Original article

Ethyl carbamate formation regulated by ornithine

transcarbamylase and urea metabolism in the

processing of Chinese yellow rice wine

Ruo-Si Fang,1 Ya-Chen Dong,1 Teng-Yang Xu,1 Guo-Qing He1 & Qi-He Chen1,2*

1 Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China

2 Food Microbiology Research Key Laboratory of Zhejiang Province, Hangzhou 310058, China

(Received 28 November 2012; Accepted in revised form 8 June 2013)

Summary 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 utilisa-

tion 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 citrul-

line 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 associa-

tion (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.

Keywords Chinese yellow rice wine, ethyl carbamate (EC), metabolic elucidation, ornithine transcarbamylase (OTCase).

Introduction

Ethyl carbamate (EC) is widely present in the fer-mented foods, especially in spirits, such as grape wine,Japanese sake and Chinese yellow rice wine (Lach-enmeier et al., 2005; Madrera & Valles, 2009; Fu et al.,2010). As a consequence, the wine industry showedgreat interest in developing effective procedures to con-trol EC content in the final products. It has beenreported that EC content could be reduced in grapewine using suitable fermentation method or adoptingthe strains with high urease activity (Chiva et al.,2009). Most notably, EC could be reduced by geneticmodification of yeasts whose metabolism will not gen-erate EC precursors, and it still has good fermentationperformance. In the manufacture of sherry, EC wasalso reduced by adjusting the baking conditions duringthe prolonged ‘baking’ periods (Hasnip et al.,2007).

Arginine was one of the major amino acids found ingrape juice and wine (Lehtonen, 1996), which could bedecomposed to the precursors of EC by yeasts andmalolactic fermentation bacteria. As reported earlier,the degradation of arginine by wine LAB occurred via

the arginine deiminase (ADI) pathway (Liu et al., 1995;Liu & Pilone, 1998), in which mainly three enzymes areinvolved: arginine deiminase (ADI), catabolic ornithinetranscarbamylase (OTCase) and carbamate kinase(CK). By means of ADI route, the arginine metabolismproduced ammonia, ATP, ornithine, carbamyl phos-phate and some citrulline (Liu et al., 1994). Researcheshad shown that citrulline was a typical indicator of thepresence of EC in grape wine (Uthurry et al., 2004,2006; Romero et al., 2009). A correlation was foundbetween citrulline production and ethyl carbamate for-mation by Lactobacillus hilgardii that was isolated fromgrape wine (Arena & Manca de Nadra, 2005).Although many studies have focused on the elucidationof the biological basis and metabolic mechanism of ECproduction in grape wine and Japanese sake, only fewstudies have been reported on Chinese yellow rice wine.Fu et al. (2010) reported that using HPLC-FLDmethod, EC concentration can be quantitatively deter-mined in Chinese yellow rice wine samples derived fromdifferent brewing origins. The highest level of EC sur-veyed in the traditional Chinese yellow rice wine sam-ples was 242.2 lg L�1. Furthermore, a positivecorrelation between EC formation and urea metabolismwas found in Chinese yellow rice wine samples (Fu*Correspondent: E-mail:chenqh@zju.edu.cn

International Journal of Food Science and Technology 2013, 48, 2551–2556

doi:10.1111/ijfs.12248

© 2013 The Authors. International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

2551

et al., 2010). Recently, EC has been identified in mostof the Chinese yellow rice wines, and it turned out thatEC concentration in the rice wines is far more than thatin grape wine (Fu et al., 2010). Therefore, it was essen-tial to carry out investigations into the cytological basisand enzymatic mechanism of EC formation during vini-fication of the traditional Chinese yellow rice wine. Ourobjective was to provide an important theoretical prooffor practical regulation in improving the traditionalChinese yellow rice wine’s safety.

Materials and methods

Reagents and standards

L-citrulline, L-ornithine hydrochloride and L-argininewere obtained from Sangon Biotech Corporation(Shanghai, China) with the purity of at least 98%.EC and 9-xanthydrol were bought from Sigma-AldrichChemical Corporation (St Louis, MO, USA) with aminimum purity of 99%. Standard urease was obtainedfrom Sigma-Aldrich with activity of 150 00–50 000units g�1 solid. Otherwise pointed out, reagents used inthis study were of analytical grade.

