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Formation of ethyl carbamate and changes during fermentation and storage of yellow rice wine
Food Chemistry 152 (2014) 108–112
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Formation of ethyl carbamate and changes during fermentation and storage of yellow rice wine Pinggu Wu a,⇑, Chenggang Cai b,⇑, Xianghong Shen a, Liyuan Wang a, Jing Zhang a, Ying Tan a, Wei Jiang a, Xiaodong Pan a a b
Zhejiang Provincial Center for Disease Control and Prevention, No. 630 Xincheng Road, Hangzhou 310051, China College of Biology and Environmental Engineering, Zhejiang Shuren University, No. 8 Shuren Street, Hangzhou 310015, China
a r t i c l e
i n f o
Article history: Received 8 May 2013 Received in revised form 6 November 2013 Accepted 23 November 2013 Available online 1 December 2013 Keywords: Ethyl carbamate Yellow rice wine Change Fermentation Storage Urea
a b s t r a c t Ethyl carbamate (EC) was analyzed during yellow rice wine production and storage. EC increased slowly during fermentation and rapidly after frying and sterilization. Less amount of EC was formed when cooled rapidly to 30 °C than when cooled naturally. High temperature and long storage time increased EC formation. After 400 days storage, EC increased from 74.0 to 84.2, 131.8 and 509.4 lg/kg at 4 °C, room temperature and 37 °C, respectively, and there was significantly difference between the fried wine and the wine on sale from 2011 (p < 0.01). Urea increased during yellow rice wine fermentation and was above 20 mg/ kg after the wine was fried; urea contributed to EC formation when the fried wine was cooled slowly. These results indicate that it is necessary for industry to optimize the wine frying conditions, such as temperature, time and cooling process in order to decrease EC formation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Ethyl carbamate (EC) is genotoxic and carcinogenic in animal species, such as mice, rats, hamsters, and monkeys (Beland et al., 2005). The World Health Organization’s International Agency for Research on Cancer (IARC) re-classified EC as a group 2A carcinogen (IARC, 2010), which suggested a potential carcinogenic risk to human. EC has been detected in many alcoholic beverages (Alberts, Stander, & De Villiers, 2011; De Melo Abreu, Alves, Beatriz, & Herbert, 2005; Fu et al., 2010; Júnior, Mendonca, & Pereira, 2011; Lachenmeier, Kanteres, Kuballa, López, & Rehm, 2009; Lachenmeier, Schehl, Kuballa, Frank, & Senn, 2005; Liu et al., 2011; Wang, Ke, Wang, Yin, & Song, 2012; Wu & Chen, 2004; Wu, Pan, Wang, Shen, & Yang, 2012; Wu et al., 2012) and several countries such as Canada, USA, Brazil and others have set specific EC standard (Lachenmeier et al., 2010) to improve the safety of wines. Every year, large numbers of fermented foods and beverages are produced and consumed in China, and EC was detected in over 73% of the tested fermented samples, with a highest concentration of 650 lg/kg (Wu et al., 2012). The yellow rice wine is a traditional fermented alcohol drink in China. Until now, two processes, the traditional one (human handing) and the mechanical one (using equipment) have been applied for yellow rice wine production. The traditional process (Fig. 1) is ⇑ Corresponding authors. Tel.: +86 571 87115276; fax: +86 571 87115263. E-mail addresses: [email protected] (P. Wu), [email protected] (C. Cai). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.135
more time consuming than the mechanical method. The typical mechanical production process includes rice soaking, steaming, addition of starter culture, pre-fermentation, post-fermentation, squeezing, addition of caramel color, frying, package and storage; in this production process, the processes of rice steaming, addition of starter culture, fermentation, squeezing, frying and packaging are mechanically operated. The fermentation process lasts for 25–40 days, and then the yellow rice wines are stored for 6– 12 months. To understand the critical factors influencing EC formation during yellow rice wine fermentation and storage, a number of yellow rice wines from 3 factories in different cities of Zhejiang province were selected and the critical procedures and conditions influencing EC formation during yellow rice wine production and storage were studied. 2. Materials and methods 2.1. Samples Yellow rice wines produced by the traditional and mechanical methods were collected and used for analysis in this experiment. The No. 1 and No. 2 yellow rice wines were from a Jiaxing factory produced by mechanical and traditional processes, respectively. The No. 3 yellow rice wine was from a Hangzhou factory produced by mechanical process. Some other yellow rice wines bought from a local supermarket in 2011 were also analyzed.
P. Wu et al. / Food Chemistry 152 (2014) 108–112
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Rice → Immerse and cooking → Cooked rice → Temperature drop → Addition of starter → Pre-fermentation (3-7 days) → Canned and Post-fermentation (30-60 days) → Wine squeezing → Caramel colored → Clarification (1-3 days) → Wine frying (86-92 °C) → Cool to room temperature → Storage (as semi-product for over 6 months) → Sterilization → Packaging and sale. Fig. 1. Diagram of the traditional yellow rice wine production process.
2.2. EC analysis EC was determined according to the AOAC (2000) first action method with minor modifications (Wu et al., 2012). In brief, the d5-ethyl carbamate was used as an internal standard. A 2.0-g wine sample containing 100-lL 1.0 lg/mL d5-ethyl carbamate was added to a centrifuge tube and vortexed for 1 min. Using a diatomite solid-phase extraction column, the analyte was eluted from the mixture with 10 mL of 5% ethyl acetate after 10 min of short static stretches. The collected elutate was 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. All samples were measured three times and the data were presented as the average of the three measured values.
increased slightly and was less than 30 lg/kg (Fig. 2). The wines produced by the mechanical process contained 11.0 (Fig. 2A) and 13.7 lg/kg (Fig. 2C) of EC respectively, which was lower than that by the traditional process (31.6 lg/kg) (Fig. 2B). This may be due to the difference in factors such as change in fermentation time and temperature between the two conditions. Both the traditional and the mechanical processes of yellow rice wine production included the steps of rice soaking, steaming, starter addition, pre-fermentation, post-fermentation, squeezing, caramel color addition, frying, packaging, and storage. In the mechanical process, steaming, addition of starter, fermentation, squeezing, frying, and packaging were all mechanically operated. The entire fermentation process was required approximately 25–40 days, which was lesser than that for the traditional process.
