A rapid and improved method for the determination of ethyl carbamate in foodstuffs of different matrices.

This work deals with the rapid and simple determination of the probable carcinogen ethyl carbamate (EC), which is naturally present in fermented food products. An undemanding, robust, and rapid pre-column derivatization utilizing a 9-xanthydrol reagent has been developed. The resulting derivative was subsequently analysed by reversed-phase high-performance liquid chromatography coupled with fluorescence detection. As a result of the thorough optimisation of the chromatographic conditions, the run was completed in just 5 minutes, considerably speeding up the usual time of EC separation (30-60 min). Thanks to the fast separation, satisfactory yields (around 90%), negligible matrix effects, no interfering peaks, very low detection limit, and simple sample pre-treatment (for the very first time, the derivatization was performed in the presence of light and without any extraction step), the proposed method represents a significant improvement of the EC determination protocol used so far. After method validation, a total of fifty food samples were subjected to analysis without any additional sample pre-treatment despite their diverse matrix. Due to its robustness, simplicity, and low time, cost, and manual demands, this method is suitable for rapid screening of EC in both final food products and during their production.

Intelligent onsite dual-modal assay based on oxidase-like fluorescence carbon dots-driven competitive effect for ethyl carbamate detection.

Intelligent onsite accurate monitoring ethyl carbamate (EC, a group 2 A carcinogen) in environment is of great significance to safeguard environmental health and public safety. Herein, we reported an intelligent dual-modal point-of-care (POC) assay based on the bimetallic Mn and Ce co-doped oxidase-like fluorescence carbon dots (Ce&MnCDs) nanozyme-driven competitive effect. In brief, the oxidase-like activity of Ce&MnCDs was inhibited by thiocholine (TCh, originating from the hydrolysis of acetylcholinesterase (AChE) to acetylthiocholine (ATCh)), preventing the oxidation of o-phenylenediamine (OPD) to 2,3-diaminophenothiazine (DAP). However, with the aid of Br2 + NaOH, EC inactivated AChE to prevent TCh generation for re-launching the oxidase-like activity of Ce&MnCDs to trigger the oxidation of OPD into DAP, thereby outputting an EC concentration-dependent ratiometric fluorescence and colorimetric readouts by employing Ce&MnCDs and OPD as the optical signal reporters. Interestingly, these dual-modal optical signals could be transduced into the gray values that was linearly proportional to the residual levels of EC on a smartphone-based portable platform, with a detection limit down to 1.66 µg/mL, qualifying the requirements of analysis of EC residues in real samples. This opened up a new avenue for onsite assessment of the risk of residues of EC, safeguarding environmental health and public safety.

Conjugated molecularly imprinted polymers based on covalent organic frameworks: Fluorescent sensing platform for specific capture of urea and elimination of ethyl carbamate.

This study described the preparation of an azide covalent organic framework-embedded molecularly imprinted polymers (COFs(azide)@MIPs) platform for urea adsorption and indirect ethyl carbamate (EC) removal from Chinese yellow rice wine (Huangjiu). By modifying the pore surface of COFs using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, COFs(azide) with a high fluorescence quantum yield and particular recognition ability were inventively produced. In order to selectively trap urea, the COFs(azide) were encased in an imprinted shell layer via imprinting technology. With a detection limit (LOD) of 0.016 µg L-1 (R2 = 0.9874), the COFs(azides)@MIPs demonstrated a good linear relationship with urea in the linear range of 0-5 µg L-1. Using real Huangjiu samples, the spiking recovery trials showed the viability of this sensing platform with recoveries ranging from 88.44 % to 109.26 % and an RSD of less than 3.40 %. The Huangjiu processing model system achieved 38.93 % EC reduction by COFs(azides)@MIPs. This research will open up new avenues for the treatment of health problems associated with fermented alcoholic beverages, particularly Huangjiu, while also capturing and removing hazards coming from food.

Molecularly imprinted electrochemical sensor for ethyl carbamate detection in Baijiu based on "on-off" nanozyme-catalyzing process.

Ethyl carbamate (EC) is a carcinogen widely found in the fermentation process of Baijiu. Herein, we construct a molecularly imprinted polymers/MXene/cobalt (II) based zeolitic imidazolate frameworks (MIP/MXene/ZIF-67) nano-enzyme sensor for the detection of EC during Baijiu production. The ZIF-67 is synthesized in situ on the MXene nanosheets to provide a superior catalytic activity to H2O2 and amplify the electrochemical signal. The MIP is prepared by the polymerization reaction to recognize EC. Owing to the interaction between EC and EC-MIP, the interferences are effectively eliminated, greatly improving the accuracy of the expected outcome. This approach attains an ultrasensitive assay of EC ranging from 8.9 µg/L to 44.5 mg/L with detection limit of 0.405 µg/L. The accuracy of this method is confirmed by the recovery experiment with good recoveries from 95.07% to 107.41%. This method is applied in natural EC analyses, and the results are consistent with certified gas chromatograph- mass spectrometer.

