Precursor Of Ethyl Carbamate Research Articles

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.

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.

Expression of an Acid Urease with Urethanase Activity in E. coli and Analysis of Urease Gene.

Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2.1U/mg for urease and 0.6U/mg for urethanase, respectively. However, apourease exhibited good resistance toward ethanol and acidic conditions. The relative activities of urease and urethanase remained over 80% in the buffers within pH 4-7. And the recoveries of both urease and urethanase activities were more than 50% in 5-25% ethanol solution. Apourease was utilized to eliminate urea in wine, and the residual urea in model wine was less than 50% after treatment with apourease for 30h. Then 3D structure of UreC was predicted, and it was docked with urea and EC, respectively. The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease, whereas only one hydrogen bond can be formed between EC and the active center. Moreover, EC exhibited greater steric hindrance than urea when combined with the active site. Due to the low specific activities of apourease, both structural genes and accessory genes of urease were co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities.

Combined Use of S. pombe and L. thermotolerans in Winemaking. Beneficial Effects Determined Through the Study of Wines' Analytical Characteristics.

The most common way to produce red wine is through the use of Saccharomyces cerevisiae strains for alcoholic fermentation and lactic acid bacteria for malolactic fermentation. This traditional winemaking methodology produces microbiologically stable red wines. However, under specific conditions off-flavours can occur, wine quality can suffer and human health problems are possible, especially after the second fermentation by the lactic acid bacteria. In warm countries, problems during the malolactic fermentation arise because of the high pH of the must, which makes it very difficult to properly control the process. Under such conditions, wines with high acetic acid and histamine concentrations are commonly produced. This study investigates a recent red wine-making technology that uses a combination of Lachancea thermotolerans and Schizosaccharomyces pombe as an alternative to the conventional malolactic fermentation. This work studies new parameters such as aroma compounds, amino acids, ethanol index and sensory evaluation. Schizosaccharomyces pombe totally consumes malic acid while Lachancea thermotolerans produces lactic acid, avoiding excessive deacidification of musts with low acidity in warm viticulture areas. This methodology also reduces the malolactic fermentation hazards in wines with low acidity. The main products are wines that contain less acetic acid, less biogenic amines and precursors and less ethyl carbamate precursors than the traditional wines produced via conventional fermentation techniques.

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.

The overexpression ofDUR1,2and deletion ofCAR1in an industrialSaccharomyces cerevisiaestrain and effects on nitrogen catabolite repression in Chinese rice wine production

Urea is acknowledged as the predominant precursor of ethyl carbamate (EC) in Chinese rice wine. During Chinese rice wine fermentation, urea accumulates owing to the nitrogen catabolite repression effect when preferred nitrogen sources are available. In previous research, two metabolically engineered strains were constructed with overexpression of DUR1,2 and deletion of CAR1 from an industrial Chinese rice wine yeast N85. The decreasing effect of urea and EC was demonstrated during small-scale Chinese rice wine fermentations. The present study compared the urea utilization rate of the parental and metabolically engineered yeast strains, using a preferred and non-preferred nitrogen source culture media, leading to alleviated urea accumulation and thus EC formation. The qRT-PCR results showed that, in all of the culture media, DUR1,2 was overexpressed with the inserted strong promoter PGK1p. During pilot scale fermentations, the urea and EC content decreased with the engineered strains. These results confirmed that the engineered strains could resist the nitrogen catabolite repression effect. Copyright © 2016 The Institute of Brewing & Distilling

Assessing the Potential Content of Ethyl Carbamate in White, Red, and Rosé Wines as a Key Factor for Pursuing Urea Degradation by Purified Acid Urease.

The ethyl carbamate (EC) content of a wine after a given temperature-time storage was theoretically predicted from the potential concentration of ethyl carbamate (PEC), as determined via an accelerated EC formation test. Such information was used to decide whether an enzymatic treatment was needed to reduce the wine urea level before bottling/aging. To this end, 6 white, red, and rosé wines, manufactured in Italy as such or enriched with urea, were tested for their PEC content either before or after enzymatic treatment using a purified acid urease preparation derived from Lactobacillus fermentum. The treatment was severely affected by the total phenolic content (TP) of the wine, the estimated pseudo-first-order kinetic rate constant for NH3 formation reducing by a factor of approximately 2000 as the TP increased from 0 to 1.64 g L(-1) . Such a sensitivity to TP was by far greater than that pertaining to a killed cell-based enzyme preparation used previously. Urea hydrolysis was successful at reducing EC concentration in wines with low levels of TP and other EC precursors.

Changes of ethyl carbamate and its precursors in maesil (Prunus mume) extract during one-year fermentation

The contents of ethyl carbamate (EC), cyanide, and ethanol were determined in maesil extracts that are liquids generated from fermentation of maesil fruit and brown sugar at 25°C or 15°C for one year. EC was detected from day 150 with a maximum value of 9.7μg/kg. The cyanide levels increased with prolonged soaking time of maesil and decreased at day 150 where EC was firstly detected, indicating that cyanide is a precursor of EC. Ethanol slowly increased at 25°C, while it fluctuated at 15°C. The contents of EC, cyanide, and ethanol were higher in the extracts fermented at 25°C compared with those at 15°C. EC contents had a higher positive correlation with cyanide contents (R=0.658) than ethanol contents (R=0.351). These results indicate that fermenting temperature gave a rise of EC precursors and consequently led to the increase of EC in the maesil extract.

