Ethyl Carbamate Production Kinetics during Wine Storage

Transcription

1 Ehyl Carbamae Producion Kineics during Wine Sorage J. Xue,*, F. Fu, M. Liang, C. Zhao, D. Wang, Y. Wu * () China Naional Research Insiue for Food and Fermenaion Indusries, Beijing 7, China () Deparmen of Food Science and Pharmacy, XinJiang Agriculural Universiy, Urumchi 85, China Submied for publicaion: December Acceped for publicaion: March 5 Key words: Wine, ehyl carbamae, producion ineics, urea, emperaure, cirulline The ineics of ehyl carbamae (EC) formaion was invesigaed during wine sorage o evaluae he poenial ris of EC formaion in wine. The sudy moniored he EC, urea and cirulline concenraion a differen sorage emperaures and wine ph. We found ha emperaure and iniial urea conen had significan effecs on EC formaion. The decay of urea and cirulline fi a firs-order reacion approaching equilibrium, bu he ph has lile effec on he conen of ehyl carbamae. Based on hese resuls, we consruced an equaion o forecas he conen of ehyl carbamae during wine sorage. INTRODUCTION Ehyl carbamae, also nown as urehane (C COONH ), has been recognised as probably carcinogenic o humans by he IRAC (Inernaional Agency for Research on Cancer) since 7 (Lachenmeier e al., 9; Larcher e al., ). I has been lined o lung cancer, lymph cancer, liver cancer, sin cancer and many ohers. Ehanol promoes he carcinogeniciy of EC (Miller e al., ; Beland e al., 5). The mean inae of EC from fermened foods and drins can reach several milligrams per lire (Ough, 976). This excludes he conribuion of alcoholic beverages. A he 6h meeing of he JECFA (Join Exper Commiee on Food Addiives) i was suggesed ha reduced consumpion of ehyl carbamae would proec human healh (Liang e al., 9). In recen years, he consumpion of wine in China has experienced srong growh, wih special aenion being given o he amoun of ehyl carbamae in hese producs (Par e al., 9; Shijia Wu, 9). Uhurry e al. () found ha, in newly produced wines, he concenraion of EC is o μg/l, alhough he sorage condiions and ime deermine he final concenraion a he ime of consumpion (Hasnip e al., ). The major precursors of EC are urea and cirulline (Ough e al., 988; Sevens & Ough, 99; Liu e al., 99). EC is formed from he reacion of urea and ehanol (Eq. ) or urea and cirulline (Eq. ) aing place during alcohol producion and preservaion (Delledonne e al., ; Wang e al., 7; Weber & Sharypov, 9). Several sudies have shown ha he formaion of EC is significanly acceleraed by high concenraions of ehanol, urea and cirulline. Sevens and Ough (99) sudied he relaionship beween urea, ehanol and he formaion of EC a differen emperaures and showed ha he concenraion of urea in wine should no exceed mg/l. Higher concenraions ris poenial concenraions of EC ha exceed he American non-preempive limi of 5 μg/l (Sevens & Ough, 99). NH CONH + C OH NH COOC + NH () H NCONH (CH ) CH(NH )COOH + C OH NH COOC + H N(CH ) CH(NH )COOH () Here we sudy changes o ehyl carbamae s concenraion in wine during sorage, including he influence of sorage emperaure, ph and he conen of urea and cirulline in wine on he final concenraion of EC. We consruced simple mehods o predic he concenraion of EC during sorage and hus hope o reduce he amoun of EC in wine. These ools may also form he heoreical basis for fuure limi sandards of EC in Chinese wine. MATERIALS AND METHODS Maerials Two red wines and one whie wine were produced on a laboraory scale ( L) from Caberne Sauvignon, Merlo and Chardonnay in. Red wines were fermened a 5 ± C using Lallemand D5 yeas srain and Lallemand VP lacobacillus. The whie wine was fermened a ± C using Lallemand DV yeas srain. Afer fermenaion he wines were sulphied (6 mg/l), and hey here were subjeced o baonnage a o 5 C hree imes a wee for.5 monhs, serile filered (.5 μm), and he red wine from Caberne Sauvignon was sored in a gallon carboy during sorage o remove he sample from he boom. The bole headspace was limied o minimise ehanol evaporaion. The oher wines were boled (5 ml). A he ime of sorage or boling, he wines had he following composiion (Caberne Sauvignon, Merlo and Chardonnay respecively): alcohol conen.9,.6 and.8% (v/v); ph.,.67 and.; residual sugars.8,. and.6 g/l; oal SO 75, 9 and 86 mg/l; urea *Corresponding auhor: Jie Xue, address: lxxuejie@6.com; Yun Wu, address: 766@qq.com Acnowledgemens: This wor was suppored by he Naional Naural Science Foundaion (66) projec of he Xin Jiang Auonomous Region Science and Technology Plans (), and by he Minisry of Science and Technology, People s Republic of China under gran no. AA8 S. Afr. J. Enol. Viic., Vol. 6, No., 5 77

