Ethyl Carbamate Content Research Articles

New Approach of QuEChERS and GC-MS Triple-Quadrupole for the Determination of Ethyl Carbamate Content in Brazilian cachaças.

Cachaça is a popular spirit produced in Brazil, obtained by distillation of fermented sugar cane. Among the contaminants arising from production, ethyl carbamate is a carcinogenic compound that occurs naturally in fermented foods and beverages; in Brazil, the maximum limit established by current legislation is 150 µg L−1. Quality control is usually performed using gas chromatography; however, robustness and reproducibility of quantitative results may be severely impaired, as the addition of 6–30 g L−1 of sucrose is a common procedure for taste standardization, directly interfering in the results. This work describes the development of a novel method to improve ethyl carbamate quantification in cachaças using a new approach of QuEChERS extraction based on salting-out phenomenon, to effectively separate ethanol from sugar-containing water. Eighteen different brands of cachaça were analyzed. The proposed methodology was able to eliminate components that contaminate the sample flow path in the gas chromatography system, while improving precision and accuracy by using a triple-quadrupole approach, in comparison with the methodology usually employed: direct analysis of cachaça samples with no sample prep. Results indicate that this approach is more effective due to the removal of sugar content, with no impact in costs per analysis.

Evaluation of ethyl carbamate formation in Luzhou-flavor spirit during distillation and storage processes

Ethyl carbamate (EC) is a toxic contaminant that poses hazard to humans due to its potential carcinogenicity. This compound has been widely detected in fermented foods and alcoholic beverages such as Chinese spirits. Clarifying the mechanism of EC formation is difficult but necessary for reducing EC levels in Chinese spirits because of their complex fermentation procedures. The formation of ethyl carbamate during distillation and spirit aging were evaluated in this study. All grains used as materials for producing Luzhou-flavor spirit contained ethyl carbamate precursors, urea and citrulline. The highest levels of urea (93 μg/kg) and citrulline (83 μg/kg) were detected in wheat and sorghum, respectively. EC and its precursors were introduced into the raw spirit of Luzhou-flavor spirit during distillation. EC, which was present at 92 μg/l concentration in raw spirit, came from the distillation process. EC content in the raw spirit increased to 66% during the first year of storage because of spontaneous formation of EC from its precursors mainly urea in the raw spirit.

Biodegradation of Ethyl Carbamate and Urea with Lysinibacillus sphaericus MT33 in Chinese Liquor Fermentation.

It is important to reduce the concentration of ethyl carbamate (EC) in fermented foods. However, controlling the formation of EC and its precursor urea is difficult in spontaneous food fermentation because urea is a natural product of nitrogen metabolism. Biodegradation is a better solution to reduce the concentration of EC. This study aimed to reduce the concentration of EC in Chinese liquor via an indigenous strain Lysinibacillus sphaericus MT33. This strain produced urethanase (940 U/L) and urease (1580 U/L) and degraded 76.52% of EC and 56.48% of urea. After inoculation in liquor fermentation, the maximal relative abundance of Lysinibacillus increased from 0.02% to 8.46%, the final EC and urea contents decreased by 41.77% and 28.15%. Moreover, the concentration of EC decreased by 63.32% in liquor. The negative correlation between abundance of Lysinibacillus and contents of EC and urea indicated the effect of L. sphaericus on EC and urea degradation.

Effect of citrulline metabolism inSaccharomyces cerevisiaeon the formation of ethyl carbamate during Chinese rice wine fermentation

Urea, as the main precursor of ethyl carbamate (EC), has received extensive attention. Here, we have metabolically engineered an industrial yeast strain – Saccharomyces cerevisiae N85 – to investigate the contribution of the EC precursor citrulline to the concentration of EC in Chinese rice wine. The results showed that the citrulline biosynthetic pathway of the modified strain N85-arg3 was completely suppressed by deletion of ARG3, encoding ornithine carbamoyltransferase. However, there were no significant differences in the levels of citrulline or EC between N85-arg3 and the parental strain N85 during fermentation. In addition, we over-expressed ARG1 (encoding argininosuccinate synthase) and ARG4 (encoding argininosuccinate lyase) to construct the engineered strains N85ARG1,4 and N85ARG1,4-arg3. The citrulline contents in Chinese rice wine fermented with N85ARG1,4 and N85ARG1,4-arg3 were respectively 24.1 and 20.4% less than that of N85. However, the contents of EC were 23.8 and 28.5% more than that of N85. These results suggested that reducing the formation of EC during Chinese rice wine fermentation by genetically engineering citrulline metabolism in S. cerevisiae was not a viable proposition. Copyright © 2018 The Institute of Brewing & Distilling

Ethyl Carbamate Formation Regulated by Lactic Acid Bacteria and Nonconventional Yeasts in Solid-State Fermentation of Chinese Moutai-Flavor Liquor.

