ETHYL CARBAMATE FORMATION IN GRAIN SPIRITS: PART IV—RADIOCHEMICAL STUDIES

The formation of radioactive ethyl carbamate from added H¹⁴CN was studied in a sample of new spirit from a grain distillery. Reaction conditions were at ambient temperature in the dark. Cupric ions added to spirit promoted the incorporation of cyanide into ethyl carbamate.     

ETHYL CARBAMATE FORMATION IN GRAIN BASED SPIRITS: PART II THE IDENTIFICATION AND DETERMINATION OF CYANIDE RELATED SPECIES INVOLVED IN ETHYL CARBAMATE FORMATION IN SCOTCH GRAIN WHISKY

Ion chromatographic procedures are described for the identification and determination of a series of cyanide related precursors involved in ethyl carbamate formation in maturing grain whisky produced by the continuous distillation Coffey still process. These precursors include cyanide and copper cyanide complex anions, lactonitrile and isobutyraldehyde cyanohydrin (collectively determined as “measurable cyanide” or MC) and cyanate and thiocyanate anions. The components comprising MC may be determined individually using a pre-chromatographic separation procedure based on anion exchange resins. These techniques have been shown to be applicable for monitoring precursors involved in post-distillation ethyl carbamate formation in maturing grain whisky. Similar ion chromatographic procedures are described for the detection of a range of copper salts, incorporating cyanide, cyanate and thiocyanate, which are naturally occurring deposits within the copper Coffey still. These results are correlated with those based upon X-ray diffraction and secondary ion mass spectrometric techniques. Species identified included cuprous cyanide, cuprous thiocyanate, cuprous oxide, cuprous and cupric sulphide and copper cyanide cluster ions such as CuCN^?, Cu(CN)₂^?, Cu₂(CN)₃^? and Cu₃(CN)₄^? and mixed species Cu(CN)₂S^?, CuSCN^? and CuCNO⁺. In addition, an ion chromatographic procedure developed to investigate the role of carbamyl phosphate in ethyl carbamate formation, showed that conversion took place by means of its thermal decomposition into cyanate ion.     

ETHYL CARBAMATE FORMATION IN THE PRODUCTION OF POT STILL WHISKY

The formation and distribution of ethyl carbamate (urethane) during pot still distillation was investigated. Formation only occurred if the distillation was carried out in the presence of copper. Removal or chelation of dissolved or suspended copper prevented ethyl carbamate formation. When copper was present, during and subsequent to distillation, formation of ethyl carbamate was time-dependent. The degree of formation was maximised between pH 4 and 6. During the second or low wines distillation only 1–2 per cent of the total available ethyl carbamate was collected with the potable spirit fraction. The remainder was distributed between the feints (15 per cent) and the spent lees (84 per cent).     

ETHYL CARBAMATE FORMATION IN GRAIN-BASED SPIRITS: PART III. THE PRIMARY SOURCE

Low concentrations of ethyl carbamate, which may occur in distilled spirits are shown to originate from traces of cyanide, produced in distillates by thermal decomposition of the cyanohydrin of isobutyraldehyde (IBAC) which is present in fermented wash. This compound arises during fermentation by the hydrolytic action of yeast beta-glucosidase on a naturally-occurring cyanogenic glycoside, identified as epiheterodendrin (EPH). EPH, which is a heat-stable precursor of IBAC is located in the acrospires of malted barley and is readily transferred to worts during mashing. Although many barley varieties display the ability to produce volatile cyanide, some low-yielders have been identified. EPH is absent from unmalted barley and its development is strongly influenced by malting conditions. Conversion of measurable cyanide (MC) into ethyl carbamate takes place during- or after distillation but, apparently, not before.     

THE POSSIBLE INVOLVEMENT OF Cu2+ PEPTIDE/PROTEIN COMPLEXES IN THE FORMATION OF ETHYL CARBAMATE

Ethanolic solutions of bovine serum albumin or wheat gluten were shown to form ethyl carbamate subsequent to distillation or reflux in the presence of copper. Substitution of propanol for ethanol resulted in the formation of propyl carbamate. The reflux of an ethanolic solution of essential amino acids, in the presence of copper, did not result in significant formation of ethyl carbamate.     

