黄酒中氨基甲酸乙酯和尿素的处理方法与工艺条件

采用自主新研制的黄酒特种助剂,以期有效去除氨基甲酸乙酯(EC)和尿素(EC的前驱物).助剂处理的工艺条件为:添加量为0.8‰～1‰(m),在室温条件下,搅拌60 min,然后静置24 h后过滤.结果表明,EC去除率为42.0％～68.4％;尿素去除率为38.8 ％～51.8％,处理酒样中EC含量均低于100 μg/L,酒体的风味口感及主要理化指标基本保持不变.在同样条件下,助剂处理发酵液比处理煎酒液,在成品酒中的EC含量可减少30.8％;与处理成品酒相比,EC含量可减少52.1％;与对照酒样相比,EC含量可减少73.4％.强化试验表明,经助剂处理的酒样在存放陈酿的过程中可能会减少其EC的生成.     

尿素吸收型工业黄酒酵母单倍体工程菌的构建

氨基甲酸乙酯是黄酒生产中的副产物,具有致癌性。尿素是氨基甲酸乙酯(EC)的主要前体物质,为降低黄酒中EC的含量,通过提高尿素转运蛋白基因DUR3的表达水平,构建了尿素吸收型工业黄酒酵母单倍体工程菌。采用融合PCR技术,将DUR3基因置于强启动子PGK1p后,构建过表达组件"URA3-PGK1p-DUR3-PGK1tURA3",转化工业黄酒酵母单倍体Na(MATa),获得单倍体工程菌Na-uD。通过实验室黄酒发酵,工程菌所酿黄酒发酵液中尿素含量降低了55.0%,EC含量降低了10.9%,其他理化指标无明显差异。因此过表达DUR3基因的工程菌Na-uD具有"吸收尿素"的能力,能够降低发酵液中尿素的含量,且无外源抗性基因的引入。     

酿酒酵母对黄酒酿造中氨基甲酸乙酯代谢及黄酒品质的影响

氨基甲酸乙酯(Ethyl carbamate, EC)是存在于黄酒中的一种潜在致癌物质。本文通过研究酿酒酵母代替酒药发酵对黄酒品质的影响,为应用实验室菌株探究黄酒发酵体系中氨基甲酸乙酯形成的代谢基础提供依据。利用本实验室分离的野生型酿酒酵母(Saccharomyces cerevisiae,SC)和纯酿酒酵母(BY4741)替代酒药,并且按照工业化黄酒酿造工艺进行发酵实验。经高效液相色谱分析,BY4741和SC发酵组发酵液中尿素浓度和氨基甲酸乙酯浓度与酒药发酵组差异不大;酿造后期添加硫酸铵作为补充氮源,能在一定水平上减少氨基甲酸乙酯的浓度;发酵结束并煎酒后的成品经检测分析,酒精度、风味物质组成及氨基酸组成差异不大,说明应用BY4741和SC替代酒药进行酿造以及酿造过程中添加硫酸铵不影响产品的质量。本研究结果表明,酿酒酵母替代酒药进行发酵对黄酒品质影响较小,可应用于模拟体系中探索氨基甲酸乙酯形成的调控因子。     

低产尿素黄酒酵母工程菌的酿造特性

为考察酵母工程菌在黄酒酿造过程中的发酵性能及其降低发酵液中尿素和氨基甲酸乙酯(ethyl carba-mate,EC)的能力,以前期构建的降低黄酒中尿素和EC效果最好的酵母工程菌N85^DUR1,2-c为研究对象,利用单因素试验考察黄酒发酵工艺对其降低发酵液中尿素和EC能力的影响,并对其在生产试验过程中的发酵性能进行研究。结果表明,酵母接种量、发酵温度以及麦曲添加量等工艺参数对工程菌N85^DUR1,2-c低产尿素和EC的性能没有明显的影响,且含量低于亲本菌株。50 kL生产试验表明,工程菌N85^DUR1,2-c所酿黄酒中理化指标含量正常,符合黄酒国标的要求。而N85^DUR1,2-c发酵液中尿素和EC的含量分别为(2.4±0.2)mg/L和(14.9±0.6)μg/L,较亲本菌株分别降低了90.7%和54.6%,且贮存过程中EC含量增加缓慢。说明酵母工程菌N85^DUR1,2-c在不改变黄酒优良品质的前提下,能够显著地降低发酵液中尿素的含量,可以从根源上减少黄酒中EC的积累,提高饮用...     