Selected Schizosaccharomyces pombe Strains Have Characteristics That Are Beneficial for Winemaking.

At present, wine is generally produced using Saccharomyces yeast followed by Oenococus bacteria to complete malolactic fermentation. This method has some unsolved problems, such as the management of highly acidic musts and the production of potentially toxic products including biogenic amines and ethyl carbamate. Here we explore the potential of the fission yeast Schizosaccharomyces pombe to solve these problems. We characterise an extensive worldwide collection of S. pombe strains according to classic biochemical parameters of oenological interest. We identify three genetically different S. pombe strains that appear suitable for winemaking. These strains compare favourably to standard Saccharomyces cerevisiae winemaking strains, in that they perform effective malic acid deacidification and significantly reduce levels of biogenic amines and ethyl carbamate precursors without the need for any secondary bacterial malolactic fermentation. These findings indicate that the use of certain S. pombe strains could be advantageous for winemaking in regions where malic acid is problematic, and these strains also show superior performance with respect to food safety.

Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation.

Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine.

Cyanate as an Active Precursor of Ethyl Carbamate Formation in Sugar Cane Spirit.

The thermodynamic and kinetic aspects of ethyl carbamate (EC) formation through the reaction between cyanate and ethanol were investigated. The rate constant values for cyanate ion decay and EC formation are (8.0 ± 0.4) × 10(-5) and (8.9 ± 0.4) × 10(-5) s(-1), respectively, at 25 °C in 48% aqueous ethanolic solution at pH 4.5. Under the investigated experimental conditions, the rate constants are independent of the ethanol and cyanate concentrations but increase as the temperature increases (ΔH1(⧧) = 19.4 ± 1 kcal/mol, ΔS1(⧧) = −12.1 ± 1 cal/K, and ΔG1(⧧) = 23.0 ± 1 kcal/mol) and decrease as the solution pH increases. According to molecular modeling (DFT) that was performed to analyze the reaction mechanism, the isocyanic acid (HNCO) is the active EC precursor. The calculated ΔG1(⧧), ΔH1(⧧), and ΔS1(⧧) values are in very good agreement with the experimental ones.

Determination and formation of Ethyl Carbamate in Chinese spirits

This study aims to develop HPLC-FLD method and verify its applicability for Ethyl Carbamate (EC) determination in Chinese spirits, and to investigate the mechanism for EC generation and its influencing factors. HPLC-FLD with prior derivatization with 9-xanthydrol was used for the EC detection. Optimal reaction condition and the parameters usually tested in the method validation were identified and evaluated. Most appropriate mobile phase is sodium acetate solution with pH value at 7.2. Optimal alcohol strength and reaction time for derivatization are 38–42% and 50 min respectively. Good linearity was obtained with correlation coefficient greater than 0.9998; limit of detection and quantitation were 1.82 and 5.34 μg/L; recovery rate was 96.67% (±6.59%), and method precision was less than 5% (RSD). No significant difference was found in EC determination measured by HPLC-FLD and GC/MS (p > 0.05) indicating HPLC-FLD may be a suitable alternative to GC/MS with greater convenience and lower cost. The average EC content in the Chinese spirit was 39.3 μg/L (ND. to 79.1 μg/L, n = 90), and EC was mainly produced during storage. Raw material profile, brewing time, and storage time and temperature were suggested as main factors influencing EC content; moreover, lowering the storage temperature can effectively reduce EC production. Hydrocyanic acid was concluded as the EC precursor in Chinese spirits. A preliminary multiple regression model based on the storage temperature and time was produced to forecast the EC content in Chinese spirits, and this could be further developed in future research including more influencing factors.

Enhancement of urea uptake in Chinese rice wine yeast strain N85 by the constitutive expression ofDUR3for ethyl carbamate elimination

Most of the fermented alcoholic beverages, particularly Chinese rice wine, contain the potentially human carcinogenic compound ethyl carbamate (EC). As a major EC precursor in Chinese rice wine, urea in fermentations can be transported into the yeast cell by urea permease and finally metabolized by urea carboxylase and allophanate hydrolase in vivo. To eliminate EC in Chinese rice wines, the present study constructed high urea uptake yeast strains N1-D, N2-D and N-D, by introducing a strong promoter (PGK1p) into the urea permease gene (DUR3) of the industrial Chinese rice wine yeast N85, and by the restoration of the URA3 gene at the same time. With these self-cloned, high urea uptake strains, the urea and EC in the terminal Chinese rice wine samples were reduced to different extents. With two copies of overexpressed DUR3, the N-D strain could reduce the urea and the EC by 53.4 and 26.1%, respectively. No difference in fermentation characteristics was found between the engineered strains and the parental industrial yeast strain N85. These results could help to optimize the genetic manipulation strategy for EC elimination in Chinese rice wine production. Copyright © 2015 The Institute of Brewing & Distilling