This study aimed to identify specific microorganisms related to the formation of precursors of EC (ethyl carbamate) in the solid-state fermentation of Chinese Moutai-flavor liquor. The EC content was significantly correlated with the urea content during the fermentation process (R2 = 0.772, P < 0.01). Differences in urea production and degradation were found at both species and functional gene levels by metatranscriptomic sequencing and culture-dependent analysis. Lactobacillus spp. could competitively degrade arginine through the arginine deiminase pathway with yeasts, and most Lactobacillus species were capable of degrading urea. Some dominant nonconventional yeasts, such as Pichia, Schizosaccharomyces, and Zygosaccharomyces species, were shown to produce low amounts of urea relative to Saccharomyces cerevisiae. Moreover, unusual urea degradation pathways (urea carboxylase, allophanate hydrolase, and ATP-independent urease) were identified. Our results indicate that EC precursor levels in the solid-state fermentation can be controlled using lactic acid bacteria and nonconventional yeasts.

Fluorescent silicon nanoparticles-based ratiometric fluorescence immunoassay for sensitive detection of ethyl carbamate in red wine

Abstract Herein, a ratiometric fluorescence (RF) enzyme-linked immunosorbent assay (RF-ELISA) for sensitive detection of ethyl carbamate (EC) was developed by introducing fluorescent silicon nanoparticles (Si NPs) into the chromogenic substrate system (o-phenylenediamine (OPD)/H 2 O 2 ) of a conventional horseradish peroxidase (HRP)-based ELISA platform to assemble a RF-based signal output system. For this system, the fluorescence of Si NPs at 440 nm (I 440 ) was acted as the reference signal which could be efficiently quenched by 2,3-diaminophenazine (DAP), the HRP-catalyzed oxidation product of OPD; Meanwhile, the fluorescence of DAP at 570 nm (I 570 ) was served as response signal. Therefore, variation in the amount of HRP labeled secondary antibody bound on the microplate which is associated with antibody-antigen recognition events in conventional HRP-based ELISA could be transferred into a more sensitive RF signal (I 570 /I 440 ). On the basis of monoclonal antibody (mAb) which could specifically recognize EC derivative, xanthyl ethyl carbamate (XEC), a Si NPs-based RF-ELISA for EC via a simple pre-analysis derivatization was developed. When detecting the EC content in red wine, this method exhibits a working range from 3.9 to 105.0 μg/L and a limit of detection (LOD) of 2.6 μg/L with excellent specificity, accuracy and reproducibility. The sensitivity is approximately 33-fold higher than that of traditional colorimetric ELISA. The proposed Si NPs-based RF-ELISA is not only highly suitable for screening EC in a large number of samples, but also provides a potential platform for high-throughput and sensitive determination of other analytes for food safety monitoring.

Simultaneous determination of ethyl carbamate and urea in Korean rice wine by ultra-performance liquid chromatography coupled with mass spectrometric detection.

In this study, a rapid method for simultaneous detection of ethyl carbamate (EC) and urea in Korean rice wine was developed. To achieve quantitative analysis of EC and urea, the conditions for Ultra-performance liquid chromatography (UPLC) separation and atmospheric-pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) detection were first optimized. Under the established conditions, the detection limit, relative standard deviation and linear range were 2.83μg/L, 3.75-5.96%, and 0.01-10.0mg/L, respectively, for urea; the corresponding values were 0.17μg/L, 1.06-4.01%, and 1.0-50.0μg/L, respectively, for EC. The correlation between the contents of EC and its precursor urea was determined under specific pH (3.5 and 4.5) and temperature (4, 25, and 50°C) conditions using the developed method. As a result, EC content was increased with greater temperature and lower pH. In Korean rice wine, urea was detected 0.19-1.37mg/L and EC was detected 2.0-7.7μg/L. The method developed in this study, which has the advantages of simplified sample preparation, low detection limits, and good selectivity, was successfully applied for the rapid analysis of EC and urea.