DETERMINATION OF ETHYL CARBAMATE IN DISTILLED SPIRITS USING NITROGEN SPECIFIC AND MASS SPECTROMETRIC DETECTION

Gas chromatographic methods are described for the determination of naturally occurring ethyl carbamate in distilled spirits at levels as low as 5 ppb. Bottled products, such as whisky, gin and brandy, are extracted in the presence of n-propyl carbamate as internal standard and analysed by capillary column gas chromatography with either nitrogen specific or mass spectrometric detection. A shorter extraction procedure is satisfactory when the more sensitive and selective mass spectrometric detection is used. Direct sample injection without extraction may be used for higher alcoholic strength cask and new-make samples. Analysis of 181 blended Scotch whisky samples indicated ethyl carbamate concentrations ranging from 20 to 75 ppb. Concentrations in 48 malt whiskies ranged from 15–100 ppb. Ethyl carbamate was not detected in gin, vodka and rectified neutral alcohol. All concentrations in bottled product fell below the Canadian limit of 150 ppb in distilled spirits.     

Ethyl carbamate kinetics in double distillation of sugar cane spirit. Part 2: influence of type of pot still

This study aimed to assess the influence of the pot still configuration on the reduction of ethyl carbamate content in double‐distilled sugar cane spirit, as well as to describe the effects of double distillation on this reduction. Ethyl carbamate is a potentially carcinogenic compound that may be present in high concentrations in sugar cane spirit, and therefore could become a public health problem, as well as a factor hindering Brazilian exports of this beverage. In sugar cane spirit production in Brazil, neither pot still configuration nor distillate reflux and cooling/condensation systems are standardized. In this study, ethanol, copper and ethyl carbamate contents were assessed (GC‐MS) in sugar cane spirits that were double‐distilled in pot stills with different reflux and cooling systems. Double distillation removed 94–98.5% of ethyl carbamate from sugar cane spirit. Pot stills with high reflux rates (equipped with dephlegmator or rectifying system) were more effective in reducing the ethyl carbamate content in double‐distilled sugar cane spirit. Copyright © 2013 The Institute of Brewing & Distilling     

Ethyl carbamate kinetics in double distillation of sugar cane spirit

The aim of this study was to verify the effect of a double distillation on the reduction of the ethyl carbamate content in sugar cane spirit. Ethyl carbamate is a potentially carcinogenic compound normally present at critical levels in sugar cane spirit, constituting a public health problem and therefore hindering the export of this beverage. The ethanol, copper and ethyl carbamate contents were evaluated, using gas chromatography/mass spectroscopy, during a double distillation of the fermented sugar cane juice. The distillate fraction from the first distillation accumulated 30% of the ethyl carbamate formed. In the second distillation, the ethyl carbamate and the copper content increased during the process as the alcohol content decreased, and only 3% of the ethyl carbamate formed was collected in the spirit. Double distillation decreased the ethyl carbamate content in the sugar cane spirit by 97%. Copyright © 2012 The Institute of Brewing & Distilling     

Chemical and microbiological quality of sugar cane juice influences the concentration of ethyl carbamate and volatile congeners in cachaça

The aim of this study was to assess the influence of heat treatment of sugar cane juice, supplementation with urea and double distillation on the concentration of volatile congeners (acetic aldehyde, ethyl acetate, n‐propyl, isobutyl and isoamyl alcohols and acetic acid) and contaminants (methanol, 1‐propyl and 2‐butyl alcohols, copper, and ethyl carbamate) in cachaça. Samples of fresh sugar cane juice, sugar cane juice submitted to heat treatment and contaminated sugar cane juice were supplemented (or not) with urea and fermented. The washes so obtained underwent single and double distillation. Supplementation with urea stimulated ethyl carbamate formation. The distilled products that originated from contaminated worts presented higher concentration of acetic acid and ethyl carbamate. Double distillation reduced the concentration of contaminants. The best quality pot still cachaça was obtained employing heat treatment of sugar cane juice, nonsupplementation with urea and double distillation. Copyright © 2015 The Institute of Brewing & Distilling     