2 78 Ehyl Carbamae Producion Kineics during Wine Sorage conen.8,.6 and 6.8 mg/l; and cirulline conen.56, 6.7 and. mg/l. Experimenal reamen Firs, 8 L of Caberne Sauvignon red wine were mixed in a gallon carboy and hen spli ino wo volumeric flass. One was spied wih urea and he oher was spied wih cirulline o bring he concenraions of urea and cirulline o 5. mg/l and 5.7 mg/l, respecively. These were hen spli ino four L gallon carboys and sored a 5 C, C, 5 C and C, respecively. Urea was added o confirm ha he iniial concenraion of urea in he Caberne Sauvignon red wine was. mg/l, 5. mg/l and. mg/l, respecively. These samples were sored a C. The es sudied how he iniial concenraion of urea effecs he formaion of EC. The experimen used buffer soluion o adjus he sample wine ph o.,.5, and.5, wih he iniial concenraions of urea being 5. mg/l. The samples were sored a 5 C. The oher boled wines (whie and red) were placed in emperaure-conrolled sorage a 8 and C, respecively. To help validae he model in his research, he levels of EC, urea and cirulline were measured a inervals for up o one year in he course of sorage. Major chemical reagens We purchased ehyl carbamae (EC) (puriy > 99%) and he inernal sandard propyl carbamae (npc) (puriy > 99%) from Sigma. Aceone (chromaographically pure), mehylene dichloride (analyically pure), urea (analyically pure) and cirulline (chromaographically pure) were purchased from J&K. Analysis of EC, urea and cirulline The conen of EC was deermined according o he mehod of De Melo Abre e al. (5) using a GC-MS (Perin-Elmer, USA) equipped wih a capillary WAX ETR column ( m,.5 mm,.5 um; Perin-Elmer, USA). Wine samples of. ml, wih he addiion of ml of propyl carbamae ( ng/ml in waer) as an inernal sandard, were exraced wih sraa FL-PR Florisil solid phase exracion (Anpu Science Insrumen Company, Shanghai). Eluion was performed wih ml mehylene chloride and he sample was concenraed o.5 ml a low emperaure before injecion under nirogen sream. The urea was measured afer derivaisaion wih xanhydrol. mol/l (Sigma-Aldrich, Seinheim, Swizerland) dissolved in -propanol using an Aglilen HPLC (Agilen Technologies, Waldbronn, Germany) equipped wih a fluoromeric deecor se a and 8 nm (exciaion and emission wavelengh respecively). Separaion was performed on an Eclipse XDB-C8 column (.6 5 mm, 5 um; Agilen Technologies) wih he precolumn se a a emperaure of 5 C (Jiangao Xing e al., ). The deerminaion of cirulline was based on he Edman reacion as described by Yang e al. (). The sample was measured afer derivaisaion wih riehylamine ( mol/l) and phenyl isohiocyanae (PITC) ( mmol/l). The chromaographic condiions employed were as follows: an Agilen HPLC sysem wih ulraviole deecor and a C 8 column (5.6 mm, paricle size 5 um) was obained from Agilen. The column emperaure was C wih a. ml/min flow velociy. A 5. μl aliquo of sample was used and he measuremen wavelengh was 5 nm. Saisical analysis Daa processing was performed using he parameers of he dynamic model ha were obained from non-liner maching of he dynamic equaion wih Origin 9. Graphing and Analysis Sofware (OriginLab, Norhampon, MA). RESULTS AND DISCUSSION Effecs of he sorage emperaure on he concenraion of urea Urea and ehanol can coninue o reac in wine during sorage and hus he concenraion of urea can be reduced as a funcion of ime. The firs experimen placed wines a 5 C, C, 5 C and C wih 5. mg/l of urea and followed he change in urea concenraion for days of sorage (Fig. ). The urea concenraion reduced logarihmically in he wine during sorage. The reducion in urea concenraion was very fas a he iniial sorage imes, bu slowed a laer ime poins. The change in urea concenraion had similar rends o ha of oher wines wih differen iniial urea concenraions. Neiher he concenraion nor naural logarihm of concenraion changed linearly wih ime. Thus, he degradaion of urea in wine during sorage does no fi a firs-order reacion dynamic. This is in sharp conras o he findings in he wor of Larcher e al. (). The reason could be he lower sorage emperaure and higher iniial urea concenraions. In Fig., he urea concenraion approaches equilibrium versus ime in wines during sorage. Here, he es refers o he sandard inegraed rae law (Equaion ) for a firs-order reacion approaching equilibrium, as sudied by Hasnip e al. (): [U = [U [ + e + + ) () where is he rae consan for he degradaion of urea and is he rae consan for he reformaion of urea. Using Origin 9. sofware o plo he urea concenraion in wine during sorage and equaion wih fixed [U, we can calculae many parameers (Table ). Where is he rae consan for he degradaion of urea, is he rae consan for he reformaion of urea, [u is he concenraion of urea a ime, and [u is he concenraion of urea a ime. The maching equaions a differen sorage emperaures had differen iniial concenraions of urea and reached a very significan level (. < P). Indeed, Equaion could presen he change in urea concenraion in wines during sorage. The reacion rae increased wih he emperaure in samples wih a urea concenraion of 5. mg/l. In his es, if he emperaure increased by C, he reacion rae increased.8-fold. S. Afr. J. Enol. Viic., Vol. 6, No., 5