Probabilistic dietary exposure to ethyl carbamate from fermented foods and alcoholic beverages in the Korean population

ABSTRACTThe occurrence of ethyl carbamate was investigated in fermented foods and alcoholic beverages of the Korean total diet study. The concentrations of ethyl carbamate ranged from not detected to 166.5 μg kg−1. Dietary exposure to ethyl carbamate was estimated by the probabilistic method. Estimated intakes of ethyl carbamate from foods and alcoholic beverages were 4.12 ng kg−1 body weight (bw) per day for average consumers and 12.37 ng kg−1 bw/day for 95th percentile high consumers. The major foods contributing to ethyl carbamate exposure were soy sauce (63%), followed by maesilju (plum liqueur, 30%), whisky (5%), and bokbunjaju (black raspberry wine, 2%). On the basis of the benchmark dose lower confidence limit 10% (BMDL10) of 0.3 mg kg−1 bw/day, margins of exposure were 128,000 for mean exposure and 40,000 for 95th percentile exposure. This indicates that the exposure of the Korean general population for ethyl carbamate is of low concern. However, careful vigilance should be continued for high consumers of fermented foods and alcoholic beverages.

Reduced Production of Higher Alcohols by Saccharomyces cerevisiae in Red Wine Fermentation by Simultaneously Overexpressing BAT1 and Deleting BAT2.

In red wine, the contents of higher alcohols and ethyl carbamate (EC) are two significant health concerns. To reduce the production of higher alcohols by wine yeast YZ22 with low production of EC, one BAT2 was replaced by a BAT1 expression cassette first and then another BAT2 was deleted to obtain the mutant SYBB3. Real-time quantitative PCR showed that the relative expression level of BAT1 in SYBB3 improved 28 times compared with that in YZ22. The yields of isobutanol and 3-methyl-1-butanol produced by mutant SYBB3 reduced by 39.41% and 37.18% compared to those by the original strain YZ22, and the total production of higher alcohols decreased from 463.82 mg/L to 292.83 mg/L in must fermentation of Cabernet Sauvignon. Meanwhile, there were no obvious differences on fermentation characteristics of the mutant and parental strain. This research has suggested an effective strategy for decreasing production of higher alcohols in Saccharomyces cerevisiae.

An Improved HPLC-FLD for Fast and Simple Detection of Ethyl Carbamate in Soy Sauce and Prediction of Precursors

A high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for ethyl carbamate (EC) determination in Chinese soy sauce was developed and used to predict EC precursors, and extraction, derivatization, and chromatographic conditions were optimized systematically. Under optimal conditions, the limit of detection, linear range, and recoveries were 3.91 μg/L, 12.87–274.87 μg/L, and 81.5–95.4%, respectively. The method precision was less than 8.9% (RSD) and no significant difference was found between EC determinations measured by HPLC-FLD and GC/MS. Using the proposed HPLC-FLD method, the EC contents in Chinese soy sauces ranged from not detected to 64.88 μg/L (n = 19). Except for one soy sauce brand, the EC contents in low-salt solid-state fermented (LSF) soy sauces were lower than those in high-salt liquid fermented (HLF) soy sauces and were mainly affected by alcohol content. Moreover, the free ornithine and total arginine contents were significantly correlated with EC content in the soy sauce products. The results of this work provide a foundation for further study of EC formation and inhibition in Chinese soy sauces.

The Evaluation of the Applicability of Fourier Transform Near-Infrared (FT-NIR) Spectroscopy in the Measurement of Analytical Parameters in Must and Wine

Fourier transform near-infrared (FT-NIR) spectroscopy can be used as a rapid method to measure the percentage of sugar and to discriminate between different must samples in terms of their free amino nitrogen (FAN) values. It can also be used as a rapid method to discriminate between Chardonnay wine samples in terms of their malolactic fermentation (MLF) status. By monitoring the conversion of malic to lactic acid, the samples could be classified on the basis of whether MLF has started, is in progress or has been completed. Furthermore, FT-NIR spectroscopy can be used as a rapid method to discriminate between table wine samples in terms of their ethyl carbamate (EC) content. It is claimed that high concentrations of ethyl carbamate in wine can pose a health threat and has to be monitored by determining the EC content in relation to the regulatory limits set by authorities. For each of the above-mentioned parameters QUANT+™ methods were built and calibrations were derived and it was found that a very strong correlation existed in the sample set for the FT-NIR spectroscopic predictions of the percentage of sugar (r = 0.99, SEP= 0.31 °Brix). However, the correlation for the FAN predictions (r = 0.602, SEP= 272.1 g.L-1), malic acid (r = 0.64, SEP= 1.02 g.L-1), lactic acid (r = 0.61, SEP= 1.35 g.L-1) and EC predictions (r = 0.47, SEP = 3.6 μg.kg·1) were not good. The must samples could be classified in terms of their FAN values when Soft Independent Modelling by Class Analogy (SIMCA) diagnostics and validation were applied as a discriminative method, with recognition rates exceeding 80% in all cases. When SIMCA diagnostics and validation were applied to the Chardonnay and EC wine samples, recognition rates exceeding 88% and 80% respectively were obtained. These results therefore confirm that this method is successful in discriminating between samples.

