Enhancement of urea uptake in Chinese rice wine yeast strain N85 by the constitutive expression of DUR3 for 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     

黄酒中氨基甲酸乙酯的分析与控制

氨基甲酸乙酯是一种存在于黄酒中的致癌物质。本文采用气质联用法检测了广东市场上42种黄酒样品中氨基甲酸乙酯含量,28个样品中EC含量超过30μg/kg唱,占66．67％,其中7个样品中EC含量超过了100μg/kg（日本清酒中EC的限量标准）,最高的高达1210μg/kg。研究了脲酶对黄酒处理的效果,结果发现,一定条件下添加脲酶能够去除酒里的尿素,破坏产生氨基甲酸乙酯的主要途径,从而控制氨基甲酸乙酯含量不再剧增。并在单因素试验结果的基础上,进行正交实验,得出脲酶最佳的处理条件为酶浓度150mg／L、处理温度为25℃,pH值为3．5,处理时间10d。该条件下进行处理,可使酒中氨基甲酸乙酯含量降低45．83％。     

The overexpression of DUR1,2 and deletion of CAR1 in an industrial Saccharomyces cerevisiae strain 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     

Effect of ‘wheat Qu’ addition on the formation of ethyl carbamate in Chinese rice wine with enzymatic extrusion liquefaction pretreatment

Ethyl carbamate (EC), which has been confirmed as probably carcinogenic to humans, can be formed during the alcoholic fermentation of Chinese rice wine. In this study, EC and two possible precursors (urea and arginine) were analysed in wines fermented from rice processed by enzymatic extrusion liquefaction pretreatment, with different wheat Qu content gradients from 6 to 18% (dry basis, d.b.). The EC concentration decreased 22.5% with the addition of wheat Qu reduced to 6% for the enzymatic extrusion‐processed rice wine (EE). Moreover, although the arginine was generated 6.33–8.39 times higher in the enzymatic extruded rice, instead of the traditional treated rice at the same wheat Qu level, the corresponding level of urea formed from arginine was not high in the EE. In addition, rice treated by extrusion combined with enzymatic hydrolysis was found to be more utilizable and fermentable, and the lost quality of the final traditional rice wine with the low wheat Qu level (6–14%) could be improved using enzymatically extruded rice as the fermenting material. Therefore, it may be feasible to inhibit EC accumulation in Chinese rice wine by wheat Qu control, combined with enzymatic extrusion pretreatment for rice. Copyright © 2015 The Institute of Brewing & Distilling     

Citrulline production by lactic acid bacteria in Chinese rice wine

Ethyl carbamate (EC) is a carcinogenic compound derived from the spontaneous reaction of ethanol with urea or citrulline in Chinese rice wine. Polymerase chain reaction–denaturing gradient gel electrophoresis showed that five species, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis and Lactobacillus coryniformis were the most abundant bacteria in the Chinese rice wine production process. Five strains belonging to these species can degrade arginine primarily in the exponential growth phase and accumulate citrulline in MRS‐Arg medium. In addition, an L. brevis strain was shown to be capable of assimilating citrulline, indicating the potential of this strain suggesting a potential route to reduce citrulline content and ethyl carbamate formation in Chinese rice wine fermentation. Copyright © 2018 The Institute of Brewing & Distilling     

Exposure assessment and risk characterisation of ethyl carbamate from Korean traditional fermented rice wine,Takju and Yakju

Ethyl carbamate is one of the most hazardous chemicals naturally occurring in food, and is present in alcoholic beverages. Korean traditional rice wine, Takju and Yakju, is frequently consumed in Korea, but there have been no studies characterising the risks of ethyl carbamate in these products. In order to assess and characterise the exposure risk of ethyl carbamate in Korean traditional rice wines, ethyl carbamate was investigated by means of GC-MS. The analytical methods were optimised and validated through determining linearity, detection limit, quantification limit, recovery and precision. A total of 283 traditional Korean rice wines, including 175 Takju and 108 Yakju samples, were analysed. Exposure assessment was performed by factoring in ethyl carbamate content, daily consumption and body weight. Daily exposures of ethyl carbamate were estimated for adults in four age groups, and risks of ethyl carbamate were characterised by the margin of exposure, which is more than 10 000. Based on this study, the risks of ethyl carbamate in Korean traditional rice wine were shown to be of low concern.     