Effect of citrulline, urea, ethanol, and urease on the formation of ethyl carbamate in soybean paste model system

The aim of this study was to determine the effect of urease on the formation of ethyl carbamate (EC) in the presence of previously known precursors of EC (citrulline, urea, and ethanol) using a soybean paste model system. The levels of EC were quantitatively determined by gas chromatography–mass spectrometry (GC–MS) every five days for a 30-day period. After 30days fermentation, the concentration of EC increased significantly by 135.2%, 242.2%, and 3757.1% when the precursors (citrulline, urea and ethanol) were added to the model system, respectively (p<0.05). Urease significantly decreased the level of EC by 38.4%, 18.8%, and 17.3% when citrulline, urea, and ethanol were added to the model system, respectively (p<0.05).

The arginine deiminase pathway of koji bacteria is involved in ethyl carbamate precursor production in soy sauce.

Ethyl carbamate (EC) is a group 2A carcinogen generated from a few precursors in many fermented foods and alcoholic beverages. Citrulline, urea, carbamoyl phosphate, and ethanol are common precursors detected in fermented foods. In this study, citrulline was proved to be the main EC precursor in soy sauce, which was found to be accumulated in moromi mash period and correlated with the utilization of arginine by koji bacteria. Six koji isolates belonging to three genera were identified to be able to accumulate citrulline via the arginine deiminase (ADI) pathway. Among these strains, only Pediococcus acidilactici retained high activities in synthesis and accumulation of citrulline in the presence of high concentration of sodium chloride. These results suggested that P.acidilactici is responsible for the accumulation of citrulline, one of the EC precursors, in the process of soy sauce fermentation.

Ethyl Carbamate in Fermented Beverages: Presence, Analytical Chemistry, Formation Mechanism, and Mitigation Proposals.

Ethyl carbamate (EC) commonly found in fermented beverages has been verified to be a multisite carcinogen in experimental animals. EC was upgraded to Group 2A by the Intl. Agency for Research on Cancer (IARC) in 2007, which indicates that EC is a probable carcinogen to humans. Because of its threat to human safety, the presence of EC may be a big challenge in the alcoholic beverage industry. During the past few years, thorough and systematic research has been carried out in terms of the generation of EC in order to meet the allowed limitation levels in fermented beverages. Previous studies have indicated that EC primarily results from the reaction of ethanol and compounds containing carbamyl groups. These main EC precursors are commonly generated from arginine metabolism by Saccharomyces cerevisiae or lactic acid bacteria accompanied by the fermentation process. This review comprehensively summarizes the genotoxicity, analytical methods, formation pathways, and removal strategies of EC in various beverages. The article also presents the metabolic mechanism of EC precursors and pertinent metabolites, such as urea, citrulline, and arginine.

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.

Studies on Two Exoenzymes Which Lyse Wine-Spoiling Bacteria

Microorganisms play an important role in the conversion of grape juice into wine. Different species of yeast are mainly responsible for the production of ethanol. Lactic acid bacteria also occur regularly in must or wine. They are mostly undesirable due to their capacity to produce wine-spoiling compounds. Especially strains of Lactobacillus brevis are able to produce biogenic amines as well as precursors of ethyl carbamate and different off-flavours (N-heterocycles, volatile phenols). By excessive formation of acetic acid some lactobacilli may even induce slow/stuck grape juice fermentations. In conventional winemaking, additions of sulphite or lysozyme are used to inhibit the growth of spoilage microorganisms. There is a strong interest in finding alternatives, because of the reduced activity of lysozyme in the wine milieu, a limited growth-inhibiting activity against lactic acid bacteria, and some health risks described regarding the application of sulphite. We found that a culture supernatant of Streptomyces albidoflavus B 578 lysed all bacteria previously isolated from must and wine samples (Acetobacter sp., Lactobacillus sp., Leuconostoc sp., Oenococcus oeni, Pediococcus sp., Staphylococcus sp.) including 35 strains of L. brevis. Two bacteriolytic exoenzymes were isolated and characterized from the streptomycete: a muramidase (24 kDa) and a protease (17 kDa). Both hydrolyzed cell wall components of L. brevis (peptidoglycan, S-layer proteins) and were active under wine-relevant conditions.

Determination L-Arginine from Grape Juice with Sakaguchi Reaction

Arginine in grape juice can be metabolized by wine yeasts to precursors of ethyl carbamate. The aim of this study was to develop a simple, accurate and fast method for determining arginine in grape juice by separating L-arginine with strong cation-exchange resins. The results showed that the optimized parameters as follows: 16g of pretreated resins packed in a glass column (D1.0×30cm), and 10.0mL of testing sample (pH = 2.0) added to the columns, kept for 20min at 20°C, then rinsed with pH 8.0 buffer solution. Finally, it was eluted with 300 mL 1 mol•L-1 NaOH at the rate of 2mL mol•min-1. The optimized method in combination with Sakaguchi reagents was simple, reproducible and accurate. This method can be applied for quick and fast determination of arginine in grape juice to take necessary action and avoid the forming of ethyl carbamate.