Formation and hazard of ethyl carbamate and construction of genetically engineered Saccharomyces cerevisiae strains in Huangjiu (Chinese grain wine).

Huangjiu, a well-known conventional fermented Chinese grain wine, is widely consumed in Asia for its distinct flavor. Trace amounts of ethyl carbamate (EC) may be generated during the fermentation or storage process. The International Agency for Research on Cancer elevated EC to a Class 2A carcinogen, so it is necessary to regulate EC content in Huangjiu. The risk of intake of dietary EC is mainly assessed through the margin of exposure (MOE) recommended by the European Food Safety Authority, with a smaller MOE indicating a higher risk. Interventions are necessary to reduce EC formation. As urea, one of the main precursors of EC formation in Huangjiu, is primarily produced by Saccharomyces cerevisiae through the catabolism of arginine, the construction of dominant engineered fermentation strains is a favorable trend for the future production and application of Huangjiu. This review summarized the formation and carcinogenic mechanism of EC from the perspectives of precursor substances, metabolic pathways after ingestion, and risk assessment. The methods of constructing dominant S. cerevisiae strains in Huangjiu by genetic engineering technology were reviewed, which provided an important theoretical basis for reducing EC content and strengthening practical control of Huangjiu safety, and the future research direction was prospected.

Research progress on the application of different controlling strategies to minimizing ethyl carbamate in grape wine.

Ethyl carbamate (EC) is a probable carcinogenic compound commonly found in fermented foods and alcoholic beverages and has been classified as a category 2A carcinogen by the International Agency for Research on Cancer (IARC). Alcoholic beverages are one of the main sources of EC intake by humans. Therefore, many countries have introduced a standard EC limit in alcoholic beverages. Wine is the second largest alcoholic beverage in the world after beer and is loved by consumers for its rich taste. However, different survey results showed that the detection rate of EC in wine was almost 100%, while the maximum content was as high as 100 µg/L, necessitating EC content regulation in wine. The existing methods for controlling the EC level in wine mainly include optimizing raw fermentation materials and processes, using genetically engineered strains, and enzymatic methods (urease or urethanase). This review focused on introducing and comparing the advantages, disadvantages, and applicability of methods for controlling EC, and proposes two possible new techniques, that is, changing the fermentation strain and exogenously adding phenolic compounds. In the future, it is hoped that the feasibility of this prospect will be verified by pilot-scale or large-scale application to provide new insight into the regulation of EC during wine production. The formation mechanism and influencing factors of EC in wine were also introduced and the analytical methods of EC were summarized.

A new urea adducts method for PUFA concentration using green food grade solvents and avoiding ethyl carbamate formation.

This study aimed to selectively enrich stearidonic acid (SDA) together with γ-linolenic acid (GLA) in Echium plantagineum oil by urea complexation. The complexation process at room temperature was carried out replacing common organic solvents, such as hexane and ethanol, by alternative compounds, included in Green Solvent and Food Grade categories, adapting this process towards the principles of Green Chemistry. This substitution was also intended to avoid the generation of the toxic compound ethyl carbamate. Among all the solvents studied, the mixture propionic acid and α-pinene provided the best results, leading to a final product comprised of ∼99% of PUFA, with ∼45% SDA (∼14% in the original oil), and without apparition of ethyl carbamate. The procedure was tested on other raw materials (salmon and microalgae oils). The solvent was efficiently recuperated from the liquid phase (∼87% recovery) and reutilized once with almost identical results.

Ethyl carbamate regulate esters degradation by activating hydrolysis during Baijiu ripening.

