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Changes of Ethyl Carbamate in Yellow Rice Wine during Shelf-Life and Formation in Simulated Ethanol and Urea Solutions

Pinggu Wu*, Chenggang Cai, Dajin Yang, Liyuan Wang, Yan Zhou, Xianghong Shen, Bingjie Ma, Jun Tan

Zhejiang Provincial Center for Disease Control and Prevention, No. 630 Xincheng Road, Hangzhou, China

Abstract: Changes of Ethyl carbamate (EC) in yellow rice wine during shelf life and Formation of EC in simulated ethanol and urea mixed solutions were analyzed. The results showed that EC presented in bottled yellow rice wine was more than that of bagged during storage. EC decreased in the first 200 days, then kept an upward tendency and reached 421 and 378 μg/kg till 600 days in bottled and bagged wines, respectively. High concentrations of urea and ethanol accelerated EC formation. EC increased to 591 μg/kg in solutions of 40 mg/kg urea and 40% ethanol, while for 20% ethanol, EC reached 189 μg/kg. After 61 days reaction of 35, 20 and 5 μg/mL urea with 30% and 15% ethanol, EC formed 283.1, 171 and 42.8 as well as 144, 99.1 and 23 μg/mL, respectively. The results directly validated EC formation caused by urea and ethanol, which was also the main reason for EC increasing in yellow rice wine during shelf life.

Keywords: Ethyl carbamate, yellow rice wine, food safety

Received June 03, 2014; Revised November 03, 2014; Accepted November 05, 2014

Citation: Pinggu Wu, Chenggang Cai, Dajin Yang, Liyuan Wang, Yan Zhou, Xianghong Shen, Bingjie Ma, Jun Tan, Changes of Ethyl Carbamate in Yellow Rice Wine during Shelf-Life and Formation in Simulated Ethanol and Urea Solutions. Journal of Food and Nutrition Research, 2 (12). 872-875. January 2014. doi:10.12691/jfnr-2-12-2

1. Introduction

Ethyl carbamate (EC) is genotoxic and carcinogenic in animal species [1,2], it was re-classiﬁed as a group carcinogen by the World Health Organization’s International Agency for Research on Cancer [3]. EC was widely present in the fermented foods such as alcohol beverages, soy sauce and etc, the contents in several alcohol beverages had been analyzed during production [4-17] including the yellow rice wine. The yellow rice wine is a traditional fermented alcohol drink in china and largely consumed every year. The typical production process of the yellow rice wine includes rice soaking, steaming, addition of starter culture, pre-fermentation, post-fermentation, squeezing, addition of caramel color, frying, package and storage. EC was widely presented in alcohol wines including the yellow rice wine [8,12,13,14,18,19,20], several studies showed that EC was mainly produced by reaction between urea and ethanol in the alcohol beverages [8,21,22,23] during the yellow rice wine production.

Usually the yellow rice wines in China were bottled and bagged stored in the supermarkets. The aroma and flavor components including EC in the rice wines increased with the rise of the storage time. Till now, little studies have been concerned on the change of EC in the yellow rice wines during their shelf life in different packages and there were scarcely studies on the process of EC production between urea and ethanol. To understand the change of EC during storage of the yellow rice wines and to illustrate EC formation influenced by ethanol and urea, two types packed yellow rice wines and the mixtures of several quantities of urea and ethanol were stored and measured periodically.

2. Materials and Methods

2.1. Samples

The yellow rice wine samples of over 5 years in bottled and bagged packages were bought from a local supermarket, both the two kind of wines contained 20 small individual packages. The wines were stored at room temperature in our lab for over 600 days and were used for periodical analysis.

2.2. EC Analysis

EC determination was carried out as the AOAC [24] first action method with minor modifications [13]. Briefly, the d₅-ethyl carbamate was used as an internal standard. A 2.0 g wine sample containing 100 μL 1.0 μg/mL d₅-ethyl carbamate was added into a centrifuge tube and vortexed for 1 min. A diatomite solid-phase extraction column was used and the analyte was eluted from the mixture with 10 mL of 5% ethyl acetate after 10 min of short static stretches. The resulting eluate was collected, dried by anhydrous sodium sulfate and concentrated using N₂ flow at 30°C. The analyte was further diluted with methanol to a final volume of 1 mL for GC/MS analysis. All samples were measured three times and the data were presented as the average of the three measured values.

2.3. EC Formation in Ethanol and Urea Solutions

Urea in 40 mg/kg (the general urea concentration in the yellow rice wines) was added to 20% and 40% ethanol water solutions, the mixed solutions were stored at 37°C and EC was analyzed intervals, then 5, 20, 35 μg/L urea were added to 15% (the general alcohol content in yellow rice wines) and 30% ethanol water solution, the mixed solutions were stored at 37°C and EC was measured periodically.