黄酒酵母的改良及对产物中尿素和氨基甲酸乙酯含量的影响

氨基甲酸乙酯（EC)是一种潜在致癌物,在黄酒发酵过程中尿素是它的前体物质。该研究通过紫外诱变和基因过表达筛选获得改良黄酒酵母菌种,并对黄酒产品的理化指标进行检测可知,与出发菌株相比,改造菌株的发酵性能和黄酒的出酒率、酒精度、总糖、总酸、氨基酸态氮和β-苯乙醇没有明显的差异,而诱变菌株JF501-A62发酵产物尿素含量降低了67%,EC含量降低了59%;基因过表达菌株JF501-B5发酵产物尿素含量降低了88%,EC含量降低了63%。两者均有很好的发酵性能,并取得了较好地降低产品中尿素含量、进而降低氨基甲酸乙酯含量的效果。与紫外诱变相比,基因过表达的改良方法获得了尿素含量更低的菌株,并贮存6个月之后产品中的EC含量更低。     

Application of an Acid Urease to Wine: Determination of Trace Urea in Wine

An improved method of urea detection having a sensitivity to 0.05 ppm, applicable to white wines, was developed by modifying Jansen's procedure. The urea concentration in many commercial bottled wines was found to be around 2 ppm. The method was utilized to the urea degradation by an acid urease which degraded naturally occurring urea of 0.5–2 ppm at the same reaction rate as that for the spiked urea of 30 ppm in the same white wine. Wine treatment with the acid urease also reduced ethyl carbamate formed by heating.     

Temperature and Light Effects on Ethyl Carbamate Formation in Wine During Storage

The formation and/or increase of ethyl carbamate during storage of wine was monitored at 43°C, 32°C and 22°C in the dark, at 22°C under fluorescent light and in a greenhouse with naturally fluctuating temperature and light conditions simulating commercial display of wines. The ethyl carbamate concentration of fifteen selected red and white table and dessert wines was determined initially, and after 3, 6, and 12 months storage. In most wines ethyl carbamate increased as a function of time and temperature. After 12 months in the dark at 43°C, the ethyl carbamate in two red wines increased 20 to 30 times. It increased three to nine-fold in nine of the wines, but remained unchanged in four. Light did not significantly influence ethyl carbamate formation, whether in clear or green bottles.     

慕萨莱思酒发酵过程中氨基甲酸乙酯含量的变化研究

氨基甲酸乙酯（EC）是发酵食品中存在的一种致癌物质,该研究以新疆阿瓦提慕萨莱思酒为研究对象,检测发酵过程中不同阶段EC含量、尿素含量、酒精体积分数,探究EC的形成与尿素、酒精含量的关系。结果显示,发酵结束后EC含量为25.6 μg/L,尿素与酒精含量越高,EC生成速率越快,慕萨莱思酒中EC含量越高,温度对EC的生成影响较大。     

Wine composition plays an important role in the control of carcinogenic precursor formation by Lactobacillus hilgardii X1B