DETERMINATION OF ETHYL CARBAMATE IN COMMERCIAL PROTEIN BASED CONDIMENT SAUCES BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Commercially obtained protein based condiments, including soy sauces, were analyzed for the presence of ethyl carbamate (urethane) using combined gas chromatography-mass spectrometry (GC-MS). Samples were spiked with 100 parts-per-billion (ppb) of t -butyl carbamate internal standard, extracted with methylene chloride and concentrated prior to GC-MS analysis. For quantitation, the ratio of the pseudomolecular ions produced during isobutane chemical ionization (CI) at m/z 90 (ethyl carbamate) and m/z 118 ( t -butyl carbamate) were monitored by mass chromatography. Response factors for ethyl carbamate versus the internal standard were determined in both the electron ionization (EI) and chemical ionization modes. The CI mode was used for quantitation as it was more sensitive and interference free. Samples testing positive by CI-MS were subsequently scanned by EI-MS for confirmation of identity as the CI spectra did not contain fragment ions necessary for adequate specificity. This method proved simple and reliable with detection limits less than 1 ppb. Limits of confirmation were 10 ppb. The recovery of internal standard was 86.7%± 5.6%. Only fermented products were found to contain ethyl carbamate.     

Effects of storage time and temperature on the concentration of ethyl carbamate and its precursors in wine

A 3-year study was carried out on the effects of time and temperature on the concentration of ethyl carbamate in wine. The study monitored the changing concentration of ethyl carbamate and of urea and citrulline, which are two major precursors of ethyl carbamate in wine. In addition to the formation of ethyl carbamate, both urea and citrulline decayed in other reactions. Kinetic analysis was carried out to model the formation of ethyl carbamate and its dependence on the concentrations of ethanol, urea and citrulline. This led to the development of an equation that can be used to predict the concentration of ethyl carbamate in wine at the point of consumption, resulting from any given storage time and temperature profile. The results were in good agreement with data obtained from similar studies.     

Ethyl carbamate in the production and aging of cachaça in oak (Quercus sp.) and amburana (Amburana cearensis) barrels

There are residual organic and inorganic contaminants in the cachaça production chain. Among the organic contaminants, ethyl carbamate has been widely studied. It is formed naturally during the fermentation processes and can be found in many foods and beverages such as bread, yoghurt, wine, beer and especially in fermented–distilled beverages such as whiskey, rum, vodka, grappa, cachaça and tiquira. The objective of this study was to identify and quantify ethyl carbamate during the steps of the production process and aging of cachaça in oak (Quercus sp) and amburana (Amburana cearensis) barrels. The cane used for cachaça production, as well as the process for obtaining the fermentation broth, did not affect the formation of ethyl carbamate, as it was not detected. Carbamate was detected in low concentrations, but was not quantified, in the fermentation, distillation and aging steps. Copyright © 2014 The Institute of Brewing & Distilling     

EFFECT OF PROCESSING CONDITIONS ON THE FORMATION OF BIOGENIC AMINES AND ETHYL CARBAMATE IN SOYBEAN TEMPE

Tempe is a fermented soybean product originating from Indonesia. The effects of manufacturing conditions, i.e., soaking, boiling, fermentation and home cooking by stewing or frying, and the effect of pure cultures of microorganisms commonly occurring in tempe towards their production of toxicants like biogenic amines and ethyl carbamate were investigated. The level of biogenic amines in soaked soybeans was rather low (total < 280 ppm), and not significantly affected by boiling, but considerably increased by fermentation. The functional fungus Rhizopus oligosporus mainly produced tyramine and some putrescine (total biogenic amines approximately 1800 ppm). With added inoculation of Klebsiella pneumoniae and Trichosporon beigelii, the total amount of biogenic amines increased slightly (2000, resp. 2100 ppm) with a shift towards cadaverine. With added Lactobacillus plantarum, a reduction of tyramine levels resulted in a considerably lower total level of biogenic amines (approx. 1000 ppm). Storage at 5C did not affect the level of biogenic amines, whereas at 25C, increased levels of putrescine were observed. Home cooking by stewing had little effect, but frying in oil resulted in significant decreases of both putrescine and tyramine. Preventive measures to keep biogenic amines at low levels in tempe are recommended. They include inoculation with selected lactic acid bacteria which cannot produce but can degrade biogenic amines, and frying instead of stewing of tempe. Ethyl carbamate levels were negligible (<11 ppb) in all treatments; this was attributed to the absence of significant concentrations of ethanol in the product .     