Process of the fermentation of Chinese yellow rice wine

The preparing procedure of cooked rice is given as fol-lows: 1.25 kg raw rice was soaked into water (the ratioof raw rice to distilled water was 1:1.2) for 48 hat 28 °C, then steamed for 20 min and cooled to roomtemperature. The fermentation process (2.5 g Chineseyeast, 1.5 L water together with 10% (m m�1)Chinese koji were all added to the prepared rice) lasted4–5 days at 28 °C. When the amount of alcohol in fer-mentation broth remained stable, wine went into thelatter period of fermentation that continued 16–20 days. During the brewing process, samples withfour different concentrations were prepared for thedetermination: control group was designed withoutadding any additive (designated as CG), and theexperimental group was divided into three groups:0.01 M L-ornithine hydrochloride (designated as T1),1500 U urease per 1.5 kg solid added at the sixth day(designated as T2), and 1500 U urease per 1.5 kg solidadded at the ninth day (designated as T3).

Analysis of ethyl carbamate by HPLC-FLD

The amount of ethyl carbamate (EC) was determinedby the previously designed method (Fu et al., 2010).Concerning low concentration of EC in original sam-ples, the samples were condensed to one-tenth undervacuum rotary evaporator at 55 °C. Then, 0.01 M 9-xanthydrol (200 lL) was added to the condensedsample (1 mL) or standard EC, then followed by the

addition of 100 lL of 1.5 M HCl. The mixture wasshaken for a few seconds, allowed to stand for 30 minand transferred into chromatographic vials. TheHPLC-FLD equipment used in this work consisted oftwo Waters 510 pumps (Waters, Milford, MA, USA),a Waters 2475 fluorescence detector and a reversed-phase Symmetry C18 (250 9 4.6 mm, 4 lm, Waters).The mobile phase was methanol–water. The chromato-graphic temperature was set at 35 °C, injection volumewas 20 lL, and the emission and excitation wave-lengths were set to 600 and 233 nm, respectively.

Ethanol determination

GC analyses were carried out on an Agilent 7890Autosystem gas chromatograph equipped with a flameionisation detector (FID) and a HP-88 capillary col-umn (l = 60 m, ID = 0.25 mm, df = 0.2 lm) (Playne,1985). Initially the temperature was set at 50 °C for2 min and gradually increased to 110 °C at the speedof 3 °C min�1, and this temperature was maintainedfor 5 min. Meantime, the injector was kept at 200 °Cand FID at 300 °C. Herein, methanol was used as anexternal standard, and samples were injected in thesplit-flow mode at the ratio of 5:1.

Urea analysis

The amount of urea was determined using the methodreported earlier (Fu et al., 2010). Two analysisreagents were prepared using the following procedure:reagent I consisted of 120 mL sulphate (98%, v/v),50 mL phosphate (85%, v/v), 0.05 g FeCl3 and330 mL distilled water; reagent II consisted of0.5 mg mL�1 diacetylmonoxime and 0.1 mg mL�1

thiosemicarbazide dissolved in water. Before use,reagents I and II were mixed at the ratio of 2:1. Thismixture (5 mL) was added to 1 mL sample in a testtube and heated for 15 min in boiling water. Subse-quently, the test tubes were cooled immediately toroom temperature. A red colour complex was obtainedfrom the reaction of urea with acidic diac-etylmonoxime in the presence of thiosemicarbazide,which was detected by a UV spectrophotometer at thewavelength of 526 nm.

Intracellular OTCase activity determination

Ornithine transcarbamylase activity was determinedusing the method reported by Carrasco et al. (2003).Cultured cells were collected by centrifugation proce-dure at 3000 r.p.m. (4 °C) and resuspended in 5 mM

Tris–HCl (1 mL, pH 7.5) and 10 mM MnCl2, and then,the cells were exposed to ultrasound for 15 min. Aftercentrifugation at 3000 rpm for 30 min, the supernatantwas used as the crude extract. The extract (125 lL) was

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Ethyl carbamate formation R.-S. Fang et al.2552

taken in a centrifuge tube and 10 mM MnCl2 (125 lL)was added and the resulting mixture was incubated at55 °C for 20 min to activate this enzyme and subse-quently mixed with 0.1 M carbonate buffer (750 lL, pH9.5) prewarmed at 37 °C. To initiate OTCase reaction,0.1 M arginine (pH value adjusted to 9.5 with HCl,250 lL) was added to the test tube, and then, the mix-ture was incubated at 37 °C for 55 min. The reactionwas stopped by mixing 187.7 lL aliquots with 563 lLglacial acetic acid and by adding 187.7 lL ninhydrin(70 mM in 60% glacial acetic acid and 40% phosphoricacid), and then the mixture was boiled for 30 min. Then,ornithine concentration was detected colorimetrically atthe wavelength of 515 nm. The absolute concentrationof ornithine was determined by the comparison with astandard solution (0.6 mM). In this work, one unit ofOTCase activity was defined as the amount of ornithine(lmol) formed per min and per microgram of protein.