BACKGROUND: Lactobacillus hilgardii, a wine lactic acid bacterium, is able to use arginine, through the arginine deiminase pathway with the formation of citrulline, a precursor of the carcinogen ethyl carbamate. The influence of different Argentine wine varieties (Merlot, Cabernet Sauvignon and Malbec), on bacterial growth and arginine metabolism was examined. Furthermore, the effect of different components normally present in wines on the enzyme activities of the arginine deiminase system was determined. RESULTS: Malbec wine under all conditions assayed (33, 50 and 100% supplemented wine:basal media) showed higher arginine consumption and citrulline production than the other wines, as well as the highest bacterial growth and survival of Lactobacillus hilgardii X₁B. Glucose and L ‐malic inhibited both arginine deiminase enzymes while fructose and citric acid only inhibited arginine deiminase. The red wines assayed in this study had different composition, and this is an explanation for the different behavior of the bacterium. CONCLUSION: The highest citrulline production in Malbec wine could be correlated with its lower concentrations of glucose, fructose, citric and phenolic acid than the other wines. Therefore, a wine with lower concentration of these sugars and acids could be dangerous due to the formation of ethyl carbamate precursors. Copyright © 2012 Society of Chemical Industry     

葡萄酒中氨基甲酸乙酯的含量测定及酿造工艺对其含量的影响

调查了我国干红葡萄酒(77款)和干白葡萄酒(17款)中氨基甲酸乙酯(EC)含量情况,并探讨了酿酒工艺(果胶酶、酵母、乳酸菌和发酵温度等)对葡萄酒中氨基甲酸乙酯(EC)含量的影响。研究结果显示:干红葡萄酒中EC含量范围在7.00～26.80μg/L,平均浓度为17.90μg/L;干白葡萄酒中EC含量范围在6.40～21.58μg/L,平均浓度为11.35μg/L。葡萄酒酿造过程中EC的生成主要受酵母菌和乳酸菌种类的影响,酒精发酵温度的降低和果胶酶的使用有助于减少葡萄酒中EC的含量。     

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.     

A survey of levels of ethyl carbamate in alcoholic beverages in 2009–2012, Hebei Province,China

Results of a survey of levels of ethyl carbamate (EC) (urethane) in alcoholic beverages carried out in four successive years from 2009 to 2012 by gas chromatography-mass spectrometry (GC/MS) are presented. The beverages were purchased for sampling from Hebei Province of China, including eight main areas of production. The samples comprised wines (n = 212), grain spirits (n = 143) and wine sauces (n = 164). The data show that the average EC content in these kinds of alcoholic beverages remains nearly constant over the years. The results provide valuable data for food authorities to establish maximum limits for EC in China.     

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.     

黄酒中氨基甲酸乙酯的研究进展

氨基甲酸乙酯（EC）是发酵食品和酒精饮品在发酵或贮存过程中产生的对人类健康存在潜在危害的一种物质。近年来,随着黄酒产业的兴起,在有关黄酒中氨基甲酸乙酯的控制的食品质量安全问题也越来越受到人们的关注。该文在检索近年来氨基甲酸乙酯相关文献和标准的基础上,对发酵产品中氨基甲酸乙酯的形成机理,以及黄酒食品安全性评价、氨基甲酸乙酯检测方法及其含量控制等方面的国内外研究现状进行综述,旨在为黄酒生产中降低氨基甲酸乙酯的含量、提升黄酒品质提供借鉴和参考。     

Effect of saliva esterase activity on ester solutions and possible consequences for the in‐mouth ester release during wine intake