2.3. Urea determination 3.2. Changes in EC concentration during the wine frying process Urea was analyzed by the diacetyl monoxime method (Wang, Feng, Wu, Zhang, & Pan, 2010). In brief, 10.0 mL of the yellow rice wine was eluted using the Carb solid-phase micro-extraction column (200 mg/6 mL). The primary 1 mL elution fraction was discarded and the following 2 mL eluate was collected for further analysis. Subsequently, 2-mL water, 0.5-mL 2% diacetyl monoxime water solution, and 3-mL acid reagents (see below) were added to 2 mL of the yellow rice wine eluate. The fraction was allowed to boil in water for 15 min, and the reaction mixture was cooled in ice water and analyzed using an ultraviolet–visible spectrophotometer at 525 nm. Water (2 mL) was used as a control instead of the yellow rice wine. The aforementioned acid reagent was prepared by adding 1 mL of 98% sulfuric acid and 16.5 mL of 85% phosphoric acid to 50 mL of water and further cooled to room temperature, to which 0.5 g cadmium sulfate and 12.5 mg thiosemicarbazide were added to a final volume of 250 mL with water. Urea concentration was calculated by using a standard curve. 2.4. Statistical analysis Analysis of variance (anova) was used to detect significant differences in the EC level of the yellow rice wine samples. The level of statistical significance was determined at confidence intervals of p < 0.05 and p < 0.01. 3. Results and discussion 3.1. Change in EC concentration during the process of yellow rice wine fermentation EC was initially present in the starter fractions of the yellow rice wine production, and was less than 10.0 lg/kg. During the processes of pre-fermentation, post-fermentation, and squeezing, EC
Frying is a process that sterilizes and matures the yellow rice wine. The fried wine exhibits better quality, stability, and flavor. During the frying process, high temperature may promote EC formation because of the reaction between urea and ethanol. In the traditional yellow rice wine production process, wine frying was normally performed at 86–92 °C, the wine was sterilized at 85 °C, and cooled to room temperature or the wine was sterilized by steaming process post-squeezing and then allowed to cool under natural conditions. In the mechanical method, wine frying was performed by sterilization and canning and then the wine was cooled to room temperature after 2–3 days, or the wine was sterilized by pasteurization at 88–92 °C and then rapidly cooled to 30 °C. The yellow rice wines from the different factories were fried; post-frying, EC concentration rapidly increased from 13.7 to 51.8 lg/kg (Fig. 2A), 31.6 to 88.6 lg/kg (Fig. 2B), and 11.0 to 25.3 lg/kg (Fig. 2C), respectively. The results validate that increase in EC concentration was rapid and occurred within a short period of time during wine frying. Wine frying significantly influenced EC formation, which was identical to other studies; the fried yellow rice wine had higher EC concentration than the fermented wine pre-frying (Wu, Hong, Ma, & Xu, 2011; Wu et al., 2012). The No. 1 and No. 2 wine samples were fried at over 90 °C; postfrying, the temperature of the wine samples remained stable until the sterilization process was complete, after which the wine samples were allowed to cool to room temperature. EC concentration increased from 51.8 to 81.6 lg/kg (Fig. 2A) and from 88.6 to 121.2 lg/kg (Fig. 2B), respectively, which validated that EC was formed continuously post-frying. To study the influence of the cooling process on EC formation, the No. 1 wine sample was cooled rapidly to 30 °C post-frying, EC concentration was 34.6 lg/kg, which was lower than the naturally cooled wine of 51.8 lg/kg (Fig. 2A). This result validates that rapid cooling process decreased
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and the results (Table. 1) revealed that EC concentration was significantly different in the wines pre- and post-frying (t = 2.536, p = 0.03), which demonstrated that wine frying process and product sterilization had significantly influenced the EC formation. The wine post-frying also showed significant difference (t = 8.361, p < 0.01) with the wine that were on sale in 2011, which suggested that EC concentration increased during the storage while on sale. Earlier investigations demonstrated that EC formation was primarily related to urea and ethanol reactions, which reacted under specific conditions at suitable temperature and storage time during the process of wine production (Stevens & Ough, 1993; Woo et al., 2001).
A EC Concentration (μg/kg)
120 100
Cooled naturally after frying
80 60 40
Cooled quickly to O 30 C after frying
20
3.3. Change in EC during storage
0 1
2
3
4
5
6
7
8
9
Fermentation and storage time (d)
B
140
EC Concentration (µg/kg)
120 100 80 60 40 20 0 0
1
2
3
4
5
6
7
8
9
10
Fermentation and Storage time (d)
EC Concentration (μ g/kg)
C
25 20
After fermentation, the yellow rice wine was stored for a period time and then was blended, packaged and sterilized. Usually, longer storage time enhanced the flavor of the material and improved the taste of the wine. To understand the effects of temperature and time on EC formation during storage, the No. 3 yellow rice wine was stored under various conditions such as 4 °C, room temperature and 37 °C with 70% air humidity for a given period of time. The results are shown in Fig. 3. During storage, EC formation gradually increased at 4 °C and at room temperature for 400 days, while at 37 °C, EC concentration decreased in a short period of 7–14 days, and then increased rapidly to about 6 times in 400 days. EC increased from 74.0 to 509.4 lg/kg at 37 °C, while at room temperature and 4 °C, EC increased to 131.8 and 84.2 lg/kg, respectively. This validated that EC increased during storage and was significantly influenced by storage temperature conditions. This was similar to earlier results which demonstrated that EC increased at different temperature during storage, and high temperature increased EC rapidly than 4 °C and room temperature (Wu et al., 2012). Several other studies also showed that EC formation was significantly accelerated by high temperature, as well as by high concentrations of ethanol, urea and citrulline (Canas, Joe, Diachenko, & Burns, 1994; Kodama, Suzuki, Fujinawa, De La Teja, & Yotsuzuka, 1994; Monteiro, Trousdale, & Bisson, 1989; Ough & Stevens 1993).