The effect of distillation conditions and alcohol content in ‘heart’ fractions on the concentration of aroma volatiles and undesirable compounds in plum brandies

This study investigates the effect of double‐ or single‐stage distillation and different alcohol content in ‘hearts’ (middle fractions) on the distribution of aroma volatiles and undesirable compounds (methanol, hydrocyanic acid, ethyl carbamate) during distillation of plum brandies. Irrespective of the distillation method used, the first fractions (‘heads’) included mainly aliphatic aldehydes, acetals and esters as well as higher alcohols (1‐propanol, 2‐methyl‐1‐propanol, 1‐butanol, 2‐methyl‐1‐butanol and 3‐methyl‐1‐butanol). Furfural, 1‐hexanol, benzyl alcohol, 2‐phenylethanol and ethyl carbamate occurred in relatively high concentrations in the ‘tail’ fractions. Increasing the concentration of alcohol in the heart fractions from 70 to 90% v /v resulted in a gradual decrease in the concentration of all detected volatile compounds. Compared with single‐stage distillation, double distillation produced heart fractions with lower concentration of acetaldehyde and benzaldehyde and with higher contents of furfural and esters, such as isobutyl acetate and isoamyl acetate. There was a statistically significant increase in the amounts of methanol and ethyl carbamate obtained from double distillation compared with similar fractions derived from the single‐stage process. However, in all fractions these compounds occurred in concentrations much lower than the limits specified by EU regulations. The heart fraction from the double‐stage process with 83% v /v alcohol content received the best scores for aroma and flavour. Copyright © 2017 The Institute of Brewing & Distilling     

Influence of fast and slow distillation on ethyl carbamate content and on coefficient of non‐alcohol components in Brazilian sugarcane spirits

Ethyl carbamate is an impurity present in distilled beverages. Given the risk of it being a carcinogenic substance, Brazilian legislation has determined that its presence in distilled beverages, such as ‘aguardente’ and ‘cachaça’ (two types of sugarcane spirits), should be limited to a maximum of 150 µg/L. Ordinary spirits usually contain variable amounts of ethyl carbamate, although in lower concentrations than the maximum determined by law. The finding that commercial spirits had a much lower concentration of this impurity (around 50 µg/L) led the authors to research the reasons for the differences, and these are explored in this paper, with a focus on the speed of the distillation process and its influence on the spirit's composition. The team conducted research in a sugarcane distillery producing ‘aguardente’ using a simple pot still and measured the influence of fast and slow distillation on the presence of ethyl carbamate and non‐alcohol components in the process. The results demonstrated that the speed of distillation was proportionally related to the concentration of ethyl carbamate and secondary components in the beverage's composition. Copyright © 2012 The Institute of Brewing & Distilling     

Contribution of citrulline to the formation of ethyl carbamate during Chinese rice wine production

Ethyl carbamate is a well-known carcinogen and widely occurs in Chinese rice wine. To provide more clues to minimise ethyl carbamate accumulation, the levels of possible precursors of ethyl carbamate in Chinese rice wine were investigated by HPLC. Studies of the possible precursors of ethyl carbamate in Chinese raw rice wine with various additives and treatments indicated that significant amounts of urea can account for ethyl carbamate formation. It was also recognised that citrulline is another important precursor that significantly affects ethyl carbamate production during the boiling procedure used in the Chinese rice wine manufacturing process. Besides urea and citrulline, arginine was also found to be an indirect ethyl carbamate precursor due to its ability to form urea and citrulline by microorganism metabolism.     