The aim of this study was to investigate the role of saliva esterase activity on carboxylic esters typically associated with pleasant and fruity aromas in wine. For this, ex vivo experiments using the same fresh and inactivated (without enzymatic activity) human saliva with a mixture of carboxylic esters with different aliphatic chain length (ethyl butanoate, ethyl pentanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, and isoamyl acetate) were prepared. Liquid–liquid extraction with dichloromethane and GC–MS analysis were applied to the saliva systems in order to determine the reduction in ester content and the formation of their corresponding metabolic products (carboxylic acids) in the saliva systems before and after incubation at 37C. In addition, to check if there was a relationship between the susceptibility of esters to saliva hydrolysis and the amount of in‐mouth ester release during wine intake, the remaining oral amount of each ester was determined by comparing the intraoral amount immediately after spitting out the wine and 4 min later. Ex vivo experiments showed ester degradation by saliva esterase enzymes mainly acted on long chain esters (ethyl octanoate and ethyl decanoate), which gave rise to the formation of their corresponding carboxylic acids. Nonetheless, in spite of their higher susceptibility to saliva enzymes, in vivo experiments showed that long chain carboxylic esters remained in the oral cavity long after swallowing. This confirmed that ester hydrophobicity is closely related to the in‐mouth temporal release of these odorants and, therefore, behind wine aroma persistence. In wines, esters represent a group of aromatic compounds of great interest since they are linked to pleasant fruity aroma nuances. Today wine consumers are demanding fresh and long persistent fruity aromatic wines. The present research contributes to better understanding the relationship between ester content in the wine and oral aroma release experienced during wine tasting, considering the changes in these compounds during oral processing. This is a necessary step when trying to unravel the factors involved in wine aroma perception and in consumer preferences, and it represents a necessary knowledge in promoting winemaking practices (e.g., the use of selected microorganisms) for improving the type and amount of these aroma compounds in the wine.     

The determination of urea in wine – a review

The concentration of urea in wine is not routinely measured in Australian laboratories, but has been examined in studies of yeast metabolism and the formation of ethyl carbamate, a known carcinogen. For alcoholic beverages that may contain high levels of urea, steps have been taken to reduce the concentration of urea and therefore prevent ethyl carbamate production. Methods for the determination of urea in wine can be grouped into three categories that indicate how selectivity for urea is achieved; those based on colour-forming reactions, enzymatic hydrolysis and chromatographic separation. The two dominant methods used by research groups over the past fifteen years for the determination of urea in wine are based on the urea/ammonia test kit available from Boeringer Mannheim/R-Biopharm and the reaction of urea with 1-phenyl-1,2-propanedione-2-oxime; both are time-consuming and labour-intensive, but involve relatively straightforward and well-established procedures. However, other options are available that may be better suited to the desired application and the instrumentation available in any particular laboratory.     

Ethyl carbamate formation regulated by ornithine transcarbamylase and urea metabolism in the processing of Chinese yellow rice wine

Ethyl carbamate (EC) is a potential carcinogenic compound present in most of the fermented foods. In this work, EC was inhibited through different strategies during vinification of Chinese yellow rice wine. EC can be inhibited by the use of ornithine in contrast to the control at peak point. However, the utilisation of urease resulted in little inhibitive effect on EC. The comparative data of intracellular ornithine transcarbamylase (OTCase) and arginine deiminase (ADI) among four experiments showed that EC was positively regulated by intracellular OTCase, but ADI was not determined. Extracellular urea and citrulline content was significantly increased by adding ornithine (P < 0.05), whereas ethanol played a minor role in EC formation. The correlation analysis between EC and OTCase or urea revealed a linear association (correlation coefficients above 0.8). These findings suggested that OTCase may be a required factor regulating EC formation during the brewing of Chinese yellow rice wine.     

Free radical scavenging capacity of selected red,rosé and white wines

The free radical scavenging capacity of selected red, rosé and white Spanish wines from different grape varieties was determined by the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^·) method using a new methodology developed at our laboratory. The amount of sample necessary to decrease by 50% the initial DPPH^· concentration (EC₅₀), the time needed to reach the steady state at EC₅₀ concentration (T_EC50) and the antiradical efficiency (AE = 1/EC₅₀T_EC50) were determined in the wine samples. Some differences between rosé wines made with Garnacha and Tempranillo grape varieties were observed in the UV‐vis spectra and in the free radical scavenging parameters, those from Garnacha variety having the highest antioxidant activity. The antiradical efficiency of the wines followed a decreasing order: red wines (22.44 × 10⁻⁶) > rosé wines (4.90 × 10⁻⁶) > white wines (1.88 × 10⁻⁶). There was a correlation between antiradical efficiency and total polyphenol (TP): AE = −3.33135 + 0.0180535TP; the correlation coefficient was r = 0.951454. © 1999 Society of Chemical Industry