新疆地产葡萄酒中氨基甲酸乙酯安全性控制机理研究

Ethyl Carbamate (EC) or urethane is the ethyl ester of carbamic acid. It occurs in many fermented food including wine. Ethyl carbamate is genotoxic and carcinogenic to humans,it has been classified as a group 2A carcinogen. The traditional wine producer countries has established the strict maximum limit value of EC in wine,but in china up to now there is no limit benchmark about EC.So the content of EC of wine was significantly higher than that of import wine ,in most cases,it exceeds the maximum limit value in wine. Therefore,it is an important issue of food safety that how to control EC in Chinese wine. At present time studies were mostly focused on the analysis of the commercial product of wine.The studies on following points are absent as far as the fermentation processes are concerned,inculding their biological generation and accumulation of EC ,the effects of different vintage technologies on the formation of ethyl during vinification, as well as species classification of EC yeast ioslated. In this study, we will utilize modern biotechnology, fermentation engineering and new detection methods to conduct experimental studies. In order to elaborate on EC biosynthesis mechanism, the impact of matrix effects and process parameters on EC content, species classification of low urea-producing strains,we will use the wine produced in XinJiang region, around microorganism, production technology, as well as the quality monitoring of terminal product to carry the research. What is more,it will form a practice for prevention and reduction of EC contamination in wine, which might be of possible applications to act as the theoretical and technical basis of early warning and review of the EC contamination in wine in the future.

氨基甲酸乙酯（EC）是葡萄酒中普遍存在的潜在致癌物，对人类健康危害较大，国外对葡萄酒中EC含量有严格的规定，但到目前为止我国仍没有制定有关EC的限量标准，造成葡萄酒中EC含量超标的问题非常突出，成为影响葡萄酒行业健康发展迫切需要解决的问题。本课题以新疆产区葡萄酒产业的整体技术升级为根本目标，以葡萄酒生产中质量安全控制和实现产品质量最优化为重点，采用生物技术、发酵工程、新型检测方法等现代高新技术手段，围绕EC 等的产生机理、生产过程的控制措施、脲酶的开发应用、低产尿素酵母等降低EC 含量等方面开展研究，从菌种、酿造关键控制点及终端产品质量监控方面，提出降低葡萄酒EC 含量的控制途径和关键点，降低葡萄酒中EC的含量，消除葡萄酒潜在安全隐患，突破国际贸易壁垒，在市场上树立质量高、文化底蕴深、具广泛国际认可度的民族葡萄酒品牌，使新疆传统葡萄酒行业得到保护和健康发展。

本项目通过对新疆葡萄酒中氨基甲酸乙酯控制技术的研究，内容如下：.（1）建立葡萄酒中EC 、尿素、精氨酸的检测方法。其方法分别为：GC-MS法、HPLC-FLD法、HPLC-UV法。.（2）对葡萄酒中的EC含量进行调查分析。结果显示：葡萄酒中EC含量差异很大，红葡萄酒中EC平均含量为13.89 μg/L，白葡萄酒中EC平均含量为8.11 μg/L。.（3）研究葡萄汁中精氨酸、尿素、瓜氨酸以及(NH4)2HPO4对葡萄酒中EC含量的影响。结果显示：葡萄汁中精氨酸的含量与发酵结束后尿素和瓜氨酸含量呈线性相关关系；尿素和瓜氨酸直接影响葡萄酒中的EC含量；(NH4)2HPO4对EC影响无规律。.（4）研究发酵温度、酿酒酵母种类、酵母泥陈酿技术对EC含量影响，其影响依次降低。酵母菌接种量对EC含量无影响。葡萄酒贮存过程中，温度、尿素含量以及pH对葡萄酒中EC含量的影响，温度上升10 ℃，由尿素生成的EC含量增加1.5～2倍，由瓜氨酸生成EC的速率增加1.5～2.5倍，尿素含量越高，反应速率越快，葡萄酒pH值基本与EC形成无关。.（5）通过稀释涂布法和平板划线法对微生物进行分离纯化，并进行分子生物学鉴定。结果如下：样品中分离酵母50株；细菌20株；分离菌株经WL、TTC、MLA活性测试；筛得酿酒酵母13株；酒酒球菌3株；植物乳杆菌2株。.（6）对筛选出酿酒酵母和乳酸菌进行酿酒试验，评价低产EC能力。结果显示：期间各菌株发酵液尿素含量均呈指数下降趋势；EC含量呈显著上升趋势；酿酒酵母Y44低产EC性能最优，酒精发酵结束时EC生成量1.51 μg/L；MLF中酒酒球菌LB和L66低产EC性能最优，MLF后发酵液中EC含量为0.56 μg/L和0.60 μg/L。.（7）运用实时荧光定量PCR技术，分析低产EC酿酒酵母和乳酸菌的特异性基因表达活性与EC含量间的关系，结果显示：发酵期间各菌株特异性基因表达量存在显著差异；特异性片段相对表达量和有害物质含量均呈正相关；筛选相对表达量小的酿酒菌株是降低葡萄酒中EC的有效手段。.（8）工业化生产应用结果显示：Y44 EC生成量为3.482 μg/L，较对照酵母低。酵母Y44具有良好生产性能，为低产EC优良酿酒酵母菌株。