The presence of ethyl carbamate in traditional fermented food is a public health concern for the FDA. The effect of forced photoirradiation (0-150,000 lx) and thermal stress (4, 25, 40 °C) on ethyl carbamate (0-150 µg/L) on physiological characteristics of Baijiu, such as esters content, photochemical degradation, and hydrogen-bond interaction efficiency were monitored by ultra high performance liquid chromatography quadrupole-orbitrap and the dynamic changes by digital foodomics analysis. Furthermore, 748 trace components covering 11 subclasses were identified in Baijiu and 71 esters were screened by Spearman's correlation, fold changes, P values and VIP values. A forward stepwise multiple regression and discriminant analysis were performed for predicting the content of esters from appearance characteristics obtained by foodomics analysis, reaching R-square values up to 0.91. A reduction of the present variation in ethyl lactate, ethyl caproate, diethyl succinate, ethyl oleate and ethyl linoleate concentration could possibly result in a better understanding of the ethyl carbamate effects. Ethyl carbamate was found to cause esters hydrolysis through inter-molecular interaction in various species of alcoholic drinks. It was demonstrated that the light exposure level and thermal intensity applied for ethyl carbamate-spiked Baijiu samples did not unambiguously influence esters concentration in Baijiu. Future research should focus on moderate light exposure level and thermal stress and should aim at reducing natural ethyl carbamate by more closely controlling the esters content of the ripening degree.

Correlation between the microbial community and ethyl carbamate generated during Huzhou rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen that is mainly produced by the spontaneous reaction between urea and ethanol during rice wine brewing. Huzhou rice wine (HZRW) is a traditional Chinese rice wine, but the correlation between its urea content and the microbial communities present during the fermentation process has not yet been evaluated. In this study, high-throughput sequencing technology was used to monitor the microbial community composition of HZRW in the different fermentation stages. The correlations between the microbial community and the physical and chemical properties and EC, urea and arginine contents were evaluated using the redundancy analysis (RDA) method. The metabolic profiles of key genes in the arginine and urea metabolic pathways were obtained via phylogenetic investigation of the communities by reconstruction of unobserved states (PICRUSt). The results showed that the fungal genera Saccharomyces, Issatchenkia, Torulaspora and Rhizopus were dominant during the fermentation of HZRW. Weissella and Acinetobacter were the dominant bacterial genera in the early stage, while Weissella, Staphylococcus, Leuconostoc and Streptophyta were the dominant bacterial genera in the late stage. Urea and arginine were positively correlated with Saccharomyces, Lactobacillus and Staphylococcus. In addition, the dominant genera of both fungi and bacteria were involved in the metabolism of arginine and urea. Finally, the relationships between the dominant microorganisms and key genes of the arginine and urea metabolic pathways were established. The obtained results are helpful in better understanding the mechanisms of metabolism of arginine and urea during rice wine fermentation and therefore improving the safety profile of rice wine.

Molecular mechanism of Mare Nectaris and magnetic field on the formation of ethyl carbamate during 19 years aging of Feng-flavor Baijiu.

Ethyl carbamate (EC) is carcinogen occurring naturally in fermented foods, while the EC formation pattern in Feng-flavor Baijiu during Mare Nectaris storage and magnetic field treatment remains controversial. In this work, variation of EC in Mare Nectaris and magnetic field were investigated for the first time through ultra high performance liquid chromatography quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap). Quantification results revealed that EC decreased significantly in the stage of 3-9 years and kept at 12.4 µg L-1 after 10 years of aging. Arginine succinate synthase (ASS) and urease were deemed as vital factors for EC decomposition. Degradation effetc of EC in 250 mT is simillar to that of EC in Baijiu stored in Mare Nectaris for 8 years. This is due to that aging process was accelerated by magnetic field and the content of total acid in Baijiu was increased, creating a favorable environment for decomposition of EC and urea.

Fabrication of a fluorescence probe via molecularly imprinted polymers on carbazole-based covalent organic frameworks for optosensing of ethyl carbamate in fermented alcoholic beverages.

Ethyl carbamate (EC), which is a group 2A carcinogen, is a byproduct formed in the alcohol fermentation process that can accumulate with heating, transportation, and storage. In this study, molecularly imprinted polymers (MIPs) on carbazole-based covalent organic frameworks (COFs) were prepared as a fluorescence probe for the optosensing of EC in fermented alcoholic beverages. The excellent optical properties of carbazole-based COFs coupled with the good adsorption and selectivity of MIPs provided fast and efficient recognition of EC. MIPs on carbazole-based COFs exhibited advantages of high efficiency, a good separation effect, fluorescence dependence, and reproducibility. A good linear relationship was obtained over the concentration range of 1-200 µg L-1, with a low limit of detection (LOD) of 0.607 µg L-1. The RSD precision and five-cycle reproducibility were lower than 4.91% and 6.38%, respectively, and the recoveries were 85.30%-109.49%. This optosensor was applied to quantify EC contents in several fermented alcoholic beverages, all of which were less than LOD. The results of the optosensors based on MIPs on carbazole-based COFs were then validated using standard gas chromatography-mass spectrometry (GC-MS), which gave results consistent with the proposed method.

Determination of ethyl carbamate in wine by matrix modification-assisted headspace single-drop microextraction and gas chromatography-mass spectrometry technique.