15
3.4. Changes in urea concentration during fermentation 10 5 0 1
2
3
4
5
6
7
Fermentation and storage time (d) Fig. 2. Changes of EC concentration in different yellow rice wine samples. (A) No. 1 yellow rice wine produced by mechanical fermentation process from a factory of Jiaxing. (B) No. 2 yellow rice wine produced by traditional fermentation process from a factory of Jiaxing. (C) No. 3 yellow rice wine produced by mechanical fermentation process from a factory of Hangzhou.
the EC formation post-frying and EC concentration was lower than the wine cooled under natural condition. In a survey conducted by us in 2012, the EC concentration for various yellow rice wine production revealed that the EC formed in pre-fermentation and post-fermentation and squeezing processes were far lower than the wines on sale in 2008 (average 116 lg/kg) and in 2010 (average 134.6 lg/kg). This suggested that EC was formed continuously during storage and while on sale (data not shown). We surveyed 11 other wines that were on sale in 2011
Urea in the yellow rice wines collected from various factories were analyzed. The results showed that urea concentration increased during fermentation (Fig. 4) in the starter material and microbial metabolites. After the wine was fried, urea concentration was over 20 mg/kg with EC of 20–80 lg/kg (Fig. 2A), which suggested that urea formed during fermentation was sufficient for the EC formation during the storage condition. Comparing the
Table 1 EC concentration of 11 collected yellow rice wines during frying in 2012 as well as 11 yellow rice wines on sale in 2011 (lg/kg). Numbers
Before frying
After frying
Yellow rice wine on sale of 2011
1 2 3 4 5 6 7 8 9 10 11 X±s
23.9 20.2 20.7 17.0 13.3 25.2 18.6 30.6 18.3 7.2 6.8 18.3 ± 7.2
30.0 33.5 28.9 20.5 25.0 37.5 96.9 46.1 27.3 15.4 16.8 34.4 ± 26.5
143.1 194.6 234.2 357.0 223.0 129.9 208.1 308.6 272.2 378.6 227.2 243.3 ± 79.7
P. Wu et al. / Food Chemistry 152 (2014) 108–112
509.4
EC Concentration (˩g/kg)
500 400
4 OC Room temperature 37 OC
300 200
131.8 100
74.0
84.2
0 0
100
200
300
400
Storage time (days) Fig. 3. Changes of EC concentration of No. 3 yellow rice wine during storage from a factory of Hangzhou.
111
increased EC formation, in which the wine stored at 37 °C increased about 6 times from 74 to 509.4 lg/kg after 400 days storage, while wine at room temperature and 4 °C increased to 131.8 and 84.2 lg/kg, respectively. EC increased during storage and in the sales process, there was significant difference between the fried wine compared to the wine that was on sale in 2011 (p < 0.01). Urea was an important precursor in EC formation and was present in the starter material, and in the product metabolized by microorganisms. Urea increased during fermentation and was over 20 mg/kg, which was one of the critical factors that influenced EC formation during wine frying and storage conditions. It may be necessary for the corporations to optimize the critical factors influencing EC formation such as the frying conditions for temperature and time, as well as the cooling method conditions and storage temperature. Some other methods such as the utilization of strains with low urea product or the addition of acidic urease to degrade urea in the yellow rice wine may decrease EC formation. Funding Funded by Zhejiang province science and technology innovation team project (No. 2011R50021).
Uera concentration (mg/kg )
25
Cooled rapidly 20
Acknowledgments
15
Cooled naturally
10
5 1
2
3
4
5
6
7
We would like to thank the Institute of Nutrition and Food Security, Chinese Center for Disease Control and Prevention and Health Department of Zhejiang province, and Disease Control and Prevention Centers of Zhejiang province, Shaoxing, Jiaxing, and Jinhua for their help in the completion of this project. We also thank the factories from which the yellow rice wine samples were obtained. This work was funded by Zhejiang Province Science and Technology Innovation Team Project (No. 2011R50021).
Fermentation and storage time (d) References Fig. 4. Changes of urea during rice wine fermentation of No. 1 yellow rice wine from a factory of Jiaxing.
cooling process post-wine frying, the wine that was rapidly cooled to 30 °C had high level of urea than the naturally cooled wine. This was in accordance to the result observed in case of EC, which was higher in the wine cooled under the natural condition as compared to the rapid cool process (Fig. 2A). This was probably due to the increased reaction rates found between the urea and ethanol at high temperature conditions during the natural cooling process. The results were identical to another study performed earlier, in that the urea level and EC concentration tested in the yellow rice wine samples had negative linear correlation (Fu et al., 2010). Several other studies also showed that EC formation was significantly accelerated at high temperature conditions, as well as high ethanol, urea, and citrulline concentration (Canas et al., 1994; Hasnip, Caputi, Crews, & Brereton, 2004; Kodama et al., 1994; Ough & Stevens, 1993).
