Control Strategy Of Ethyl Carbamate In Chinese Rice Wine And The Mechanism

“To the people foodstuff is all-important. To the foodstuff safe is all-important.” Traceethyl carbamate (EC) in fermented alcoholic beverages is known to be carcinogenic. Amongvarious fermentated alcoholic beverages in China, the EC problem that Chinese rice winefaces is tough as a result of its specific process. During the research on several EC preventiveactions, an effective measure was developed to scavenge urea precursor, basing on the acidurea-degrading enzyme from Enterobacter sp. R-SYB082. In addition, a creative andconvinent technique using functional materiasl was provided for directly removing EC fromChinese rice wine. An integrated strategy for the control of urea and EC was established forthe first time for satisfying the EC limitation requirement. These are vital and valuable forenriching fermenting technology and safety theory on Chinese rice wine. The main resultswere shown as follows:(1) In the determination of EC in Chinese rice wine, the time-consuming extractionprocedures, the global uncertainty and solvent-requirement represent the major disadvantagesof the present techniques. And an automated procedure using headspace solid-phasemicroextraction (HS-SPME) followed by gas chromatographic-mass specometric (GC-MS)detection is developed for fast determination of EC in Chinese rice wine. Using propylcarbamate (n-PC) as internal standard, the optimised HS-SPME extraction is45min at70Cwith Polyacrylic (PA) fibre after the addition of NaCl to0.39g mL-1. The HS-SPME-GC-MSprocedure overcame the matrix interference from Chinese rice wine. And the procedurerequires little operator effort, which is suitable for the automated, rapid, convenient, andinexpensive determination of EC in Chinese rice wine. One sample only takes altogether57min in continuous detection, which is much faster than over2h that of the standardSPE-GC-MS method. The relative standard deviation and limits of detection are lower than2.5%and1.19μg L-1, respectively. And this solvent-free procedure is recommended as one ofthe standard procedures for the determination of EC in Chinese rice wine.(2) The urea-degrading enzyme of Enterobacter sp. R-SYB082suffers from low yields.And the production of the enzyme was optimized from1100U L-1to2303U L-1by anapproach which includes the optimization of initial glucose concentration to40g L-1, theelevation of anaerobic level of the reactor by charging CO2. A similar level was obtainedwhen scaled up to30L fermentor. Interestingly, Enterobacter sp. R-SYB082enzyme withgood stability was in vitro naturally activated by simple cold storage (4°C) or theparticipation of CO2. The urea-degrading enzyme of Enterobacter sp. R-SYB082offeredadvantages in terms of the rate of urea removal in Chinese rice wine. The urea removal rate of66.47%was higher than that of the widely used Japanese commercial acid urease (58.79%).The reduction of urea content in Chinese rice wine succeeded to control the content of ethylcarbamate from headstream.(3) Cloning and expressinon of Enterobacter sp. R-SYB082enzyme were performed. Theurea-degrading enzyme of Enterobacter sp. R-SYB082was found to be ureidoglycolateamidohydrolase (UAH) inferred through gene comparisons, a isoenzyme to acid urease other than a urease. The enzyme is promising for industrial application thanks to being able toeffectively degrading both urea and EC. And to our knowledge, the nitrogen assimilationpathway with UAH was newly found in urease-negative E. coli independent of urease. Thegene was cloned and functionally expressed in recombinant E. coli strain BL21-pET28-allAwith independent intellectual property.(4) A specific L-material was selected from various absorptive materials based ondirectional removing technology and intermolecular action principle, for the direct removal ofEC from Chinese rice wine. The ion-exchange action of the strong base group,[-N+R3], mainlydrives the directional removal of EC. After directionally modification and mixing, with theaddition of a fine adjustment process on Chinese rice wine sample, the material was able toeffectively remove EC from Chinese rice wine. The removal went with heat absorption, andthe temperature should be kept higher than20C when the removal was conducted. Thesuitable flow rate for dynamic adsorption is0.25BV h-1. For Chinese rice wine samples withEC contents lower than300μg L-1, a EC content below50μg L-1was achieved, meeting theEC limitation requirement while maintaining most of the components in Chinese rice wine.After the combination of urea degradation by Enterobacter sp. R-SYB082enzyme and ECdirectional removal by functional materials, the urea content in Chinese rice wine wasreduced to the level less than10mg L-1, and for Chinese rice wine samples with EC contentslower than300μg L-1, an EC content below50μg L-1can be achieved. By both treatments, anintegrated strategy for the control of urea and EC in Chinese rice wine was established for thefirst time. These results provided a brand new applicable and convinent approach for reducingEC content in Chinese rice wine, which implies great significance for ensuring food safety,and coping with foreign technical barrier. The results also provide a new idea for the controlof EC in other alcoholic beverages.