脲基酰胺酶基因在黄酒酵母中的整合型表达

氨基甲酸乙酯是一种人类的潜在致癌物,在许多发酵酒中均有存在,主要来源于发酵过程中酵母代谢产生的尿素。以pYX212为载体,将脲基酰胺酶基因DUR1,2克隆到TPI强启动子和终止子之间的位点,再通过同源重组的方式将受强启动子调控的目的基因整合到黄酒酵母的基因组中,最终获得一株低产尿素的胞内脲基酰胺酶基因组成型高表达的黄酒酵母85#DUR1,2。在实验室规模的黄酒酿造实验中,85#DUR1,2产尿素量为8.34mg/L,比出发菌株降低了69.9%,贮存一段时间后的酒液中氨基甲酸乙酯含量比出发菌株降低了40.5%,而发酵性能、酒精度、总酸及氨基态氮与出发菌株无显著差异。     

黄酒酵母发酵过程中产尿素的变化规律研究

本论文采用二乙酰肟比色法测定了黄酒酵母发酵过程中产尿素的规律。结果显示:黄酒酵母在生长过程中,分泌大量尿素到胞外,在稳定期(培养第51 h)胞外尿素浓度达38.642 mg/L,胞内尿素浓度(培养第27 h)为6.043 mg/L。培养基初始pH3,4时,相对初始pH=5而言酵母细胞的尿素的胞外分泌较少;培养基酒精浓度为12%时,酵母胞外尿素分泌水平下降;培养温度升高胞外尿素分泌水平下降。酶制剂代替麦曲的黄酒发酵实验结果显示,发酵液中尿素含量水平随着发酵进程而升高。     

从黄酒的酿造工艺上探讨氨基甲酸乙酯

阐述了酒中氨基甲酸乙酯的形成机理,并从黄酒的整个酿造工艺--原料、菌种、发酵、煎酒、陈酿等方面入手,借鉴葡萄酒中氨基甲酸乙酯的形成及控制策略,综述了目前黄酒中氨基甲酸乙酯的形成及控制的研究,以期为黄酒生产中降低氨基甲酸乙酯的含量提供科学依据。     

谨防黄酒中氨基甲酸乙酯的危害

重点阐述了氨基甲酸乙酯的形成、致癌机理及其生产中控制，为酿酒业(尤其是黄酒工业)提供了重要的理论依据。     

黄酒酿造过程中氨基甲酸乙酯形成的细胞生物学基础及消减研究进展

氨基甲酸乙酯(EC)是发酵食品(面包、酸奶、大豆奶酪等)和酒精饮料(葡萄酒、清酒、黄酒等)在发酵过程中,含氮化合物不完全代谢产生的副产物,与乙醇自发反应产生的,对人体具有潜在遗传毒性和致癌性。黄酒是我国传统而独特的发酵酒,因独特的风味和较高的营养价值,而深受消费者的喜爱。然而,在传统黄酒中,氨基甲酸乙酯含量显著高于其它酒精饮料,对消费者健康构成潜在的安全隐患。降低黄酒中氨基甲酸乙酯含量对黄酒产业的可持续发展具有重要意义。本文综述尿素、瓜氨酸及精氨酸分别在酿酒酵母及乳酸菌细胞中代谢调控机制,以及黄酒酿造过程中EC消减的方法。     

减少麦曲用量对黄酒中尿素含量及质量的影响

试验了适当减少麦曲用量对黄酒中尿素含量、黄酒理化指标及感官指标的影响。结果表明减少麦曲用量1％，可降低约29％左右的尿素；减少麦曲用量2％，可降低约33％左右的尿素。同时减少麦曲用量1％的酒样其理化指标、感官指标与对照样比较差距不大，可以作为实际生产中降低尿素含量的途径之一加以使用。     

Enhancement of urea uptake in Chinese rice wine yeast strain N85 by the constitutive expression of DUR3 for ethyl carbamate elimination

Most of the fermented alcoholic beverages, particularly Chinese rice wine, contain the potentially human carcinogenic compound ethyl carbamate (EC). As a major EC precursor in Chinese rice wine, urea in fermentations can be transported into the yeast cell by urea permease and finally metabolized by urea carboxylase and allophanate hydrolase in vivo. To eliminate EC in Chinese rice wines, the present study constructed high urea uptake yeast strains N1‐D, N2‐D and N‐D, by introducing a strong promoter (PGK1p) into the urea permease gene (DUR3) of the industrial Chinese rice wine yeast N85, and by the restoration of the URA3 gene at the same time. With these self‐cloned, high urea uptake strains, the urea and EC in the terminal Chinese rice wine samples were reduced to different extents. With two copies of overexpressed DUR3, the N‐D strain could reduce the urea and the EC by 53.4 and 26.1%, respectively. No difference in fermentation characteristics was found between the engineered strains and the parental industrial yeast strain N85. These results could help to optimize the genetic manipulation strategy for EC elimination in Chinese rice wine production. Copyright © 2015 The Institute of Brewing & Distilling     

Contribution of citrulline to the formation of ethyl carbamate during Chinese rice wine production

Ethyl carbamate is a well-known carcinogen and widely occurs in Chinese rice wine. To provide more clues to minimise ethyl carbamate accumulation, the levels of possible precursors of ethyl carbamate in Chinese rice wine were investigated by HPLC. Studies of the possible precursors of ethyl carbamate in Chinese raw rice wine with various additives and treatments indicated that significant amounts of urea can account for ethyl carbamate formation. It was also recognised that citrulline is another important precursor that significantly affects ethyl carbamate production during the boiling procedure used in the Chinese rice wine manufacturing process. Besides urea and citrulline, arginine was also found to be an indirect ethyl carbamate precursor due to its ability to form urea and citrulline by microorganism metabolism.     