3 Ehyl Carbamae Producion Kineics during Wine Sorage 79 urea(mgl urea(mg/l) - ) Time(day) FIGURE Evoluion of urea conens in wine during sorage a differen emperaures. The parameers of he iniial wines were he same. A each emperaure, hree independen wines were analysed. Values are he mean of hree repeiions. Bars indicae sandard deviaion. TABLE The maching dynamic equaions and parameers of urea reacions in differen wines during sorage. The iniial concenraions of urea (mg/l) Temp. ( C) Equaion Maching correlaion index (R ) K K ) exp 5 [ u = [ u ) exp [ u = [ u ) exp [ u = [ u ) exp [ u = [ u ) exp [ u = [ u ).+.5exp [ u = [ u ) exp [ u = [ u ) exp [ u = [ u ) exp [ u = [ u ) exp 5 [ u = [ u ).7 +.exp [ u = [ u ) exp [ u = [ u ) exp [ u = [ u S. Afr. J. Enol. Viic., Vol. 6, No., 5

4 8 Ehyl Carbamae Producion Kineics during Wine Sorage E a The Arrhenius equaion is K = A exp (- ), where RT means rae consan, T means absolue emperaure, and R means gas consan (8. J/mol). The acivaion energy for he decay of urea was calculaed from he Arrhenius plo o be.6 J/mol. The rae of urea degradaion is differen if he iniial urea concenraion is differen, even a he same emperaure. A higher iniial urea concenraion implies a faser reacion rae. The rae consan in wine wih urea concenraions of 5. mg/l is wice ha of wine wih a urea concenraion of 5. mg/l. Indeed, no only he emperaure, bu also he reacan concenraions, affecs he degradaion of urea. Effecs of he sorage emperaure on he EC concenraion of wine To invesigae he effec of emperaure on EC concenraion during sorage, he fresh Caberne Sauvignon red wines were ep a differen emperaures (5 C, C, 5 C and C). As can be seen from Figs and, he reacion beween ehanol and urea coninued wih ime. Because urea is he major precursor for he formaion of EC, he change in urea concenraion could lead o changes in he EC concenraion. The concenraion of EC shows a logarihmic increase when he urea concenraion is decreased in wines a differen emperaures (Fig. ). The change in EC concenraion is he opposie of urea during sorage. The longer he ime, he more EC is produced. Here, he concenraion of EC increased rapidly a firs and hen increased slowly. Higher EC concenraions were found a higher emperaures. These were.5 o wo imes higher a C han hose a 5 C afer days of sorage. I shows ha he concenraion of EC does no exceed 5 μg/l, so sorage a low emperaures could limi he increase in EC concenraion. Effecs of he iniial urea concenraion on he EC concenraion in wine Consisen wih previous sudies, he concenraions of EC and urea have a direcly proporional relaionship (Moneiro e al., 989; Sevens & Ough, 99; Kodama e al., 99). Our es followed he change in EC concenraion in wines ha have differen iniial concenraions of urea (Fig. ). Figure illusraes ha he formaion of EC was rapid a firs, bu hen reached equilibrium. Wih lower iniial urea concenraions, equilibrium is reached sooner and less EC is