Analysis of arginine deiminase (ADI) activity

In this investigation, we used BioAssay Systems’ argi-nase assay kit to determine ADI activity, following pre-vious studies (Murphy et al., 2009; Weiss et al., 2009;Feola et al., 2010). This method utilised a chromogenthat formed a coloured complex specifically with ureawhich produced in the arginase reaction. Cells were col-lected as described above in OTCase determinationmethod. Two types of samples were prepared: one sam-ple consisted of 40 lL sample with 59 substrate buffer(10 lL) that was added to wells of a clear-bottom 96-well reaction plate (ODSAMPLE) and another sampleconsisted of 40 lL sample without 59 substrate buffer(sample blank control, ODBLANK). In addition, 50 lLof H2O (standard background, ODWATER) and 50 lLof 1 mM urea standard solution (ODSTANDARD) werealso added into the respective well of reaction plate andincubated at 37 °C for 2 h. To stop arginase reaction,200 lL urea was added to all wells, and then, 59 sub-strate buffer (10 lL) was added to the sample in blankcontrol well. The mixture was incubated for 60 min atroom temperature, and optical density was read at430 nm. ADI activity (units per litre sample) is calcu-lated using the following equation:

ADI activity (U/L) ¼ ðODSAMPLE

�ODBLANKÞ=ðODSTANDARD

�ODWATERÞ � ½Urea Standard�� 50� 103=ð40� tÞ

¼ ðODSAMPLE

�ODBLANKÞ=ðODSTANDARD

�ODWATERÞ � 10:4

One unit of ADI was defined as the amount of ADIthat converts 1 lmol of L-arginase to ornithine andurea per minute at pH 9.5 and 37 °C.

Determination of extracellular citrulline concentration

Citrulline was determined according to the methodreported by Boyde & Rahmatullah (1980). Tworeagents were prepared: reagent I consisted of 250 mLconcentrated sulphuric acid, 200 mL phosphoric acid,250 mg L�1 FeCl3 and 550 mL distilled water, andreagent II was prepared by dissolving 500 mg diac-etylmonoxime in 100 mL of distilled water. Before use,5 mg thiosemicarbazide was added to 50 mL ofreagent II and 100 mL of reagent I, and the final mix-ture is called reagent III. 0.1 mL supernatant wasmixed with 3 mL of regent III, boiled at 100 °C for5 min and cooled it to room temperature. The absor-bance value was measured at 530 nm.

Protein determination

Bradford method was used for the determination ofprotein concentration in samples (Bradford, 1976).First, 0, 1, 2, 3, 4, 5 and 6 lL of bovine serum albu-min (BSA) standard solution were added to 10 lL ofPBS in microtitre plates; secondly, 190 lL of BradfordCoomassie brilliant blue was added to measure OD595

to plot the standard curve. 10 lL sample and 190 lLCoomassie brilliant blue were mixed at room tempera-ture for 5–10 min, and then, OD595 was determined.

Statistical analysis

The data were analysed using Design Expert, version7.0 (Stat-Ease Inc., Minneapolis, MN, USA). TheP < 0.05 was considered as statistically significant in alltests. All data are means � standard deviations (SD) ofat least three determinations. Significant differencesbetween various treatments were determined by Tukey’spairwise comparison test at a level of P < 0.05.