4. Conclusions EC formation was slow during yellow rice wine fermentation and rapid post-wine frying. EC showed significant difference preand post-frying, which suggested that frying was a critical factor that influenced EC formation. This suggested that it may be possible to decrease EC formation by optimization of frying and sterilization conditions. Post-yellow rice wine frying, the natural cooling process increased the EC concentration, while decreased the EC formation when rapidly cooled to 30 °C. High temperature
Alberts, P., Stander, M. A., & De Villiers, A. (2011). Development of a novel solidphase extraction, LC–MS/MS method for the analysis of ethyl carbamate in alcoholic beverages: Application to South African wine and spirits. Food Additives and Contaminants, 7, 826–839. AOAC (2000). AOAC official method 994.07: Ethyl carbonate in alcoholic beverages and soy sauce. In Association of official analytical chemists, AOAC official methods of analysis, pp. 14–15). Gaithersburg, MD: AOAC International. Beland, F. A., Benson, R. W., Mellick, P. W., Kovatch, R. M., Roberts, D. W., Fang, J. L., et al. (2005). Effect of ethanol on the tumorigenicity of urethane (ethyl carbamate) in B6C3F1 mice. Food Chemistry and Toxicology, 43, 1–19. Canas, B. J., Joe, F. L., Diachenko, G. W., & Burns, G. (1994). Determination of ethyl carbamate in alcoholic beverages and soy sauce by gas chromatography with mass selective detection: Collaborative study. Journal of the Association of Official Analytical Chemists, 77, 1530–1536. De Melo Abreu, S., Alves, A., Beatriz, O., & Herbert, P. (2005). Determination of ethyl carbamate in alcoholic beverages: An interlaboratory study to compare HPLC– FLD with GC–MS methods. Analytical and Bioanalytical Chemistry, 382, 498–503. Fu, M. L., Liu, J., Chen, Q. H., Liu, X. J., He, G. Q., & Chen, J. C. (2010). Determination of ethyl carbamate in Chinese yellow rice wine using high-performance liquid chromatography with fluorescence detection. International Journal of Food Science and Technology, 45, 1297–1302. Hasnip, S., Caputi, A., Crews, C., & Brereton, P. (2004). Effects of storage time and temperature on the concentration of ethyl carbamate and its precursors in wine. Food Additives and Contaminants, 12, 1155–1161. IARC. (2010). IARC monographs on the evaluation of carcinogenic risks to humans. http://monographs.iarc.fr/ENG/Monographs/vol96/index.php. Júnior, J. C. B., Mendonca, R. C. S., De Assis Teixeira Kluge Pereira, J. M., Pereira, J. A. M., & de Fátima Ferreira Soares, N. (2011). Ethyl-carbamate determination by gas chromatography–mass spectrometry at different stages of production of a traditional Brazilian spirit. Food Chemistry, 129, 1383–1387. Kodama, S., Suzuki, T., Fujinawa, S., De La Teja, P., & Yotsuzuka, F. (1994). Urea contribution to ethyl carbamate formation in commercial wines during storage. American Journal of Enology and Viticulture, 45, 17–24. Lachenmeier, D. W., Kanteres, F., Kuballa, T., López, M. G., & Rehm, J. (2009). Ethyl carbamate in alcoholic beverages from Mexico (tequila, mezcal, bacanora, sotol) and Guatemala (cuxa): Market survey and risk assessment. International Journal of Environmental Research and Public Health, 6, 349–360. Lachenmeier, D. W., Lima, M. C. P., Nóbrega, I. C. C., Pereira, J. A. P., Kerr-Corrêa, F., Kanteres, F., et al. (2010). Cancer risk assessment of ethyl carbamate in alcoholic
112
P. Wu et al. / Food Chemistry 152 (2014) 108–112
beverages from Brazil with special consideration to the spirits cachaca and tiquira. BMC Cancer, 10, 266. Lachenmeier, D. W., Schehl, B., Kuballa, T., Frank, L., & Senn, T. (2005). Retrospective trends and current status of ethyl carbamate in German stone-fruit spirits. Food Additives and Contaminants, 5, 397–405. Liu, Y. P., Dong, B., Qin, Z. S., Yang, N. J., Lu, Y., Yang, L. X., et al. (2011). Ethyl carbamate levels in wine and spirits from markets in Hebei Province, China. Food Additives and Contaminants: Part B, 1, 1–5. Monteiro, F. F., Trousdale, E. K., & Bisson, L. F. (1989). Ethyl carbamate formation in wine: Use of radioactively labeled precursors to demonstrate the involvement of urea. American Journal of Enology and Viticulture, 40, 1–8. Ough, C. S., & Stevens, D. F. (1993). Ethyl carbamate formation: Reaction of urea and citrulline with ethanol in wine under low to normal temperature conditions. American Journal of Enology and Viticulture, 44, 309–312. Stevens, D. F., & Ough, C. S. (1993). Ethyl carbamate formation: Reaction of urea and citrulline with ethanol in wine under low to normal temperature conditions. American Journal of Enology and Viticulture, 44, 309–312. Wang, L. Y., Feng, L., Wu, P. G., Zhang, J., & Pan, T. Q. (2010). Determination of the urea content in yellow rice wine by spectrophotometric method. Chinese Journal of Health Laboratory Technology, 11, 3059–3061.
