The goal of this project was to accurately monitor and document heat exposure of wine during a significantly large and representative number of shipments from winery to distributors across the country under the most extreme conditions that can be expected for commercial freight during the months of summer. The shipping project showed quite dramatically the impact of wine shipments in regular non-refrigerated trucks with different types of insulation. During the summer months, wines shipped to or via hot geographic locations are frequently exposed to temperatures above 75°F, and often for extended periods of time. Under the most extreme shipping conditions the wine would have been exposed to temperatures above 110°F. A substantial daily fluctuation in temperature was observed with the potential consequences for the sealing capabilities of the bottle closures in use. We observed a large variation of temperatures within one shipment based on the location of the wine case within the shipping container. Different models of aging kinetics were applied to compare the heat exposure to the aging of wine under ideal cellar conditions. When applying a general rule of thumb for chemical reactions, it was calculated that the wines were exposed to heat that represented an aging time equivalent to between one month and six years. While the aging reaction of and between different components in a wine vary substantially, we obtained a best but weak fit for ethyl carbamate (EC) formation kinetics that may be used to predict maximum EC formation in wine under different shipping conditions.
In the past term we have investigated the inhibitory effect of several energy sources on the degradation of arginine and the excretion of citrulline by malolactic bacteria. It was found that increasing sugar concentrations lead to a reduced arginine degradation rate. It is likely that the inhibition is mediated by the energy status of the bacterial cells, or more specifically by the intracellular concentration of ATP that may be increased when higher sugar concentrations are present in the medium. Citrulline excretion from arginine degradation was increased at high glucose concentrations, suggesting that more citrulline was built up intracellularly during arginine degradation. Therefore, an inhibition is likely to occur at the second enzymatic step responsible for the degradation of citrulline. Degradation pathway of arginine in malolactic bacteria. L-arginine + H20 ? >? L-citrulline + NFL,* (1) L-citrulline + Pi -< £?!£ >? L-ornithine + carbamyl-P (2) carbamyl-P + ADP < £K ^ ATP + C02 + NH/ (3) The ability of different malolactic bacteria to increase their growth by degrading arginine was further investigated with 2 lactobacilli and 2 oenococci. It was found that only one lactobacilli was able to increase its growth by degradation of arginine and only at pH values of 3.5 or more. However, under these conditions the growth advantage was considerable. A cheap and simple enzymatic method for quantification of arginine was evaluated. The test is an end-point method comparable to those offered by Boehringer Mannheim (now Roche) for many substrates and fills the gap between sophisticated apparative methods and the unspecific chemical methods that are still widely used.
We have investigated factors influencing the excretion of citrulline (a precursor of carcinogenic ethyl carbamate) from arginine degradation by malolactic bacteria in laboratory-fermented wine. One strain of Lactobacillus buchneri (CUC-3) and two strains of Oenococcus oeni (MCW and Lolll) have been examined for differences in citrulline excretion during growth in wine at several initial pH-values (pH 3.3, 3.6 and 3.9), at several initial arginine concentrations (0, 500, 1000, 1500 mg/1 arginine) and in wine fermented with different yeast strains (Montrachet, Premiere Cuvee). It was shown for both Lactobacillus buchneri CUC-3 and Oenococcus oeni MCW that the citrulline excretion rate was proportional to the arginine degradation rate. Factors that increase the arginine degradation rate will lead to an increase in the excretion rate of citrulline and finally to an increase in the total amount of citrulline excreted into the wine, given that the reutilization of citrulline is small. In accordance with these findings, citrulline excretion was more significant at higher pH values, where arginine degradation proceeds at a faster rate for all three bacteria examined. However, lactobacilli and oenococci varied in their metabolic rates at several initial arginine concentrations. The arginine degradation rate of Lactobacillus buchneri CUC-3 increased during incubation and therefore, the highest citrulline concentration was produced at the highest initial arginine concentration. In the case of both oenococci, the arginine degradation rates were significantly lower than those for Lactobacillus buchneri CUC-3 and varied only slightly at different initial arginine concentrations. This resulted in similar amounts of citrulline being excreted during fermentation. Additionally, high arginine concentrations inhibit arginine degradation. Differences in the excretion of citrulline upon realization of malolactic fermentation in wines fermented with different yeast strains were found for Oenococcus oeni MCW. Wines previously fermented with strain Premiere Cuvee (previously Prise de Mousee) led to better growth, higher arginine degradation rates and thus, higher citrulline levels. Malic acid degradation was completed for both oenococci at least 12 days earlier than arginine degradation.
Ethyl carbamate is a natural fermentation byproduct but a potential carcinogen at very high doses. The major precursor to ethyl carbamate in wine is arginine, a natural amino acid in grapes. In order to minimize ethyl carbamate levels in wine, we developed a simple, rapid method for determining the arginine concentration in grape juice, that is suitable for use at a commercial winery. The new spectrophotometry assay for arginine utilizes techniques adapted from ion exchange chromatography. In our method development, we combined the application of a selective strong cation exchange resin with the NOPA assay that we developed in the previous AVF project “Rapid Analysis for Yeast Available Nitrogen Compounds in Must”. We have been able to demonstrate the isolation and quantification of arginine from various model and natural juices. The accuracy and reliability of our assay was evaluated by correlation to a HPLC reference method, and by measuring the recovery of arginine in spiked model and natural juices. The assay will be tested on commercial musts during the 1998 crush, and an analytical procedure will be made available to the California wine industry as soon as our method validation is complete. Recommendations on critical arginine levels in juices have already been made in our “Ethyl Carbamate Preventative Action Manual”.