yielded. Wih higher iniial urea concenraions, high EC levels were found afer sorage. According o he daa in Fig., he change in EC concenraion a differen imes (d [EC/d), combined wih d [urea/d from Fig., yields a sraigh line. Indeed, he formaion of EC has a direc and proporional relaionship beween he changes in urea concenraion. The slope of he sraigh line is he rae consan, Ku, for he formaion of EC from urea. Similarly, he rae consan, Kc, for he formaion of EC from cirulline could be calculaed (Table ). A C, he Ku and Kc were. and., respecively. These differ slighly from he figures obained in Hasnip e al. s sudies (), which showed Ku and Kc o be. and.8 respecively a 8 C. The sorage EC(ugL EC(μg/L) - ) ime(day) FIGURE Evoluion of EC conens in wine during sorage a differen emperaures. The parameers of he iniial wines were he same. A each emperaure, hree independen wines were analysed. Resuls are expressed as mean values ± sandard errors. TABLE The differen rae consans, Ku (he formaion of EC from urea) and Kc (he formaion of EC from cirulline) Temperaure C 5 C C 5 C K R K R K R K R Ku Kc S. Afr. J. Enol. Viic., Vol. 6, No., 5

5 Ehyl Carbamae Producion Kineics during Wine Sorage EC(μg/L) EC(ugL - ) mg/L 5.mg/L.mg/L.mg/L 5 5 ime(day) FIGURE Evoluion of EC conens in wine during sorage a C. The iniial concenraion of urea in he wine was 5. mg/l,. mg/l, 5. mg/l and. mg/l, respecively. Experimens were carried ou in riplicae. Daa shown correspond o average and sandard deviaions cirulline(mgl cirulline(mg/l) - ) ime(day) FIGURE Evoluion of cirulline conens in wine during sorage a differen emperaures. The parameers of he iniial wines were he same. A each emperaure, hree independen wines were analysed. Values are he mean of hree repeiions. Bars indicae sandard deviaion. TABLE The maching dynamic equaions and parameers of he cirulline reacion a differen emperaures during sorage Maching correlaion Temperaure ( C) Equaion K index (R ) ) exp 5 [ c = [ c ) exp [ c = [ c ) exp 5 [ c = [ c ) exp [ c = [ c K S. Afr. J. Enol. Viic., Vol. 6, No., 5

6 8 Ehyl Carbamae Producion Kineics during Wine Sorage.. EC(μg/L) EC(ugL - ) ph=. ph=.5 ph= ime(day) Time(day) FIGURE 5 Evoluion of EC conens in wine during sorage a 5 C wih iniial concenraion of urea being 5. mg/l. The ph of he wines was.,.5 and.5 respecively. A each ph, hree independen wines were analysed. Daa shown correspond o average and sandard deviaions. EC(μg/L) EC(ugL - ) C 8 C ime(day) Time(day) FIGURE 6 Formaion of EC over ime observed in Merlo wine (- -) and he corresponding formaion prediced by equaion (- -) a various emperaures. Values given are he mean of hree deerminaions. 7 6 EC(μg/L) EC(ugL - ) 5 C 8 C Time(day) ime(day) FIGURE 7 Formaion of EC over ime observed in Chardonnay wine (- -) and he corresponding formaion prediced by equaion (- -) a various emperaures. Values given are he mean of hree deerminaions. S. Afr. J. Enol. Viic., Vol. 6, No., 5