Wang, L. J., Ke, R. H., Wang, B., Yin, J. J., & Song, Q. H. (2012). Direct determination of ethyl carbamate in Chinese rice wine and grape wine by ultra performance liquid chromatography–electrospray ionization tandem mass spectrometry. Chinese Journal of Chromatography, 9, 903–907. Woo, I. S., Kim, I. H., Yun, U. J., Chung, S. K., Rhee, I. K., Choi, S. W., et al. (2001). An improved method for determination of ethyl carbamate in Korean traditional rice wine. Journal of Industrial Microbiology and Biotechnology, 26, 363–368. Wu, P., & Chen, Z. D. (2004). Determination of ethyl carbamate in alcoholic beverages by GC/MS with solid-phase extraction. Journal of Hygiene Research, 38, 627–628. Wu, H. M., Chen, L., Pan, G. S., Tu, C. Y., Zhou, X. P., & Mo, L. Y. (2012). Study on the changing concentration of ethyl carbamate in yellow rice wine during production and storage by gas chromatography/mass spectrometry. European Food Research Technology, 235, 779–782. Wu, J. M., Hong, R. Z., Ma, L. H., & Xu, H. Q. (2011). Determination and control of ethyl carbamate in the production of rice wine. Liquor Making, 3, 65–67. Wu, P. G., Pan, X. D., Wang, L. Y., Shen, X. H., & Yang, D. J. (2012). A survey of ethyl carbamate in fermented foods and beverages from Zhejiang, China. Food Control, 23, 286–288.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Formation of ethyl carbamate and changes during fermentation and storage of yellow rice wine Pinggu Wu a,⇑, Chenggang Cai b,⇑, Xianghong Shen a, Liyuan Wang a, Jing Zhang a, Ying Tan a, Wei Jiang a, Xiaodong Pan a a b
Zhejiang Provincial Center for Disease Control and Prevention, No. 630 Xincheng Road, Hangzhou 310051, China College of Biology and Environmental Engineering, Zhejiang Shuren University, No. 8 Shuren Street, Hangzhou 310015, China
a r t i c l e
i n f o
Article history: Received 8 May 2013 Received in revised form 6 November 2013 Accepted 23 November 2013 Available online 1 December 2013 Keywords: Ethyl carbamate Yellow rice wine Change Fermentation Storage Urea
a b s t r a c t Ethyl carbamate (EC) was analyzed during yellow rice wine production and storage. EC increased slowly during fermentation and rapidly after frying and sterilization. Less amount of EC was formed when cooled rapidly to 30 °C than when cooled naturally. High temperature and long storage time increased EC formation. After 400 days storage, EC increased from 74.0 to 84.2, 131.8 and 509.4 lg/kg at 4 °C, room temperature and 37 °C, respectively, and there was significantly difference between the fried wine and the wine on sale from 2011 (p < 0.01). Urea increased during yellow rice wine fermentation and was above 20 mg/ kg after the wine was fried; urea contributed to EC formation when the fried wine was cooled slowly. These results indicate that it is necessary for industry to optimize the wine frying conditions, such as temperature, time and cooling process in order to decrease EC formation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Ethyl carbamate (EC) is genotoxic and carcinogenic in animal species, such as mice, rats, hamsters, and monkeys (Beland et al., 2005). The World Health Organization’s International Agency for Research on Cancer (IARC) re-classified EC as a group 2A carcinogen (IARC, 2010), which suggested a potential carcinogenic risk to human. EC has been detected in many alcoholic beverages (Alberts, Stander, & De Villiers, 2011; De Melo Abreu, Alves, Beatriz, & Herbert, 2005; Fu et al., 2010; Júnior, Mendonca, & Pereira, 2011; Lachenmeier, Kanteres, Kuballa, López, & Rehm, 2009; Lachenmeier, Schehl, Kuballa, Frank, & Senn, 2005; Liu et al., 2011; Wang, Ke, Wang, Yin, & Song, 2012; Wu & Chen, 2004; Wu, Pan, Wang, Shen, & Yang, 2012; Wu et al., 2012) and several countries such as Canada, USA, Brazil and others have set specific EC standard (Lachenmeier et al., 2010) to improve the safety of wines. Every year, large numbers of fermented foods and beverages are produced and consumed in China, and EC was detected in over 73% of the tested fermented samples, with a highest concentration of 650 lg/kg (Wu et al., 2012). The yellow rice wine is a traditional fermented alcohol drink in China. Until now, two processes, the traditional one (human handing) and the mechanical one (using equipment) have been applied for yellow rice wine production. The traditional process (Fig. 1) is ⇑ Corresponding authors. Tel.: +86 571 87115276; fax: +86 571 87115263. E-mail addresses: [email protected] (P. Wu), [email protected] (C. Cai). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.11.135
more time consuming than the mechanical method. The typical mechanical production process includes rice soaking, steaming, addition of starter culture, pre-fermentation, post-fermentation, squeezing, addition of caramel color, frying, package and storage; in this production process, the processes of rice steaming, addition of starter culture, fermentation, squeezing, frying and packaging are mechanically operated. The fermentation process lasts for 25–40 days, and then the yellow rice wines are stored for 6– 12 months. To understand the critical factors influencing EC formation during yellow rice wine fermentation and storage, a number of yellow rice wines from 3 factories in different cities of Zhejiang province were selected and the critical procedures and conditions influencing EC formation during yellow rice wine production and storage were studied. 2. Materials and methods 2.1. Samples Yellow rice wines produced by the traditional and mechanical methods were collected and used for analysis in this experiment. The No. 1 and No. 2 yellow rice wines were from a Jiaxing factory produced by mechanical and traditional processes, respectively. The No. 3 yellow rice wine was from a Hangzhou factory produced by mechanical process. Some other yellow rice wines bought from a local supermarket in 2011 were also analyzed.
P. Wu et al. / Food Chemistry 152 (2014) 108–112
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Rice → Immerse and cooking → Cooked rice → Temperature drop → Addition of starter → Pre-fermentation (3-7 days) → Canned and Post-fermentation (30-60 days) → Wine squeezing → Caramel colored → Clarification (1-3 days) → Wine frying (86-92 °C) → Cool to room temperature → Storage (as semi-product for over 6 months) → Sterilization → Packaging and sale. Fig. 1. Diagram of the traditional yellow rice wine production process.
2.2. EC analysis EC was determined according to the AOAC (2000) first action method with minor modifications (Wu et al., 2012). In brief, the d5-ethyl carbamate was used as an internal standard. A 2.0-g wine sample containing 100-lL 1.0 lg/mL d5-ethyl carbamate was added to a centrifuge tube and vortexed for 1 min. Using a diatomite solid-phase extraction column, the analyte was eluted from the mixture with 10 mL of 5% ethyl acetate after 10 min of short static stretches. The collected elutate was 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. All samples were measured three times and the data were presented as the average of the three measured values.
increased slightly and was less than 30 lg/kg (Fig. 2). The wines produced by the mechanical process contained 11.0 (Fig. 2A) and 13.7 lg/kg (Fig. 2C) of EC respectively, which was lower than that by the traditional process (31.6 lg/kg) (Fig. 2B). This may be due to the difference in factors such as change in fermentation time and temperature between the two conditions. Both the traditional and the mechanical processes of yellow rice wine production included the steps of rice soaking, steaming, starter addition, pre-fermentation, post-fermentation, squeezing, caramel color addition, frying, packaging, and storage. In the mechanical process, steaming, addition of starter, fermentation, squeezing, frying, and packaging were all mechanically operated. The entire fermentation process was required approximately 25–40 days, which was lesser than that for the traditional process.
