Abstract:
The maximum amount of ethyl carbamate (EC), a known animal carcinogen produced by
the reaction of urea and ethanol, allowed in alcoholic beverages is regulated by
legislation in many countries. Wine yeast produce urea by the metabolism of arginine,
the predominant assimilable amino acid in must. This action is due to arginase (encoded
by CARl). Regulation of CARl, and other genes in this pathway, is often attributed to a
well-documented phenomenon known as nitrogen catabolite repression. The effect of
the timing of di-ammonium phosphate (DAP) additions on the nitrogen utilization,
regulation of CARl, and EC production was investigated. A correlation was found
between the timing of DAP addition and the utilization of nitrogen. When DAP was
added earlier in the fermentations, less amino nitrogen and more ammonia nitrogen was
sequestered from the media by the cells. It was also seen that early DAP addition led to
more total nitrogen being used, with a maximal difference of ~25% between
fermentations where no DAP was added versus addition at the start of the fermentation.
The effect of the timing ofDAP addition on the expression of CARJ during fermentation
was analyzed via northern transfer and the relative levels of CARl expression were
determined. The trends in expression can be correlated to the nitrogen data and be used
to partially explain differences in EC formation between the treatments. EC was
quantified at the end of fermentation by GC/MS. In Montrachet yeast, a significant
positive correlation was found between the timing of DAP addition, from early to late,
and the final EC concentration m the wine (r = 0.9226). In one of the fermentations, EC
levels of 30.5 ppb was foimd when DAP was added at the onset of fermentation. A twofold
increase (69.5 ppb) was observed when DAP was added after 75% of the sugars were metabolized. When no DAP was added, the ethyl carbamate levels are comparable
at a value of 38 ppb. In contrast, the timing of DAP additions do not affect the level EC
produced by the yeast ECU 18 in this manner. The study of additional yeast strains
shows that the effect of DAP addition to fermentations is strain dependent. Our results
reveal the potential importance of the timing of DAP addition to grape must with respect
to EC production, and the regulatory effect of DAP additions on the expression of genes
in the pathway for arginine metabolism in certain wine yeast strains.