Results and discussion

Effect of different additives on EC formation duringChinese yellow rice wine fermentation

Ornithine transcarbamylase could be inhibited by theaddition of ornithine (Langley et al., 2000). Further-more, urea, one of the precursors of EC formation(Wang et al., 2007), could be degraded by urease.Hence, in this study the inhibitors of OTCase and ureawere considered to investigate the regulatory influenceon EC formation during the fermentation of Chineseyellow rice wine. As shown in Fig. S1, the effects ofinhibitors added in the processing of yellow rice winewere evaluated in EC formation. The use of ornithinehydrochloride in the fermentation of Chinese yellowrice wine resulted in the best inhibitive effect on ECproduction, whereas the group treated with urease at

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Ethyl carbamate formation R.-S. Fang et al. 2553

6 days of cultivation generally caused the significantinhibition. But after 20 days of brewing time, theinducing effect is significant when urease was added at9 days of fermentation time in contrast to other treat-ments. It could be speculated that the presence of orni-thine possibly controlled the metabolism of arginineand EC. Relevant report insisted that ornithine couldinduce the expression of OTCase (Langley et al.,2000), while the present finding showed an oppositeconclusion. Urease could decompose urea, which wasalready admitted as the prominent precursor of ECformation, was in accordance with this study. There-fore, further investigation should focus on the inhibi-tion of arginine metabolism enzymes (OTCase andADI) and other metabolites in the process of Chineseyellow rice wine.

Change in intracellular OTCase and ADI during theprocessing of Chinese yellow rice wine

The degradation of arginine played an importantrole in EC formation, which mainly provided the pre-cursors of EC in grape wine brewing and spiritsproduction (Schehl et al.,2007; Spano et al.,2007).Nevertheless, few researches presently focused on elu-cidating the relationship between these two enzymesand EC formation. As a key urea cycle enzyme, OT-Case usually catalysed the reaction that convertedornithine and carbamoyl phosphate into citrulline.Ornithine was the deamination product of arginine,whereas carbamoyl phosphate was the condensationproduct of ammonium generated by amino aciddeamination and carbon dioxide. Thus, OTCase wasimportant for cellular ammonium secretion (as a formof urea) and amino acid catabolism (Scaglia et al.,2004). In the present work, OTCase and ADI werecomparatively determined between the control andthe treatments by urease and ornithine. The resultsdemonstrated in Fig. S2 revealed that the presence ofornithine leads to the best production of OTCase ascompared to the control group at 18 days of culturetime. Additionally, urease generally resulted in theinducing effect on OTCase biosynthesis in comparisonwith the control. Combining the data of EC formationand OTCase, this finding confirmed that the increase inOTCase activity leads to a positive effect on ECformation in the processing of Chinese yellow rice wine.

Comparative analysis of urea metabolism and ethanolformation in Chinese yellow rice wine

It is generally accepted that urea is the major precur-sor of EC production in spirits production (Schehlet al., 2007). Uthurry et al. (2006) insisted that moreEC would be produced along with the number of pre-cursors rising. Certainly, the concentration of urea

should also be paid more attention. The variation inurea amount in fermentation broth during the wholeprocess of Chinese yellow rice winemaking is pre-sented in Fig S3. Compared with other treatmentsand the control group, ornithine added in the fermen-tation of yellow rice wine produced significant urealevel in fermentation broth, so the amount of ureaused to generate EC was lower. T3 group producedthe lowest concentration of urea in rice wine, whichmeans that the amount of urea used to produce ECis highest. Specially noted, T2 group led to the signifi-cant formation of urea in comparison with the con-trol. Considering the formation of EC, the amount ofurea detected in fermentation broth means that theurea used to form EC is low. Based on the presentresults, urea is determined as the major substrate forEC production, which is in agreement with thereported conclusion in spirits production (Schehlet al.,2007). Moreover, Fu et al. (2010) investigatedthe correlation between EC level and urea presentedin Chinese yellow rice wine and revealed that the con-centration of EC had a negative linear correlationwith urea content in Chinese yellow rice wine sam-ples. However, this phenomenon is not observed dur-ing the processing period of Chinese yellow rice winein the present researches.As reported by Hasnip et al. (2004), ethanol

present in grape wine may be beneficial for EC for-mation at the storage period. In contrast, relevant lit-erature was rarely concerned on the correlationanalysis between ethanol and EC. The data presentedin Fig. S3(b) further confirmed that there was insig-nificant difference for ethanol formation between thecontrol and the treated group by ornithine or ureasebefore 24 days. After that time, the brewing processof Chinese yellow rice wine without inhibitors or addi-tives showed the lowest ethanol content in contrast toother treatments. Arena et al. (1999) reported thatEC can be spontaneously formed in the presenceof ethanol and urea or the relevant compoundscontaining guanidyl group. The present data didnot verify the above-mentioned finding. Futureresearch should be conducted to examine the detailedmechanism of EC formation.