A novel method for the analysis of ethyl carbamate in wine has been developed by coupling matrix modification-assisted headspace single-drop microextraction and gas chromatography-mass spectrometry (GC-MS) techniques. The method was developed by optimizing the matrix modifier and extraction parameters. The calibration method was followed by quantifying the internal isotope standard. The results suggested that the method was linear in the concentration range of 2-1000 ng/mL (R2 = 0.9996). The method presents a detection limit of 1.5 ng/mL, and the quantification limit is 5 ng/mL. The accuracy ranged between 94.9 and 99.9%, and the precision of the method was less than 5%. The method was applied for the detection of wine samples, and the results exhibited no significant difference when compared to the solid phase extraction method.

Occurrence of Ethyl Carbamate in Foods and Beverages: Review of the Formation Mechanisms, Advances in Analytical Methods, and Mitigation Strategies.

ABSTRACT: Ethyl carbamate (EC) is a process contaminant that can be formed as a by-product during fermentation and processing of foods and beverages. Elevated EC concentrations are primarily associated with distilled spirits, but this compound has also been found at lower concentrations in foods and beverages, including breads, soy sauce, and wine. Evidence from animal studies suggests that EC is a probable human carcinogen. Consequently, several governmental institutions have established allowable limits for EC in the food supply. This review includes EC formation mechanisms, occurrence of EC in the food supply, and EC dietary exposure assessments. Current analytical methods used to detect EC will be covered, in addition to emerging technologies, such as nanosensors and surface-enhanced Raman spectroscopy. Various mitigation methods have been used to maintain EC concentrations below allowable limits, including distillation, enzymatic treatments, and genetic engineering of yeast. More research in this field is needed to refine mitigation strategies and develop methods to rapidly detect EC in the food supply.

Ethyl carbamate in retail market condiments and risk assessment of its dietary exposure for the Korean population.

Ethyl carbamate (EC), a potential human dietary carcinogen, is found in fermented foods including the fermented soybean-based condiments, the major part of the Korean diet. Therefore, it is expected that their EC contents might pose health risks. Herein, we collected 111 condiments and estimated their EC contents via gas chromatography-mass spectrometry. Further, dietary intake of EC was evaluated, and the risk levels were assessed via the margin of exposure (MOE) approach and excess cancer risk assessment. EC contents of the condiments ranged from not detectable to 39.47 µg/kg, and the daily EC exposure ranged from 1.4 to 2.0 ng/kg BW per day, depending on gender and age groups in Korea. Of the condiments, soy sauce was the largest contributor to EC exposure. MOE and excess cancer risks for the average consumer were 166,300 and 9.0 × 10-8, respectively, and those for the consumers in the 95th percentiles (P95) were 53,504 and 2.8 × 10-7, respectively, indicating that the risk of exposure to EC is of lower concern in average consumers than heavy consumers. However, the EC exposure from condiments was higher than that in other Asian countries.Abbreviations: EC: ethyl carbamate; GC-MS: gas chromatography-mass spectrometry; MOE: margin of exposure; MRL: maximum residue level; IDL: instrumental detection level; IQL: instrumental quantification level; MDL: method detection level; MQL: method quantification level; EDI: estimated daily intakes; BMDL10: benchmark dose lower confidence limit.

Ethyl carbamate in Chinese liquor (Baijiu): presence, analysis, formation, and control.

Ethyl carbamate (EC) is a genotoxic and carcinogenic compound that is also a by-product of fermented foods (bread, sour milk, soy cheese, etc.) and alcoholic beverages (wine, sake, distilled liquor, etc.). Studies have showed that ethyl carbamate is ingested by humans primarily through the consumption of alcoholic beverages. Many countries have thus established EC limits for alcoholic beverages. Chinese liquor (Baijiu) is a traditional and unique distilled liquor, which has a huge consumption in China due to its excellent color, flavor, and taste. Therefore, the control of EC in Chinese liquor is of great significance. This review summarized for the first time the progress in presence level, analysis method, formation mechanism, and elimination strategy of EC of Chinese liquor in recent decades. KEY POINTS: • GC-MS and HPLC are the main methods to quantify EC in Chinese liquor. • EC is formed in the fermentation, distillation, and storage stage. • EC content can be reduced from raw material, microorganism, and production process.

Fluorescent sensor based on quantum dots and nano-porphyrin for highly sensitive and specific determination of ethyl carbamate in fermented food.