3. Results and Discussion

3.1. EC Changes during Shelf Life

The bottled and bagged rice wines were stored at room temperature for over 600 days. The results in Figure 1 showed that EC concentration in the bottle and bag packed rice wines decreased during the beginning to 250 days, EC decreased from 210 μg/kg to 184 μg/kg in the bottled wines, while the bagged wines were from 196 μg/kg to 158 μg/kg, then EC in bottled and bagged wines reached 421 and 378 μg/kg till 600 days, respectively.

Some studies had surveyed EC contents in the yellow rice wines [8,12,13,14,18,19,20], but little had been concerned about EC changes during shelf life. The results in this study showed that EC had a total rise tendency during its shelf life with a little drop in the first 250 days. Similar result was reported in the red wine stored from 1997 to 2001, EC in the newly produced red wines decreased firstly, then increased in the next two years during storage [25].

Figure 1. EC changes in yellow rice wine during shelf life

3.2. EC formation in the Simulated Solutions

3.2.1. Effects of Ethanol on EC Formation

Urea was added to 20% and 40% ethanol water solutions to 40 mg/kg (the generally urea concentration in the yellow rice wines), then the mixed solutions were stored at 37°C and EC was analyzed intervals. EC formation and changes in the mixture solutions were shown in Figure 2.

Figure 2. EC formation in ethanol and 40 μg/mL urea solutions at37 °C

The results indicated that EC formed continuously from the beginning, after 82 days, EC reached 189 μg/kg and 591 μg/kg in 20% and 40% ethanol, respectively, which showed that EC was formed by the reaction of ethanol and urea, and 40% ethanol produced more EC than that of 20% ethanol.

3.2.2. Effects of Urea on EC formation

Different quantities of urea were added to 15% (the generally alcohol content in yellow rice wines) and 30% ethanol solution to the level of 5, 20 and 35 μg/mL, then the mixed solutions were stored at 37°C and EC was analyzed intervals. The results were indicated in Figure 3 and Figure 4.

Figure 3. EC formation in solutions of urea and 15% ethanol at 37°C

The results showed a rise tendency in EC concentration, 35 μg/mL urea formed more EC than 20 and 5 μg/mL after reacted with 15% and got 144, 99.1 and 23 μg/mL, respectively (Figure 3).

Figure 4. EC formation in urea solutions and 30% ethanol at 37°C

As for 30% ethanol, 35, 20 and 5 μg/mL urea formed 283.1, 171 and 42.8 μg/mL EC after 61 days, respectively (Figure 4). EC formed quickly in high concentrations of urea and ethanol compared to that of low concentrations.

To the best of our known, there were little reports about the simulated studies on EC formation in ethanol and urea mixtures. Usually the ethanol and urea in yellow rice wines were of 15% and 40 μg/mL, respectively. Four different urea concentrations of 5, 20, 30 and 40 μg/mL as well as two ethanol concentrations of 15, 30% were mixed at 37°C for over 60 days in this study. The results indicated that higher concentrations of urea and ethanol promoted EC formation. Naturally occurring EC in wines was once confirmed [21,26], which stated that EC was mainly from naturally reaction of ethanol and carbamyl chemicals, such as urea, citrulline, carbamoyl phosphate, carbanyl aspartate and etc, in which the main substrate was ethanol [23,27,28,29,30].

As for the probably genotoxic and carcinogenic effects, several ways such as utilization of urea-deficiency yeast [31], addition of acidic urease and optimization of production and storage conditions had been suggested in yellow rice wine production.

4. Conclusions

EC increased with the storage time in the bottled and bagged yellow rice wines during storage, EC kept an upward tendency in the first 200 days and reached 421 and 378 μg/kg till 600 days in the bottled and bagged wines, respectively. Urea and ethanol in high concentrations accelerated EC formation. EC increased to 591 and 189 μg/kg in 40 mg/kg urea solution with 40% and 20% ethanol, respectively. After 61 days reaction of 35, 20 and 5 μg/mL urea with 30% and 15% ethanol, EC formed 283.1, 171 and 42.8 as well as 144, 99.1 and 23 μg/mL, respectively. The results directly validated EC formation caused by urea and ethanol, which was also the main reason for EC increasing in yellow rice wine during shelf life.

Acknowledgements

This work is supported by the National High Technology Research and Development Program of China (863 Program, No. 2012AA101603), the program For Zhejiang Leading Team of Science and Technology Innovation (2011R50021) and Project supported by the Open Foundation from Top Key Discipline of Mordern Agricultural Biotechnology and Biological Control of Crop Diseases in Zhejiang Provincial Colleges (2011KFJJ006).

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