Comparative analysis of citrulline metabolism

One of the major concerns about arginine metabolismby wine LAB was the formation of EC precursors (Liu& Pilone, 1998). Thus, the leading extracellular aminoacid of arginine catabolism was comparativelydetermined among different experimental designs. Theresults in Fig. S4 made clear that the use of ornithineadded at the culture broth induced the best formationof extracellular citrulline. Besides, urease treated atculture time of 9 days led to the significant decrease in

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Ethyl carbamate formation R.-S. Fang et al.2554

citrulline, which confirmed that urea was degraded byurease and thus affected the overall metabolism ofarginine. In the process of grape wine vinification, thepotential formation of EC was well correlated withcitrulline production in the study of Oenococcus oenistrains and was higher in those conditions where afaster growth and metabolism were observed. It isadmitted that citrulline was a good indicator of the ECconcentrations, particularly at the prolonged storageperiod (Romero et al., 2009). Citrulline and relevantcompounds containing guanidyl group were reported tobe leading amino acids, which were responsible for theformation of EC in grape wine brewing. The amount ofcitrulline in fermentation broth is shown in Fig. S4.Higher level of citrulline detected in the wine impliedthat citrulline used to form EC was lower,demonstrating the consistent conclusion on the basis ofthe formation of EC and extracellular citrulline inChinese yellow rice wine.

Correlation analysis of EC formation with intracellularOTCase or extracellular urea concentration

As found earlier by Schehl et al. (2007), urea is themain component involved in EC formation in grapewinemaking. On the basis of present results, we couldobserve that there is a close correlation between ECand extracellular urea during the processing of Chineseyellow rice wine. Furthermore, the statistical analysiswas used to determine the correlation between EC andextracellular urea or intracellular OTCase. As indi-cated in Table S1, no direct linear relationshipbetween EC and OTCase or extracellular urea wasobserved. But interesting results also presented inTable S1, EC concentration at T1 group was linearlyassociated with intracellular OTCase or extracellularurea. In contrast to T1 group, the treatments (T2 andT3 groups) by urease added at different cultivationtime lead to an insignificant correlation between ECformation and OTCase or urea in view of correlationcoefficient (R2 < 0.8000). We may conclude that thepresence of extracellular urea or intracellular OTCasesynthesis played an important role in the EC forma-tion of Chinese yellow rice wine brewing. It is wellknown that OTCase was a reversible enzyme that con-trols the reaction steps of citrulline and ornithine (Liu& Pilone, 1998). As reported by Carrasco et al. (2003,arginase activity generally was a useful marker ofnitrogen limitation during alcoholic fermentations. Ithas been shown that citrulline produced in experimentsin a model wine system with 15% (v/v) ethanol corre-lated linearly with EC. A linear correlation observedcan be used to anticipate potential EC levels accordingto citrulline production during bacterial metabolism infortified wines (Azevedo et al., 2002). Nevertheless,grape wines with higher residual arginine content did

not produce a clear tendency to higher EC concentra-tion after incubation with LAB strains (Su�arez Lepe &Uthurry, 2007). Thus, the use of ornithine in the fer-mentation of rice wine might favour the accumulationof citrulline. As postulated by the known conclusionsregarding grape and sake wine fermentation, the gen-eral metabolism pathway of arginine is presented inFig. S5. OTCase was a key enzyme in urea cycle todetoxify ammonium produced from amino acid catab-olism. Lys88 acetylation in OTCase was regulated byboth extracellular glucose and amino acid availability,indicating that OTCase activity may be controlled bycellular metabolic status (Yu et al., 2009). However,the detailed regulatory mechanism still needed furtherstudy for the processing of Chinese yellow rice wine.

Conclusions

In summary, this study provided the basic data foruncovering the metabolism and mechanism of ECformation in the processing of Chinese yellow ricewine on the basis of the survey of the presence of ECand elucidation of metabolism processes. These resultsmade clear that EC formation was positively associ-ated with the presence of intracellular OTCase, whichconfirmed that the increase in OTCase could lead tothe formation of extracellular EC. The extracellularurea present in fermentation broth or intracellularOTCase expression played an important role in ECformation of Chinese yellow rice wine. Furtherinvestigation should focus on the elucidation ofOTCase and ADI participating in the regulation ofEC in Chinese yellow rice wine by means of molecularbiology so as to make the above-mentioned conclu-sions stronger.

Acknowledgments

This work was financially supported by the NationalNatural Science Foundation of China (31171734) andby State Key Laboratory Breeding Base, Key Labora-tory for Plateau Crop Germplasm Innovation andUtilization of Qinghai Province. We thank Dr ChenJi-cheng for the language revision of this work.