An Efficient Assay for Simultaneous Quantification of Ethyl Carbamate and Phthalate Esters in Chinese Liquor by Gas Chromatography-Mass Spectrometry

An efficient assay was developed by gas chromatography-mass spectrometry (GC-MS) for simultaneous quantifying of ethyl carbamate (EC) and phthalate esters (PAEs, dibutylphthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP)) in Chinese liquor. Samples were directly detected using ethyl-d5 carbamate (D5-EC) and diisoamyl phthalate (DIPP) as internal standards without any pretreatment. The detection limits were 0.56, 0.70, and 1.13 μg/L for EC, DBP, and DEHP, respectively. The average recoveries were 97.0–104.0, 96.9–105.0, and 85.0–124.2% for EC, DBP, and DEHP with relative standard deviations of 0.5–1.3, 1.6–4.2, and 2.2–5.3%, respectively. The proposed method is suitable for routine analysis of EC and PAEs in liquor due to its shorter detection time and simple pretreatment. With assistance of the established method, the contents of EC, DBP, and DEHP in Chinese liquor during the distillation process were assayed and a higher level of EC was found in the head and tail of liquor distillation.

Effects of different distillation patterns on main compounds of ChineseLuzhou-flavour raw liquors

This study aimed to investigate the effects of different distillation patterns on main compounds of Chinese Luzhou-flavour liquor. Results showed that more volatiles of raw liquor might originate from fresh grains for a newly constructed pit. A modest (p = 0.05) increase in the total amount of volatiles was found in the heart liquor from fermented grains added to fresh grains and rice hulls to distillate (AG pattern), reaching a maximum value of 1012.22 mg/L. The percentage of esters in the AG pattern ranged from 81.22 to >95.03% compared with samples from fermented grains only added to rice hulls to distillate (NG pattern). Acids in the heart liquor obtained by the AG pattern were higher than those for NG, but the corresponding proportion was opposite. Alcohols were detected at ~1.62% in NG pattern but were 1.42% in AG. Cyanide and ethyl carbamate content in the head liquor were far higher than those in the heart and tail liquor. In particular, cyanide content dramatically increased after adding fresh grains during the distillation process. Copyright © 2017 The Institute of Brewing & Distilling

Effect of gallic and protocatechuic acids on the metabolism of ethyl carbamate in Chinese yellow rice wine brewing

It was studied that gallic and protocatechuic acids played important roles in ethyl carbamate (EC) forming. Gallic and protocatechuic acids can reduce the arginine consumption through inhibiting the arginine deiminase enzyme. Therefore, they are generally added to regulate EC catabolism in the course of yellow rice wine leavening at the third day. In this work, gallic and protocatechuic acids made little influence on the growth of Saccharomyces cerevisiae. Besides, the addition of 200mg/L gallic or protocatechuic acid could prevent the transformation from urea/citrulline to EC. Gallic acid showed better inhibiting effect that the content of EC could be reduced by 91.9% at most. Furthermore, the production of amino acids and volatile flavor compounds are not markedly affected by phenolic compounds. The discoveries reveal that EC can be reduced by supplying gallic acid or protocatechuic acid while yellow rice wine leavening.

A survey of ethyl carbamate in beer from Chinese market

Result of a survey of ethyl carbamate (EC) in beer was carried out by gas chromatography-mass spectrometry (GC/MS). 217 samples were purchased from supermarkets in five cities of China. The concentrations of EC ranged from n.d. to 19.6 μg/kg with an average level of 2.2 μg/kg, and positive rate was 95.4%. Mean in domestic beer (N = 142, incidence 97.3%) was 1.8 μg/kg, lower than the 2.9 μg/kg in imported beer (N = 75, incidence 94.4%). The result of domestic beer showed that EC level in canned beer (N = 73, mean = 1.5 μg/kg) was lower than that in bottled beer (N = 69, mean = 2.1 μg/kg). Additionally, EC level of beer from different countries was varied. Furthermore, a strong positive correlation of EC content in beer with alcohol content, storage time, temperature and light exposure was observed.