2.3. Urea determination 3.2. Changes in EC concentration during the wine frying process Urea was analyzed by the diacetyl monoxime method (Wang, Feng, Wu, Zhang, & Pan, 2010). In brief, 10.0 mL of the yellow rice wine was eluted using the Carb solid-phase micro-extraction column (200 mg/6 mL). The primary 1 mL elution fraction was discarded and the following 2 mL eluate was collected for further analysis. Subsequently, 2-mL water, 0.5-mL 2% diacetyl monoxime water solution, and 3-mL acid reagents (see below) were added to 2 mL of the yellow rice wine eluate. The fraction was allowed to boil in water for 15 min, and the reaction mixture was cooled in ice water and analyzed using an ultraviolet–visible spectrophotometer at 525 nm. Water (2 mL) was used as a control instead of the yellow rice wine. The aforementioned acid reagent was prepared by adding 1 mL of 98% sulfuric acid and 16.5 mL of 85% phosphoric acid to 50 mL of water and further cooled to room temperature, to which 0.5 g cadmium sulfate and 12.5 mg thiosemicarbazide were added to a final volume of 250 mL with water. Urea concentration was calculated by using a standard curve. 2.4. Statistical analysis Analysis of variance (anova) was used to detect significant differences in the EC level of the yellow rice wine samples. The level of statistical significance was determined at confidence intervals of p < 0.05 and p < 0.01. 3. Results and discussion 3.1. Change in EC concentration during the process of yellow rice wine fermentation EC was initially present in the starter fractions of the yellow rice wine production, and was less than 10.0 lg/kg. During the processes of pre-fermentation, post-fermentation, and squeezing, EC
Frying is a process that sterilizes and matures the yellow rice wine. The fried wine exhibits better quality, stability, and flavor. During the frying process, high temperature may promote EC formation because of the reaction between urea and ethanol. In the traditional yellow rice wine production process, wine frying was normally performed at 86–92 °C, the wine was sterilized at 85 °C, and cooled to room temperature or the wine was sterilized by steaming process post-squeezing and then allowed to cool under natural conditions. In the mechanical method, wine frying was performed by sterilization and canning and then the wine was cooled to room temperature after 2–3 days, or the wine was sterilized by pasteurization at 88–92 °C and then rapidly cooled to 30 °C. The yellow rice wines from the different factories were fried; post-frying, EC concentration rapidly increased from 13.7 to 51.8 lg/kg (Fig. 2A), 31.6 to 88.6 lg/kg (Fig. 2B), and 11.0 to 25.3 lg/kg (Fig. 2C), respectively. The results validate that increase in EC concentration was rapid and occurred within a short period of time during wine frying. Wine frying significantly influenced EC formation, which was identical to other studies; the fried yellow rice wine had higher EC concentration than the fermented wine pre-frying (Wu, Hong, Ma, & Xu, 2011; Wu et al., 2012). The No. 1 and No. 2 wine samples were fried at over 90 °C; postfrying, the temperature of the wine samples remained stable until the sterilization process was complete, after which the wine samples were allowed to cool to room temperature. EC concentration increased from 51.8 to 81.6 lg/kg (Fig. 2A) and from 88.6 to 121.2 lg/kg (Fig. 2B), respectively, which validated that EC was formed continuously post-frying. To study the influence of the cooling process on EC formation, the No. 1 wine sample was cooled rapidly to 30 °C post-frying, EC concentration was 34.6 lg/kg, which was lower than the naturally cooled wine of 51.8 lg/kg (Fig. 2A). This result validates that rapid cooling process decreased
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and the results (Table. 1) revealed that EC concentration was significantly different in the wines pre- and post-frying (t = 2.536, p = 0.03), which demonstrated that wine frying process and product sterilization had significantly influenced the EC formation. The wine post-frying also showed significant difference (t = 8.361, p < 0.01) with the wine that were on sale in 2011, which suggested that EC concentration increased during the storage while on sale. Earlier investigations demonstrated that EC formation was primarily related to urea and ethanol reactions, which reacted under specific conditions at suitable temperature and storage time during the process of wine production (Stevens & Ough, 1993; Woo et al., 2001).
A EC Concentration (μg/kg)
120 100
Cooled naturally after frying
80 60 40
Cooled quickly to O 30 C after frying
20
3.3. Change in EC during storage
0 1
2
3
4
5
6
7
8
9
Fermentation and storage time (d)
B
140
EC Concentration (µg/kg)
120 100 80 60 40 20 0 0
1
2
3
4
5
6
7
8
9
10
Fermentation and Storage time (d)
EC Concentration (μ g/kg)
C
25 20
After fermentation, the yellow rice wine was stored for a period time and then was blended, packaged and sterilized. Usually, longer storage time enhanced the flavor of the material and improved the taste of the wine. To understand the effects of temperature and time on EC formation during storage, the No. 3 yellow rice wine was stored under various conditions such as 4 °C, room temperature and 37 °C with 70% air humidity for a given period of time. The results are shown in Fig. 3. During storage, EC formation gradually increased at 4 °C and at room temperature for 400 days, while at 37 °C, EC concentration decreased in a short period of 7–14 days, and then increased rapidly to about 6 times in 400 days. EC increased from 74.0 to 509.4 lg/kg at 37 °C, while at room temperature and 4 °C, EC increased to 131.8 and 84.2 lg/kg, respectively. This validated that EC increased during storage and was significantly influenced by storage temperature conditions. This was similar to earlier results which demonstrated that EC increased at different temperature during storage, and high temperature increased EC rapidly than 4 °C and room temperature (Wu et al., 2012). Several other studies also showed that EC formation was significantly accelerated by high temperature, as well as by high concentrations of ethanol, urea and citrulline (Canas, Joe, Diachenko, & Burns, 1994; Kodama, Suzuki, Fujinawa, De La Teja, & Yotsuzuka, 1994; Monteiro, Trousdale, & Bisson, 1989; Ough & Stevens 1993).