顶空-固相微萃取-气质联用技术快速测定黄酒中氨基甲酸乙酯的方法研究

黄酒中含有微量氨基甲酸乙酯(Ethyl Carbamate,EC),对人体健康有潜在不利影响,对其含量的准确测定是监管与控制的基础。采用顶空-固相微萃取-气质联用技术(HS-SPME-GC-MS),建立了完全自动化、快速简便测定黄酒中EC的方法。结果表明,以氨基甲酸丙酯为内标,优化的参数为:添加NaCl至0.39g/mL酒样,采用聚丙烯酸酯(PA)萃取头70℃下萃取45min。此方法线性相关系数R为0.9983,回收率为90.21%～99.07%,相对标准偏差小于2.5%,检测限低至1.19μg/L。此EC快速测定方法无需繁琐的预处理过程,自动快速高效,连续分析中单个样品平均总需时只要50min,且无有机溶剂危害,推荐作为黄酒中EC含量测定的标准方法之一。     

黄酒中氨基甲酸乙酯吸附去除的动力学和热力学研究

有效控制黄酒中氨基甲酸乙酯含量(EC)对于黄酒食品安全和行业发展具有重要意义。本研究通过筛选得到能够有效吸附黄酒中EC的吸附树脂,对EC吸附去除率在70%以上,该树脂具有良好的吸附性能和再生能力。对其吸附动力学和热力学特性进行考察,结果表明该树脂吸附黄酒中EC的过程符合准二级动力学模型,树脂的表观吸附活化能Ea为8.89×103k J/mol。热力学研究表明,该树脂吸附EC符合Freundlich方程,该吸附过程是自发地、熵增加的吸热过程。研究结果为高效、简便去除黄酒中EC的工业化应用提供了依据。      

江西省产黄酒中氨基甲酸乙酯残留量的GC-MS分析

采用气相色谱-质谱法调查60份江西省内生产的黄酒产品中氨基甲酸乙酯(EC)残留量。酒样添加d5-EC同位素内标,经硅藻土层析住吸附,乙醚洗脱,用GC-MS测定,内标法定量。色谱柱为DB-INNOWAX石英毛细管柱(30m×0.25mm×0.25μm)。加标回收试验的平均回收率为107.5%,相对标准偏差为1.33%(n=2),黄酒中EC的污染水平中,EC的中位数为24.4μg/kg,均值为35.0μg/kg,检出率为70%,含量值范围为6.0~123.7μg/L。通过此次调查江西产黄酒中氨基甲酸乙酯残留量,将为我国制定国家标准和对黄酒中氨基甲酸乙酯的污染水平评估提供数据支持。     

慕萨莱思酒发酵过程中氨基甲酸乙酯含量的变化研究

氨基甲酸乙酯（EC）是发酵食品中存在的一种致癌物质,该研究以新疆阿瓦提慕萨莱思酒为研究对象,检测发酵过程中不同阶段EC含量、尿素含量、酒精体积分数,探究EC的形成与尿素、酒精含量的关系。结果显示,发酵结束后EC含量为25.6 μg/L,尿素与酒精含量越高,EC生成速率越快,慕萨莱思酒中EC含量越高,温度对EC的生成影响较大。     

Effect of citrulline metabolism in Saccharomyces cerevisiae on the formation of ethyl carbamate during Chinese rice wine fermentation

Urea, as the main precursor of ethyl carbamate (EC), has received extensive attention. Here, we have metabolically engineered an industrial yeast strain – Saccharomyces cerevisiae N85 – to investigate the contribution of the EC precursor citrulline to the concentration of EC in Chinese rice wine. The results showed that the citrulline biosynthetic pathway of the modified strain N85‐arg3 was completely suppressed by deletion of ARG3, encoding ornithine carbamoyltransferase. However, there were no significant differences in the levels of citrulline or EC between N85‐arg3 and the parental strain N85 during fermentation. In addition, we over‐expressed ARG1 (encoding argininosuccinate synthase) and ARG4 (encoding argininosuccinate lyase) to construct the engineered strains N85^ARG1,4 and N85^ARG1,4‐arg3. The citrulline contents in Chinese rice wine fermented with N85^ARG1,4 and N85^ARG1,4‐arg3 were respectively 24.1 and 20.4% less than that of N85. However, the contents of EC were 23.8 and 28.5% more than that of N85. These results suggested that reducing the formation of EC during Chinese rice wine fermentation by genetically engineering citrulline metabolism in S. cerevisiae was not a viable proposition. Copyright © 2018 The Institute of Brewing & Distilling