Conflict of Interest

The authors declare that they have no conflict ofinterest.

References

Arena, M.E. & Manca de Nadra, M.C. (2005). Influence of ethanoland low pH on arginine and citrulline metabolism in lactic acidbacteria from wine. Research in Microbiology, 156, 858–864.

Arena, M.E., Saguir, M.C. & Manca de Nadra, M.C. (1999). Argi-nine, citrulline, ornithine metabolism by lactic acid bacteria fromwine. International Journal of Food Microbiology, 52, 155–161.

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Ethyl carbamate formation R.-S. Fang et al. 2555

Azevedo, Z., Couto, J.A. & Hogg, T. (2002). Citrulline as the mainprecursor of ethyl carbamate in model fortified wines inoculatedwith Lactobacillus hilgardii: a marker of the levels in a spoiled for-tified wine. Letters in Applied Microbiology, 34, 32–36.

Boyde, T.R.C. & Rahmatullah, M. (1980). Optimization of condi-tions for the calorimetric determination of citrulline, using diacetylmonoxime. Analytical Biochemistry, 107, 424–431.

Bradford, M.M. (1976). A rapid and sensitive method for the quanti-fication of microgram quantities of protein utilizing the principleof protein-dye binding. Analytical Biochemistry, 72, 248–254.

Carrasco, P., P�erez-Ort�ın, J.E. & del Olmo, M. (2003). Arginaseactivity is a useful marker of nitrogen limitation during alcoholicfermentations. Systematic and applied Microbiology, 26, 471–479.

Chiva, R., Baiges, I. & Mas, A. (2009). The role of GAP1 gene inthe nitrogen metabolism of Saccharomyces cerevisiae during winefermentation. Journal of Applied Microbiology, 107, 235–244.

Feola, D.J., Garvy, B.A., Cory, T.J. et al. (2010). Azithromycinalters macrophage phenotype and pulmonary compartmentaliza-tion during lung infection with Pseudomonas. Antimicrobial Agentsand Chemotherapy, 54, 2437–2447.

Fu, M.L., Liu, J., Chen, Q.H. et al. (2010). Determination of ethylcarbamate in Chinese yellow rice wine using high-performanceliquid chromatography with fluorescence detection. InternationalJournal of Food Science and Technology, 45, 1297–1302.

Hasnip, S., Caputi, A. & Crews, C. (2004). Effects of storage timeand temperature on the concentration of ethyl carbamate and itsprecursors in wine. Food additives and Contaminants, 21, 1155–1161.

Hasnip, S., Crews, C., Potter, N. et al. (2007). Survey of ethyl carba-mate in fermented foods sold in the United Kingdom in 2004.Journal of Agricultural and Food Chemistry, 55, 2755–2759.

Lachenmeier, D.W., Schehl, B., Kuballa, T. et al. (2005). Retrospec-tive trends and current status of ethyl carbamate in German stone-fruit spirits. Food Additives and Contaminants, 22, 397–405.

Langley, D.B., Templeton, M.D., Fields, B.A. et al. (2000). echanismof inactivation of ornithine transcarbamoylase by Nd-(N′-Sulfodia-minophosphinyl)-l-ornithine, a true transition state analogue? Jour-nal of Biological Chemistry, 275, 20012–20019.

Lehtonen, P. (1996). Determination of amines and amino acids inwine. American Journal of Enology and Viticulture, 47, 127–133.

Liu, S.Q. & Pilone, G.J. (1998). A review: Arginine metabolism inwine lactic acid bacteria and its practical significance. Journal ofApplied Microbiology, 84, 315–327.

Liu, S.Q., Pritchard, G.C., Hardman, M.J. et al. (1994). Citrullineproduction and ethyl carbamate (urethane) precursor formationfrom arginine degradation by wine lactic bacteria Leuconostoc oe-nos and Lactobacillus buchneri. American Journal of Enology andViticulture, 45, 235–242.

Liu, S.Q., Pritchard, G.C., Hardman, M.J. et al. (1995). Occurrenceof arginine deiminase pathway enzymes in arginine catabolism inwine lactic acid bacteria. Applied Environmental Microbiology, 61,310–316.

Madrera, R.R. & Valles, B.S. (2009). Determination of ethyl carba-mate in cider spirits by HPLC-FLD. Food Control, 20, 139–143.