Acid Urease: Reduction of Ethyl Carbamate Formation in Sherry under Simulated Baking Conditions

Acid urease derived from Lactobacillus fermentum was applied to reduce ethyl cthe target level (90 μg/L). However, complete prearbamate (EC) formation in California sherry production. Commercial sherry and sherry base were treated with urease under different conditions. Amounts of urea were determined and EC amounts were monitored by gas chromatography. Urea removal by acid urease reduced EC formation during the baking process and effectively maintained EC concentrations below vention of EC was not achieved because of precursors other than urea.     

Determination of ethyl carbamate in cachaça stored in newly made oak,amburana, jatobá, balsa and peroba vats and in glass containers

Ethyl carbamate was quantified in cachaça using high‐performance liquid chromatography with fluorescence detection during the production process and during storage in different types of wooden casks and glass containers, with and without the presence of light. According to the physicochemical analysis, the head and tail fractions are unacceptable for consumption. Ethyl carbamate was detected in sugarcane juice and during fermentation. During distillation, ethyl carbamate levels ranged from the limit of detection to 17.1 μg L⁻¹. Ethyl carbamate increased during storage in wooden casks. The presence (or absence) of light did not affect the formation of ethyl carbamate in cachaça stored in glass containers. Copyright © 2017 The Institute of Brewing & Distilling     

高粱中蜀黍氰苷与浓香型白酒中氰化物及氨基甲酸乙酯关系的研究

为研究高粱中蜀黍氰苷与浓香型白酒中氰化物及氨基甲酸乙酯(EC)之间的关系,以不同高粱为研究对象,分别进行水解试验、模拟蒸酒试验和酿酒试验。水解试验结果表明：高粱中蜀黍氰苷含量与其水解所生成的氰化物总量呈正相关;模拟蒸酒试验结果表明：实验组(以S1高粱为原料)的各段馏分酒中氰化物含量显著低于对照组(以S2高粱为原料)(P<0.01);酿酒试验结果表明：实验组原酒中氰化物初始含量低于对照组,浓香型白酒中绝大部分EC是在贮存过程中生成的,原酒中氰化物初始含量和达到相对稳定状态时EC含量呈正相关(R²=0.966 9),氰化物是浓香型白酒中EC的重要前体物。该研究从生产水平上证实了浓香型白酒生产企业采用蜀黍氰苷含量低的高粱作为酿酒原料,可有效降低产品中EC含量。     

FRUIT BRANDY PRODUCTION BY BATCH COLUMN DISTILLATION WITH REFLUX

The relationship between the operating parameters of batch fruit spirits column stills with reflux and the congener (trace compounds that provide flavors and aromas) concentrations in resulting fruit spirits has not been widely studied. Congener concentrations were determined in three different collection fractions, or “cuts,” during batch distillation. Acetaldehyde and ethyl acetate were found in higher concentrations in the head cut, first overhead fraction, of the distillation and have lower boiling points relative to ethanol. 1‐Propanol and isoamyl alcohol (isopentanol) were present in higher concentrations in the tail cut, third or final fraction, of the distillation and have boiling points that are higher than ethanol. Methanol has a unique concentration profile as it has higher concentrations in both the head and tail cuts, but a lower concentration in the heart cut, the middle fraction which is the desired product of the distillation. Methanol was of particular interest because the distillate must adhere to governmental regulations that limit its concentration in the product. Operating‐condition parameters that were studied include the number of trays used in the distillation as well as the use of a “catalytic converter,” a high surface, copper‐packing material thought to catalyze formation of cyanide‐containing compounds allowing them to be separated from the distillate. The effect of the number of trays used in a distillation on the concentration of ethanol and the congeners, methanol, acetaldehyde, ethyl acetate, 1‐propanol and isoamyl alcohol in the final distilled spirits product is presented. An additional result of acetaldehyde production at the copper surface of the catalytic converter was also discovered in the analysis of the data.