Ethyl Carbamate

In the past several years much attention has been directed to ethyl carbamate (urethane), a compound suspected of being a mild carcinogen that may be naturally present in fermented foods as a consequence of the metabolic activity of microorganisms. Table 8-1 shows concentrations re- ported in wines by BATF in 1986 to 1987. The concern about ethyl carbamate may result in governmentally imposed limits and the testing and regulatory compliance that go with such limits.
Table 8-1.
Survey of Ethyl carbamate levels (g/L) in commercial wines.
(BATF 1986-1987).

Product Range Average
U.S. Wines    
Table 0 - 102 10
Port 7 - 254 93
Sherry 18 - 209 82
Imported Wines    
Table 0 - 80 12
Port/Madiera 17 - 108 55
Sherry 23 - 82 62

The U.S. wine industry has established a voluntary target for ethyl carbamate of 15 ppb (g/L) or less in table wines and less than 60 ppb in fortified wines. Additionally, the U.S. Food and Drug Administration. has notified all countries exporting wines to the United States that they must meet targeted levels established by the American wine industry.

Known precursors of ethyl carbamate are urea, citrulline, carbamyl phosphate and n-carbamyl amino acids (Monteiro et al. 1989; Ough (et al. 1988). Figure 8-3 shows the reaction sequence leading to ethyl carbamate.

The formation of ethyl carbamate is related to the concentrations of urea and ethanol, to time, and increases exponentially with increases in temperature. Because urea is the principal precursor of ethyl carbamate, controlling the urea concentration may be important in limiting ethyl carbamate levels. Factors influencing urea in wines include the arginine content of the grape, yeast strain, method of yeasting, fortification, timing of fortification, temperature, and duration of wine storage.

The amino acid arginine is the main precursor of urea. The majority of the urea formed comes from arginase-catalyzed degradation of arginine during fermentation. High urea levels can occur in wines produced from grapes of high (>400 rng/L) arginine content. Such grapes tend to come from vineyards heavily fertilized or displaying high vigor.

Urea is often formed during the early or middle stages of fermentation with subsequent yeast generations utilizing it during the latter stages. Fortified wines, which are made by arresting fermentation, may contain high concentrations of urea if the fermentation is stopped at the point of great- est urea production. With many yeasts the maximum extraction occurs at about 12 to 16 Brix; followed by metabolism of the remaining arginine and reabsorption of the urea (Ough 1993b). Wineries that inoculate by pouring fresh juice over recently fermented tank bottoms may produce wines with elevated urea concentrations.

Yeast strains differ in their urea excretion and uptake during fermentation. Therefore, yeast selection may play a role in minimizing the potential for ethyl carbamate formation.

C2H5

NH2O

C==O + C2H5OH C==O + NH3

NH2 NH2

Urea Ethanol Ethyl Ammonia

Carbamate

Fig. 8-3. Ethyl carbamate formation in wine.

The yeasts 71B (Lallenmand), SD 1120 (Red Star), and Prise de Mousse have been shown to release fairly low levels of urea during fermentation (An and Ough 1993). Yeasts that excrete little urea have slight but important differences in their arginine transport system and urea metabolizing enzymes.

Stevens and Ough (1993) and Ough (1993) reviewed several wine production factors influencing ethyl carbamate formation. Storage temperature is the single most important variable influencing the rate of formation, with wine type and pH having less effect. The concentrations of ethyl carbamate are proportional to the urea concentration during storage. Therefore, knowing the urea content and wine storage temperature allows for an estimation of the ethyl carbamate level that will be formed. The relationship between urea content and ethyl carbamate formation at 24°C (75.5F) was established by Ough (1993). Storage of wine at temperatures greater than 24°C (75.5F) with urea concentrations over 5 mg/L should be avoided.

Reductions in the level of urea formed can be achieved by minimal fertilization, utilizing yeast strains that release less urea, and by fortification when urea concentrations are low. Reducing the concentrations of urea immediately after fermentation, however, likely, presents the best alternative to limiting ethyl carbamate production. (See discussion of ureases below.)

Ureases

Concerns about high ethyl carbamate levels in sake (a high urea rice wine usually heated prior to serving) resulted in the development of bacterially produced (Lactobacillus fermentatum) commercial urease enzymes. (Yoshizawa and Takahashi 1988). The products of urea are ammonia and carbon dioxide. A urease with activity within the pH range of wine has been approved for use in the United States. The enzyme is added after fermentation and before final filtration. Use levels are governed by alcohol, pH, urea concentration, enzyme contact period, and temperature. No perceptible effects on wine flavor or aroma have been noted at enzyme addition levels up to 500 mg/L; a level far greater than normally required (Caputi 1993).

Trioli and Ough (1989) reported minor inhibition of urease from excessive Fe+++,Ca+++, P04, S02, and phenolic compounds. Inhibition was more pronounced with L-lactic, acetic, pyruvic, and keto-glutaric and particularly L-malic acid.

The presence of fluoride at greater than 1 mg/L irreversibly inhibits the action of urease (Kodama et al. 1991). The mineral cryolite (sodium alu- minum fluoride, AIF6Na) is used in the United States (California) to help control the grape leaf skeletonizer (Archer and Gauer 1979). Famu)iwa and Ough (1991) showed that fluoride present injuice at 1 mg/L inactivates 10 mg/L added urease. Ureases can reduce ethyl carbamate formation, however, because urea is not the only precursor, complete elimination may not be seen.