Ethyl carbamate (urethane, C(2)H(5)OCONH(2)) is a known genotoxic carcinogen of widespread occurrence in fermented food and beverages with highest concentrations found in stone-fruit spirits. Between 1986 and 2004, 631 cherry, plum or mirabelle (yellow plum) spirits were analysed for ethyl carbamate using gas chromatography in combination with mass spectrometry after extrelut extraction. The ethyl carbamate concentration of the samples ranged between 0.01 mg l(-1) and 18 mg l(-1) (mean 1.4 mg l(-1)). After exposure of the samples to UV light, significantly (p=0.001) higher concentrations between 0.01 mg l(-1) and 26 mg l(-1) (mean 2.3 mg l(-1)) were found. The ethyl carbamate concentration increased on average by 1.3 mg l(-1). A linear correlation between the year of sampling and ethyl carbamate concentration showed a statistically significant but very slight decrease (R=-0.10, p=0.024). However, if only samples which officially were non-compliant were considered exceeding the upper limit of 0.4 mg l(-1) more than twice, a significant reduction (R =-0.56, p=0.018) of the quota was evident. This shows that measures to reduce ethyl carbamate were successfully introduced in many distilleries. However, nearly 20 years after the first warnings about ethyl carbamate in spirit drinks, the problem persists especially in products derived from small distilleries. During experimental production of stone-fruit spirits using state-of-the-art technologies, it was shown that the occurrence of ethyl carbamate in stone fruit spirits is preventable. Even for small distilleries, simple possibilities like destoning exist to minimize the ethyl carbamate content.

A top fermented beer was brewed from 100% buckwheat malt and sensory and analytical characterisation was carried out. Difficulties with lautering and filtration were encountered during the brewing process, which resulted in problems during fermentation and beer filtration. The beer was evaluated in the fresh and forced aged state for the following attributes: odour, purity of taste, mouthfeel, tingling, and bitterness. Analytical results indicated that the buckwheat beer compared quite closely to a typical wheat beer with regard to pH, FAN, fermentability and total alcohol. However, the extract of the buckwheat wort was lower, resulting in a final extract yield of 54.5%. GC analysis of the resultant beer revealed commonly encountered levels of the esters that give beer a fruity character. A low level of fusel alcohols, in comparison to a typical wheat beer, was detected. A high level of ethyl caprinate (coconut flavour) and lauric acid (fatty odour) was detected. Sensory analysis indicated that the buckwheat beer was acceptable with regard to odour, purity of taste, mouthfeel, tingling and bitterness. In conclusion, results of this study prove buckwheat's qualification as a gluten-free brewing material and with process optimisation, its readiness for marketing.

To evaluate the influence of stone content on spirit quality from stone fruit, cherry and plum mashes were prepared and fermented with a commercial and a diploid laboratory yeast strain. Fermentation parameters such as sugar content and ethanol production were followed. Despite an initial lag phase in cherry spirits, both yeast strains performed similarly, as substantiated by the determination of specific flavor compounds, ethyl carbamate, and methanol in the mashes and after distillation. The spirits produced were subjected to sensory analyses by trained panels of at least 25 judges. Although mashes retaining the stones could be clearly distinguished from those where the stones had been removed, no significant preference could be attributed to either spirit, indicating that qualities added by the presence of stones during fermentation are largely a matter of personal taste. Interestingly, the yeast strain used for fermentation seemed to have little influence on the spirit quality.

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Fermented fruit and beverages frequently contain ethyl carbamate (EC), a potentially carcinogenic compound that can be formed by the reaction of urea with ethanol. Both are produced by the yeast Saccharomyces cerevisiae with ethanol as the major end product of hexose fermentation and urea as a by-product in arginine catabolism. In spirit production, EC can also be derived from cyanide introduced by stone fruit. To determine the relative contribution of yeast metabolism to EC production, we genetically engineered a diploid laboratory strain to reduce the arginase activity, thus blocking the pathway to urea production. For this purpose, strains with either a heterozygous CAR1/car1 deletion or a homozygous defect (car1/car1) were constructed. These strains were compared to the parental wild type and to an industrial yeast strain in cherry mash fermentations and spirit production. The strain with the homozygous car1 deletion showed a significant reduction of EC in the final spirits in comparison to the non-engineered controls. Nevertheless, using this strain for fermentation of stoneless cherry mashes did not completely impede EC formation. This indicates another, as yet unidentified, source for this compound.

No abstract

Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. Here we used two haploid laboratory strains with suitable auxotrophic markers for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Three different fruit mashes (cherries, plums and pears) were fermented in a 90 kg scale. These were then subjected to distillation and used for the production of spirits with a final ethanol content of 40% (v/v). Fermentation parameters assayed included growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols and glycerol. The spirits were also tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques for adjusting yeast physiology to special production schemes.

The influence of two factors, total concentration and fraction of three pairs of commercial enzymes, which showed statistical significance (Biocellulase W with Hitempase 2XL, Biocellulase W with Amylo 300 and Amylo 300 with Hitempase 2XL), were studied for their overall effect on buckwheat wort quality using response surface methodology (RSM). This study revealed that the addition of increasing levels of Hitempase 2XL to the buckwheat mash increased colour, extract levels, wort filtration, fermentability and total fermentable extract (TFE), along with decreasing viscosity values. Results also determined a high level of fermentability when an enzyme combination of 30% Biocellulase and 70% Hitempase was added to the mash. The addition of increasing levels of Amylo 300 to buckwheat mashes resulted in increases in fermentability and total fermentable extract (TFE), along with increases in total soluble nitrogen (TSN), free amino nitrogen (FAN) and Kolbach index (KI). With regard to the proposed optimal regime, although no synergistic effect was found when the three enzymes were used together, the optimum conditions for the production of buckwheat wort with lowest viscosity, highest extract and optimal fermentability were achieved using a joint model. Overall, the findings of this study demonstrate the feasibility of producing wort suitable for the brewing of glutenfree beer from 100% malted buckwheat with careful optimisation of enzyme types and dosage levels.