BACKGROUND: Ethyl carbamate (EC) is a potentially toxic carcinogen produced during fermentation and storage of fermented foods, and many countries have set thresholds for its content in food. Therefore, sensitive, rapid and accurate detection of EC is meaningful to ensure the quality of fermented food. RESULTS: This study introduces a CdTe quantum dots/nano-5,10,15,20-tetrakis (4-methoxyphenyl)-porphyrin (nano TPP-OCH3 ) fluorescence sensor system detection of EC. The specificity of this sensing mainly relies on a photo-induced electron transfer and electrostatic force interaction between EC and nano TPP-OCH3 . This sensor presented a linear range of 10 to 1000 µg L-1 (R2  = 0.9903) with a low detection limit of 7.14 µg L-1 . Meanwhile, the recovery (91.19-101.09%) and precision [relative standard deviation (RSD) = 0.64-3.05%] of the sensor for the analysis of fermented food (yellow rice wine, soy sauce, Chinese spirits, Pu-erh tea) samples were good and could meet the requirements of practical detection. Moreover, the detection results of fermented food (yellow rice wine, soy sauce, Chinese spirits, Pu-erh tea) samples by this sensor are basically consistent with those of high-performance liquid chromatography with fluorescence detector (HPLC-FLD). CONCLUSION: This method was expected to provide a potential platform for sensitive and accurate detection of EC in food safety monitoring, which would provide knowledge of the flavor and quality related to fermented food. © 2021 Society of Chemical Industry.

Inhibition of ethyl carbamate accumulation in soy sauce by adding quercetin and ornithine during thermal process.

Ethyl carbamate (EC), a genotoxic and carcinogenic compound in soy sauce accumulated during thermal processes, has raised public health concern for its multipoint potential carcinogenic risk to human. In this work, based on the analysis of EC accumulation during thermal processes of soy sauce, ornithine and quercetin were added before thermal processes to reduce EC accumulation. A reduction rate of 23.7-63.8% in simulated solution was founded. Kinetic studies indicated that ornithine was a byproduct of alcoholysis reaction when EC formed, while quercetin could compete with the precursor ethanol and react with carbamyl compounds, which therefore preventedEC accumulation. A maximum of 47.2% decrease of EC in soy sauce was achieved, and no remarkable changes in volatile compounds profile and color of soy sauce were found. In conclusion, the addition of quercetin and ornithine before thermal processes may be preferable for the controlling of EC content in soy sauce.

Effects of the pre-fermentative addition of chitosan on the nitrogenous fraction and the secondary fermentation products of SO2 -free red wines.

BACKGROUND: Different red winemaking were carried out to evaluate the effects of the prefermentative addition of chitosan, as an alternative to the use of SO2 , on the secondary products of alcoholic fermentation, yeast available nitrogen (YAN), biogenic amines and ethyl carbamate. RESULTS: The wines made with chitosan presented higher total acidity and higher content of tartaric and succinic acids than those made only with SO2 . The use of chitosan in winemaking resulted in wines with higher glycerol and diacetyl content without increasing the concentration of ethanol, acetic acid, acetaldehyde or butanediol. YAN was lower in wines made with chitosan, which may mean an advantage for the microbial stability of the wines. Furthermore, the use of chitosan at the beginning of alcoholic fermentation did not increase the concentration of biogenic amines or the formation of ethyl carbamate in SO2 -free red wines. CONCLUSION: The total or partial substitution of SO2 for chitosan at the beginning of the alcoholic fermentation gives rise to quality red wines without negatively affecting their nitrogen fraction or their very important secondary fermentation products such as acetic acid or acetaldehyde. © 2020 Society of Chemical Industry.

Development of Cu(II)/Cu(I)-induced quantum dot-mediated fluorescence immunoassay for the sensitive determination of ethyl carbamate.

A series of haptens were rationally designed for producing monoclonal antibodies specific for EC and a simple fluorescence immunoassay platform was developed for the sensitive determination of EC based on alkaline phosphatase (ALP)-triggered Cu+ quenching of CdSe quantum dots (QDs). It was noted that Cd as a fluorescence substrate in CdSe QDs can be selectively substituted by Cu+ that resulted in a more significant fluorescence quenching in comparison with Cu2+. Meanwhile, because ALP catalyzed ascorbic acid phosphate and then assisted the transformation of Cu2+ to Cu+, the change in fluorescence intensity was found to be proportional to ALP concentration. After simple magnetic separation, the sensitivity and linear range of the established assay were improved approximately 53-fold and an order of magnitude, respectively, when compared with the conventional ELISA. The proposed platform was able to both amplify the signal and eliminate matrix interferences, making it a promising to determine EC as well as other contaminants in complex food matrix in a highly sensitive and simple manner. Graphical abstract.