The study shows the results of duplicate experiments where the production of ethyl carbamate upon accelerated storage respectively on a white, and a red wine control were compared with the same wine to which urea, the glucose ureide, and the glucose monoureide were individually added at 2 ppm. The results for both types of wine show that, as expected, the glucose diureide does release some of its urea increasing the ethyl carbamate content upon accelerated storage. Thus, this compound could be a source of post-bottling ethyl carbamate in wines. It was surprising however, that the monoureide did not similarly release urea, indicating that, contrary to what was expected, the monoureide is not a precursor of ethyl carbamate in wine. Apparently in the latter compound, urea is tenaciously held by glucose even in the acidic conditions of the wine. Our results also indicate that under prolonged urease contact, no additional ethyl carbamate was produced from the diureide. We believe this was due to the diureide reaching an equilibrium between it and the glucose plus urea, thereby allowing the urease to destroy the freed urea. As urea is destroyed, the equilibrium eventually shifts, resulting in the complete destruction of the diureide. At this time, it is not known whether the diureide is susceptible to destruction by the urease directly. From the results obtained herewith, it is obvious that the sugar ureides are not a serious source of post-bottling ethyl carbamate in wines when these wines are properly treated with urease. Thus, work on these compounds as potential sources of ethyl carbamate in wines is hereby discontinued.
Post-bottling formation of ethyl carbamate in wines that have tested negative for both urea and ethyl carbamate upon leaving the winery, continues to pose a problem for the industry. This is of particular concern in the case of dessert wines. During this year’s study (1995-96), we examined the possibility that urea-carbonyl adducts formed prior to fermentation might be responsible. We had previously reported (AVF, 1994-95) that diacetyl-urea adducts did not seem to be responsible for post-bottling ethyl carbamate formation. We have since reexamined the behavior of 3a, 6a-dimethylglycoluril (one of the adducts of urea and diacetyl) and found that indeed this compound might be a precursor of ethyl carbamate in wines. We also synthesized several glucose-urea adducts (diureides and monoureides). When subjected to acid hydrolysis under conditions similar to those found in wine, these urea adducts also released urea with the production of ethyl carbamate. This behavior indicates that these compounds, that could form prior to fermentation, might also be responsible for post-bottling ethyl carbamate formation in wine.
The prevailing thought regarding the origin of ethyl carbamate in wines seems to be centered on the interaction of urea (and immediate urea precursors such as arginine and citrulline) with ethanol under the acidic conditions found in wine. Thus, appropriate remedial steps have been taken by industry which have considerably lowered levels of ethyl carbamate in wines. However, a seemingly paradoxical situation exists by which wines which have tested negative for the presence of both urea and ethyl carbamate upon leaving the winery, develop considerable levels of ethyl carbamate within a relatively short time. This situation seems to be more prevalent in the case of dessert wines. We have examined the possibility that urea-carbonyl adducts formed prior to fermentation might be responsible. We focused initially on urea-diacetyl adducts. Our research proved conclusively that these diacetyl-urea adducts are not responsible for post-bottling ethyl carbamate formation. We are currently investigating glucose- and fructose-urea adducts.
Arginine is one of the major amino acids found in grapes and wine. Certain strains of malolactic wine lactic acid bacteria can degrade this amino acid with the formation of ornithine, carbon dioxide and ammonia. The degradation of arginine by two strains of malolactic bacteria, Leuconostoc oenos MU2 and Lactobacillus buchneri CUC-3, was investigated to assess the potential formation of ethyl carbamate precursors in wine. Both strains degraded arginine and surprisingly, excreted citrulline, a known precursor of ethyl carbamate. The excreted citrulhne reacts with alcohol to form substantial amounts of ethyl carbamate with heat treatment (71°C/48 hours). The formation of ethyl carbamate correlated well with arginine degradation and citrulline production. From a winemaking point of view, the production of citrulhne is a problem, since it can react with alcohol over time to form ethyl carbamate even at low storage temperatures. However, only very small amounts of ethyl carbamate were formed without heat treatment (<10 ng/g or ppb). 7-1 Our results show that malolactic bacteria can indeed be a potential source of ethyl carbamate precursor (at least citrulline). Thus, care must be exercised in selection of starters to conduct malolactic fermentation in wine. Ideally, those strains unable to degrade arginine should be chosen in order to minimize precursor formation. In addition, spontaneous malolactic fermentation by undefined indigenous strains should be discouraged, as this may result in formation of ethyl carbamate precursors.
The prediction of the changes in ethyl carbamate are directly proportional to the ethanol concentration and to the urea concentrations. The rate at which the formation occurs follows the Arrhenius equation. It is simply a chemical reaction (no enzymes involved). The higher the storage temperature the faster the reactions proceed. Essentially, only a small percentage of the urea is lost with time. At the temperatures of normal storage, the hydrolysis rate is very, very slow. Citrulline also reacts, but much more slowly with ethanol to form ethyl carbamate.