7 Ehyl Carbamae Producion Kineics during Wine Sorage 8 emperaure increased by C and he formaion of EC from urea could hus increase by.5 o wo imes. Acivaion energy for he formaion of EC from urea was calculaed from he Arrhenius plo o be.7 J/mol. The acivaion energy for he formaion of EC from cirulline was similarly calculaed as 66.9 J/mol. Effecs of sorage emperaure on he concenraion of cirulline Cirulline is also a major facor ha affecs he formaion of EC in wines. Here, we sudied changes in cirulline concenraion a differen emperaures (Fig. ). The concenraion of cirulline in wine during sorage showed a downward rend (Fig. ). I iniially decreased rapidly during sorage, and hen gradually slowed. This rend is similar o ha seen for urea concenraion during sorage. Using Equaion o plo he cirulline concenraions in wines during sorage agains ime, we find he daa shown in Table. where is he rae consan for he degradaion of cirulline, is he rae consan for he reformaion of cirulline, [c is he concenraion of cirulline a ime and [c is he concenraion of cirulline a ime. Here we used 5.7 mg/l. According o, emperaure has an effec on he decay rae of cirulline. If he emperaure increases by C, he decay rae of cirulline would increase.5 o.5 imes. The effec of emperaure on he decay rae of cirulline was greaer han he effec on he decay rae of urea. The acivaion energy for he degradaion of cirulline was calculaed o be 59.7 J/mol from he Arrhenius equaion according o he rae consan of cirulline a differen emperaures. This sudy used he rae consan for he formaion of EC from cirulline in Table. Effecs of ph on he formaion of EC in wines during sorage The reacion of ehanol wih urea or cirulline is acid mediaed. Thus, we sudied he effec of ph on he formaion of EC using ph.,. and.5 (Fig. 5). The scaer diagram shows ha he rend in EC concenraion is similar o ha seen previously, wih lile difference for he differen ph values. This is consisen wih he observaions made by Sevens and Ough (99), who found ha he ph of a wine does no significanly affec he rae of EC formaion. However, he lower ph wine had a slighly faser formaion rae for EC because he mechanism of EC formaion is acid caalysed. DISCUSSION The daa shows ha he formaion of EC in wine depends on he change in urea and cirulline concenraions during sorage. This assumes ha he urea and cirulline in reacion wih ehanol are he wo major ways o form EC and ha he wo reacions are independen of each oher. The es suggess ha he concenraion of ehanol remains effecively consan hroughou he whole sorage ime, so he concenraion of EC a ime could be expressed as [EC =[EC + u [EOH [U =[U [ [ c [ u d = = [ c [ c d = + e + + ) For example, if he calculaions were based on a wine being sored for days a C, [EC =[EC +[EOH(.8[u +.78[c ). [ u d + c [EOH [ c d, + ) [ u ( e [ ) + e [, + + ) [ c ( e [ [EC =[EC + [EOH{ [ u + [ u ( e ) ) }+ ) ) + ) [ c ( e ) c [EOH{ [ } () Equaion describes he empirical daa well for he formaion of EC from urea and cirulline in he wines used in his sudy. To really validae his model, he oher boled wines (whie and red) were placed in emperaure-conrolled sorage a 8 and C respecively. A similar one-year experimen was carried ou o predic he EC concenraion using he model, and he predicion hen was confirmed wih chromaography. The resuls showed ha here was good agreemen beween he repored daa and he daa predicaed by he equaion (Figs 6 and 7). However, i was no possible o es he equaion fully, since he experimens in his sudy were only one year in duraion. CONCLUSIONS This sudy has shown how he concenraions of he major precursors of EC vary during he sorage of wine and how his affecs he formaion of EC. The concenraion of urea and cirulline in wine decreases during sorage rapidly a firs, and hen more slowly. The rae consans were measured for he formaion of EC from he urea and cirulline precursors. Temperaure is anoher major facor ha affecs he formaion of EC. When emperaure increased by C, he formaion of EC from urea and cirulline increased by.5 o wo and.5 o.5 imes, respecively. We concluded ha equaion may be used o accuraely predic EC levels for a given ime/emperaure profile. The resuls are in good agreemen wih daa obained from similar sudies (Hasnip e al., ). LITERATURE CITED Beland, F.A., Wayne, B.R., Mellic, P.W., Kovach, R.M., Robers, D.W., Fang, J.L. & Doerqe, D.R., 5. Effec of ehanol on he umorigeniciy of urehane (ehyl carbamae) in B6CF mice. Food Chem. Toxicol., -9. S. Afr. J. Enol. Viic., Vol. 6, No., 5

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