Ethyl carbamate control by genomic regulation of arginase inSaccharomyces cerevisiaeEC1118 in sugarcane juice fermentation

Ethyl carbamate (EC) is one of the harmful byproducts which occurs in alcohol drinks, such as Wine, Rum, Whisky, Saki and other fruit wines as a result of a spontaneous reaction between ethanol and urea in the fermentation process. In this work, the relationship between arginase CAR1 gene in Saccharomyces cerevisiae EC1118 and EC generation in sugarcane juice fermentation was investigated. Results indicate that the content of EC can be controlled by regulating the expression of arginase CAR1 gene. Urea and arginase exhibited expression of arginase CAR1 gene, which resulted in an increase of EC content. Besides arginine and urea, the expression of arginase CAR1 gene was also significantly influenced by the content of ethanol, salt and culture temperature. Low fermentation temperature, (below 25°C) and lower ethanol and salt content showed some benefits in terms of controlling the EC content by decreasing the expression of arginase.

Determination of Ethyl Carbamate (EC) by GC-MS and Characterization of Aroma Compounds by HS-SPME-GC-MS During Wine Frying Status in Hakka Yellow Rice Wine

Wine frying status is one of the most important processes in the brewing of Chinese yellow rice wine (CYW) and has a great influence on the quality and flavor of CYW. The present work determined the ethyl carbamate (EC) concentration by gas chromatography-mass spectrometry (GC-MS) and aroma compounds by headspace solid-phase microextraction GC-MS (HS-SPME-GC-MS) during wine frying status in CYW. In addition, effects of wine frying conditions on the changes of EC and aroma compounds were also investigated. Results indicated that EC concentration increased along with the increase of wine frying temperature and time. Pearson test indicated that EC had highly significant correlation (p < 0.01) with temperature and time. Esters, alcohols, and ethers consisted of the most aroma components and constituted the main flavor of CYW. Considering EC concentration, main flavor, unique ingredients, and proportions of volatile aroma compounds, 70 °C and 11–20 min are the most appropriate temperature and time for wine frying of CYW. It is necessary to control wine frying conditions influencing EC formation and aroma compounds in CYW brewing.

Assessment of Ethyl Carbamate Contamination in Cachaça (Brazilian Sugar Cane Spirit)

Cachaça is a sugar cane spirit produced in Brazil. Ethyl carbamate (EC), a potential carcinogenic compound, may be present in cachaça above the limit established by law. The purpose of this study was to determine the concentration of ethyl carbamate in cachaças recently produced in Brazil in order to verify their compliance with the law. The concentration of ethyl carbamate was determined in 376 samples of cachaça through gas chromatography coupled to a mass spectrometer (GC-MS). The mean value of ethyl carbamate in the cachaças analyzed was 145 µg/L, and 24% of them were not in compliancy with the law (EC &lt; 210 µg/L). However, compared to previous studies, advances have been observed regarding the adjustment of cachaças to the legal limit. Cachaças produced in large distilleries through continuous column distillation presented a mean value of 200 µg/L of ethyl carbamate. Cachaças produced in small distilleries using pot still distillation presented a mean content of 74 µg/L. Small producers have been more engaged in using good manufacturing practices to guarantee the quality of cachaças.

Occurrence of ethyl carbamate in three types of Chinese wines and its possible reasons.

A total of 75 wines including 30 white wines, 31 red wines, and 14 sparkling wines were obtained from several regions in China (Sinkiang, Tonghua, Huailai, Yantai, Changli, Shanxi, Gansu, and Ningxia). Ethyl carbamate (EC) was detected by gas chromatography mass spectrometry. The EC concentration ranged from less than 1.16 to 38.56 μg/L, and the concentrations in 17 wines exceeded the U.S. limit for table wines (15 μg/L). The concentrations of EC increased in the order of white, red, and sparkling wines with the corresponding mean concentrations of 6.12, 9.22, and 14.03 μg/L. The relationship between EC concentration and wine type suggested that EC concentrations in wines might be affected by vinification patterns, most likely due to the difference between EC precursors in different vinification processes. This work provides a novel clue for EC contamination in different wines.

SERS-active Ag Nanostars Substrates for Sensitive Detection of Ethyl Carbamate in Wine.

A simple and sensitive surface-enhanced Raman scattering (SERS) method for the detection of ethyl carbamate (EC) is reported in this work. Star-shaped silver nanostars (Ag NSs) were used as a novel SERS substrate. In comparison to other plasmonic nanoparticles (NPs), including Au NPs, Au NSs and Ag NPs, Ag NSs exhibit best SERS activity. Raman signal of EC at a trace level can be enhanced by several orders of magnitude with the help of Ag NSs. The Raman intensity of EC increased linearly with an increase of the EC concentration in the range from 5 × 10(-9) mol L(-1) to 1.0 × 10(-4) mol L(-1) with detection limit (LOD) of 1.37 × 10(-9) mol L(-1) (S/N = 3). The developed SERS approach also has the advantages of being simple, fast and requiring less amount of the sample. It could serve as a useful technology for the rapid determination of EC in both alcoholic beverages and fermented food.