15
3.4. Changes in urea concentration during fermentation 10 5 0 1
2
3
4
5
6
7
Fermentation and storage time (d) Fig. 2. Changes of EC concentration in different yellow rice wine samples. (A) No. 1 yellow rice wine produced by mechanical fermentation process from a factory of Jiaxing. (B) No. 2 yellow rice wine produced by traditional fermentation process from a factory of Jiaxing. (C) No. 3 yellow rice wine produced by mechanical fermentation process from a factory of Hangzhou.
the EC formation post-frying and EC concentration was lower than the wine cooled under natural condition. In a survey conducted by us in 2012, the EC concentration for various yellow rice wine production revealed that the EC formed in pre-fermentation and post-fermentation and squeezing processes were far lower than the wines on sale in 2008 (average 116 lg/kg) and in 2010 (average 134.6 lg/kg). This suggested that EC was formed continuously during storage and while on sale (data not shown). We surveyed 11 other wines that were on sale in 2011
Urea in the yellow rice wines collected from various factories were analyzed. The results showed that urea concentration increased during fermentation (Fig. 4) in the starter material and microbial metabolites. After the wine was fried, urea concentration was over 20 mg/kg with EC of 20–80 lg/kg (Fig. 2A), which suggested that urea formed during fermentation was sufficient for the EC formation during the storage condition. Comparing the
Table 1 EC concentration of 11 collected yellow rice wines during frying in 2012 as well as 11 yellow rice wines on sale in 2011 (lg/kg). Numbers
Before frying
After frying
Yellow rice wine on sale of 2011
1 2 3 4 5 6 7 8 9 10 11 X±s
23.9 20.2 20.7 17.0 13.3 25.2 18.6 30.6 18.3 7.2 6.8 18.3 ± 7.2
30.0 33.5 28.9 20.5 25.0 37.5 96.9 46.1 27.3 15.4 16.8 34.4 ± 26.5
143.1 194.6 234.2 357.0 223.0 129.9 208.1 308.6 272.2 378.6 227.2 243.3 ± 79.7
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509.4
EC Concentration (˩g/kg)
500 400
4 OC Room temperature 37 OC
300 200
131.8 100
74.0
84.2
0 0
100
200
300
400
Storage time (days) Fig. 3. Changes of EC concentration of No. 3 yellow rice wine during storage from a factory of Hangzhou.
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increased EC formation, in which the wine stored at 37 °C increased about 6 times from 74 to 509.4 lg/kg after 400 days storage, while wine at room temperature and 4 °C increased to 131.8 and 84.2 lg/kg, respectively. EC increased during storage and in the sales process, there was significant difference between the fried wine compared to the wine that was on sale in 2011 (p < 0.01). Urea was an important precursor in EC formation and was present in the starter material, and in the product metabolized by microorganisms. Urea increased during fermentation and was over 20 mg/kg, which was one of the critical factors that influenced EC formation during wine frying and storage conditions. It may be necessary for the corporations to optimize the critical factors influencing EC formation such as the frying conditions for temperature and time, as well as the cooling method conditions and storage temperature. Some other methods such as the utilization of strains with low urea product or the addition of acidic urease to degrade urea in the yellow rice wine may decrease EC formation. Funding Funded by Zhejiang province science and technology innovation team project (No. 2011R50021).
Uera concentration (mg/kg )
25
Cooled rapidly 20
Acknowledgments
15
Cooled naturally
10
5 1
2
3
4
5
6
7
We would like to thank the Institute of Nutrition and Food Security, Chinese Center for Disease Control and Prevention and Health Department of Zhejiang province, and Disease Control and Prevention Centers of Zhejiang province, Shaoxing, Jiaxing, and Jinhua for their help in the completion of this project. We also thank the factories from which the yellow rice wine samples were obtained. This work was funded by Zhejiang Province Science and Technology Innovation Team Project (No. 2011R50021).
Fermentation and storage time (d) References Fig. 4. Changes of urea during rice wine fermentation of No. 1 yellow rice wine from a factory of Jiaxing.
cooling process post-wine frying, the wine that was rapidly cooled to 30 °C had high level of urea than the naturally cooled wine. This was in accordance to the result observed in case of EC, which was higher in the wine cooled under the natural condition as compared to the rapid cool process (Fig. 2A). This was probably due to the increased reaction rates found between the urea and ethanol at high temperature conditions during the natural cooling process. The results were identical to another study performed earlier, in that the urea level and EC concentration tested in the yellow rice wine samples had negative linear correlation (Fu et al., 2010). Several other studies also showed that EC formation was significantly accelerated at high temperature conditions, as well as high ethanol, urea, and citrulline concentration (Canas et al., 1994; Hasnip, Caputi, Crews, & Brereton, 2004; Kodama et al., 1994; Ough & Stevens, 1993).
