY4743 Δspe2 Δmri1 double disruptants revealed that the DMTS producing potential was further decreased as compared with the Δspe2 or Δmri1 single disruptant. These results suggest that MRI1, MDE1, and SPE2 are promising targets for breeding yeast to suppress the formation of DMTS during storage of sake.

Lignin is linked to ethyl-carbamate formation in ume (Prunus mume) liqueur.

Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. The extracts were loaded onto a C18 column, washed with water, and eluted with methanol. The (13)C-NMR spectra for the main constituents of the methanol elution fractions were consistent with those for lignin or fragments thereof. The methanol fractions were added to ume liqueur which was stored for 3 months. Relative to a control, the ethyl carbamate concentrations in the 3-month old liqueurs were found to be 1.2 and 4.6 higher for the untoasted oak-powder and the toasted oak-powder respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.

The concentration of ethyl carbamate in commercial ume (Prunus mume) liqueur products and a method of reducing it.

The ethyl carbamate concentration of commercial ume liqueur products was studied, and a method of reducing it was examined from the viewpoint of antioxidation. The average ethyl carbamate concentration across 38 ume liqueur products was 0.12 mg/l (0.02-0.33 mg/l). When potassium metabisulfite was added to a concentration of 0-1,000 ppm during production, the generation of ethyl carbamate was reduced in a concentration-dependent manner, but when the amount of potassium metabisulfite added was below the maximum level allowed under the Japanese Food Sanitation Act, the reduction was only 27%. When ume liqueurs were produced under deoxygenated conditions created using an oxygen absorber, the ethyl carbamate concentration was reduced by up to 47% as compared with the control group, probably due mainly to a reduction in free hydrogen cyanide. When ume liqueur was produced in an oxygen atmosphere, the ethyl carbamate concentration increased by up to 50% as compared with the control group. Thus, oxygen may be involved in the generation of ethyl carbamate in ume liqueur production.

Contribution of 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) to the formation of dimethyl trisulfide (DMTS) during the storage of Japanese sake.

Dimethyl trisulfide (DMTS) is involved in the unpalatable aroma of stale Japanese sake, called "hineka". Recently, we isolated one of the precursor compounds of DMTS in sake and identified it as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), a previously unknown compound. In this work, the contribution of DMTS-P1 to the formation of DMTS was investigated. DMTS-P1 was chemically synthesized from methional in three steps, consisting of the Grignard reaction, followed by oxidation by MnO(2) and an immobilized osmium oxide catalyst. The formation of synthetic DMTS-P1 was confirmed by a comparison of the liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) data to that of natural DMTS-P1. Quantitative analysis of DMTS-P1 in sake was developed using LC-MS/MS, and a positive correlation was observed between the concentration of DMTS-P1 in sake and the production of DMTS during storage. These results indicate that DMTS-P1 contributes to the formation of DMTS in sake.