Murphy, B.S., Vidya, S. & Cory, T.J. (2009). Azithromycin altersmacrophage phenotype. Journal of Antimicrobial Agents and Che-motherapy, 61, 554–560.

Playne, M.J. (1985). Determination of ethanol, volatile fatty acids,lactic and succinic acids in fermentation liquids by gas chromatog-raphy. Journal of the Science of Food and Agricultural, 36, 638–644.

Romero, S.V., Reguant, C., Bordons, A. et al. (2009). Potential for-mation of ethyl carbamate in simulated wine inoculated with Oeno-coccus oeni and Lactobacillus plantarum. Journal of Food Scienceand Technology, 44, 1206–1213.

Scaglia, F., Brunetti-Pierri, N., Kleppe, S. et al. (2004). Clinical con-sequences of urea cycle enzyme deficiencies and potential links toarginine and nitric oxide metabolism. Journal of Nutrition, 134,2775S–2782S.

Schehl, B., Senn, T., Lachenmeier, D.W. et al. (2007). Contribution ofthe fermenting yeast strain to ethyl carbamate generation in stonefruit spirits. Applied Microbiology Biotechnology, 74, 842–850.

Spano, G., Massa, S., Arena, M.E. et al. (2007). Arginine metabo-lism in wine Lactobacillus plantarum: in vitro activities of theenzymes arginine deiminase (ADI) and ornithine transcarbamilase(OTCase). Annals of Microbiology, 57, 67–70.

Su�arez Lepe, J.A. & Uthurry, C.A. (2007). Incidence of nitrogenouscompounds of must on ethyl carbamate formation induced by lac-tic acid bacteria. Journal International des Sciences de la Vigneet du Vin, 41, 215–223.

Uthurry, C.A., Varela, F., Colomo, B. et al. (2004). Ethyl carbamate con-centrations of typical Spanish red wines. Food Chemistry, 88, 329–336.

Uthurry, C.A., Su�arez Lepe, J.A., Lombardero, J. et al. (2006).Ethyl carbamate production induced by selected yeasts and lacticacid bacteria in red wine. Food Chemistry, 94, 262–270.

Wang, D., Yang, B., Zhai, X. et al. (2007). Synthesis of diethyl car-bonate by catalytic alcoholysis of urea. Fuel Process Technology,88, 807–812.

Weiss, J.M., Back, T.M., Scarzello, A.J. et al. (2009). Successfulimmunotherapy with IL-2/anti-CD40 induces the chemokine-medi-ated mitigation of an immunosuppressive tumor microenvironment.Proceedings of the National Academic Sciences, 106, 19455–19460.

Yu, W., Lin, Y., Yao, J. et al. (2009). Lysine 88 acetylation negativelyregulates ornithine carbamoyltransferase activity in response tonutrient signals. Journal of Biological Chemistry, 284, 13669–13675.

Supporting Information

Additional Supporting Information may be found inthe online version of this article:Figure S1. Effect of different treatments on ethyl

carbamate (EC) formation during vinification processof Chinese yellow rice wine. CG means control group,and T1 (ORT) stands for ornithine hydrochlorideadded at day 1.Figure S2. Change in and the effect of intracellular

ornithine transcarbamylase (OTCase) under differenttreatments during the processing of Chinese yellow ricewine. CG means control group, and T1 (ORT) standsfor ornithine hydrochloride added at day 1.Figure S3. Effect of the extracellular additives (orni-

thine and urease) 27 on urea metabolism and ethanolformation. (a) Regulation of urea metabolism by theaddition of ornithine hydrochloride or urease duringthe processing of Chinese yellow rice wine. (b) Varia-tion in and comparison of ethanol formation in theprocessing of Chinese yellow rice wine under differenttreatments. CG means control group, and T1 (ORT)stands for ornithine hydrochloride added at day 1.Figure S4. Extracellular citrulline production under

different treatments during the fermentation of Chineseyellow rice wine. CG means control group, and T1(ORT) stands for ornithine hydrochloride added at day 1.Figure S5. Proposed pathway for arginine metabo-

lism during the processing of Chinese yellow rice wine.Table S1. Correlation analysis of ethyl carbamate

content (y) with ornithine transcarbamylase (OTCase)or urea during the brewing of Chinese yellow rice wineunder four designed conditions.

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2013

Ethyl carbamate formation R.-S. Fang et al.2556