4. Conclusions EC formation was slow during yellow rice wine fermentation and rapid post-wine frying. EC showed significant difference preand post-frying, which suggested that frying was a critical factor that influenced EC formation. This suggested that it may be possible to decrease EC formation by optimization of frying and sterilization conditions. Post-yellow rice wine frying, the natural cooling process increased the EC concentration, while decreased the EC formation when rapidly cooled to 30 °C. High temperature
Alberts, P., Stander, M. A., & De Villiers, A. (2011). Development of a novel solidphase extraction, LC–MS/MS method for the analysis of ethyl carbamate in alcoholic beverages: Application to South African wine and spirits. Food Additives and Contaminants, 7, 826–839. AOAC (2000). AOAC official method 994.07: Ethyl carbonate in alcoholic beverages and soy sauce. In Association of official analytical chemists, AOAC official methods of analysis, pp. 14–15). Gaithersburg, MD: AOAC International. Beland, F. A., Benson, R. W., Mellick, P. W., Kovatch, R. M., Roberts, D. W., Fang, J. L., et al. (2005). Effect of ethanol on the tumorigenicity of urethane (ethyl carbamate) in B6C3F1 mice. Food Chemistry and Toxicology, 43, 1–19. Canas, B. J., Joe, F. L., Diachenko, G. W., & Burns, G. (1994). Determination of ethyl carbamate in alcoholic beverages and soy sauce by gas chromatography with mass selective detection: Collaborative study. Journal of the Association of Official Analytical Chemists, 77, 1530–1536. De Melo Abreu, S., Alves, A., Beatriz, O., & Herbert, P. (2005). Determination of ethyl carbamate in alcoholic beverages: An interlaboratory study to compare HPLC– FLD with GC–MS methods. Analytical and Bioanalytical Chemistry, 382, 498–503. Fu, M. L., Liu, J., Chen, Q. H., Liu, X. J., He, G. Q., & Chen, J. C. (2010). Determination of ethyl carbamate in Chinese yellow rice wine using high-performance liquid chromatography with fluorescence detection. International Journal of Food Science and Technology, 45, 1297–1302. Hasnip, S., Caputi, A., Crews, C., & Brereton, P. (2004). Effects of storage time and temperature on the concentration of ethyl carbamate and its precursors in wine. Food Additives and Contaminants, 12, 1155–1161. IARC. (2010). IARC monographs on the evaluation of carcinogenic risks to humans. http://monographs.iarc.fr/ENG/Monographs/vol96/index.php. Júnior, J. C. B., Mendonca, R. C. S., De Assis Teixeira Kluge Pereira, J. M., Pereira, J. A. M., & de Fátima Ferreira Soares, N. (2011). Ethyl-carbamate determination by gas chromatography–mass spectrometry at different stages of production of a traditional Brazilian spirit. Food Chemistry, 129, 1383–1387. Kodama, S., Suzuki, T., Fujinawa, S., De La Teja, P., & Yotsuzuka, F. (1994). Urea contribution to ethyl carbamate formation in commercial wines during storage. American Journal of Enology and Viticulture, 45, 17–24. Lachenmeier, D. W., Kanteres, F., Kuballa, T., López, M. G., & Rehm, J. (2009). Ethyl carbamate in alcoholic beverages from Mexico (tequila, mezcal, bacanora, sotol) and Guatemala (cuxa): Market survey and risk assessment. International Journal of Environmental Research and Public Health, 6, 349–360. Lachenmeier, D. W., Lima, M. C. P., Nóbrega, I. C. C., Pereira, J. A. P., Kerr-Corrêa, F., Kanteres, F., et al. (2010). Cancer risk assessment of ethyl carbamate in alcoholic
112
P. Wu et al. / Food Chemistry 152 (2014) 108–112
beverages from Brazil with special consideration to the spirits cachaca and tiquira. BMC Cancer, 10, 266. Lachenmeier, D. W., Schehl, B., Kuballa, T., Frank, L., & Senn, T. (2005). Retrospective trends and current status of ethyl carbamate in German stone-fruit spirits. Food Additives and Contaminants, 5, 397–405. Liu, Y. P., Dong, B., Qin, Z. S., Yang, N. J., Lu, Y., Yang, L. X., et al. (2011). Ethyl carbamate levels in wine and spirits from markets in Hebei Province, China. Food Additives and Contaminants: Part B, 1, 1–5. Monteiro, F. F., Trousdale, E. K., & Bisson, L. F. (1989). Ethyl carbamate formation in wine: Use of radioactively labeled precursors to demonstrate the involvement of urea. American Journal of Enology and Viticulture, 40, 1–8. Ough, C. S., & Stevens, D. F. (1993). Ethyl carbamate formation: Reaction of urea and citrulline with ethanol in wine under low to normal temperature conditions. American Journal of Enology and Viticulture, 44, 309–312. Stevens, D. F., & Ough, C. S. (1993). Ethyl carbamate formation: Reaction of urea and citrulline with ethanol in wine under low to normal temperature conditions. American Journal of Enology and Viticulture, 44, 309–312. Wang, L. Y., Feng, L., Wu, P. G., Zhang, J., & Pan, T. Q. (2010). Determination of the urea content in yellow rice wine by spectrophotometric method. Chinese Journal of Health Laboratory Technology, 11, 3059–3061.
Wang, L. J., Ke, R. H., Wang, B., Yin, J. J., & Song, Q. H. (2012). Direct determination of ethyl carbamate in Chinese rice wine and grape wine by ultra performance liquid chromatography–electrospray ionization tandem mass spectrometry. Chinese Journal of Chromatography, 9, 903–907. Woo, I. S., Kim, I. H., Yun, U. J., Chung, S. K., Rhee, I. K., Choi, S. W., et al. (2001). An improved method for determination of ethyl carbamate in Korean traditional rice wine. Journal of Industrial Microbiology and Biotechnology, 26, 363–368. Wu, P., & Chen, Z. D. (2004). Determination of ethyl carbamate in alcoholic beverages by GC/MS with solid-phase extraction. Journal of Hygiene Research, 38, 627–628. Wu, H. M., Chen, L., Pan, G. S., Tu, C. Y., Zhou, X. P., & Mo, L. Y. (2012). Study on the changing concentration of ethyl carbamate in yellow rice wine during production and storage by gas chromatography/mass spectrometry. European Food Research Technology, 235, 779–782. Wu, J. M., Hong, R. Z., Ma, L. H., & Xu, H. Q. (2011). Determination and control of ethyl carbamate in the production of rice wine. Liquor Making, 3, 65–67. Wu, P. G., Pan, X. D., Wang, L. Y., Shen, X. H., & Yang, D. J. (2012). A survey of ethyl carbamate in fermented foods and beverages from Zhejiang, China. Food Control, 23, 286–288.