绍兴黄酒氨基甲酸乙酯的检测与控制

2007年国际癌症研究总署(IARC)将氨基甲酸乙酯(EC)归为人类可能致癌物质(2A类).在黄酒中含量较高.应用GC/MS方法对绍兴黄酒EC含量进行研究,对煎酒前后进行氨基甲酸乙酯监测分析,发现煎酒前和煎酒后EC平均含量分别为16.1μg/kg、63.7μg/kg、其中在煎酒过程中EC增加了47.6μg/kg.在贮存过程中,对年份加饭酒分析,发现EC含量在不断地增加.第一年增加量最多,但后几年平均增加量大约每年15μg/kg左右.所以需加强对黄酒生产工艺和贮存条件的管理和改进,以减少EC的产生,确保绍兴黄酒的竞争性及安全.     

Citrulline production by lactic acid bacteria in Chinese rice wine

Ethyl carbamate (EC) is a carcinogenic compound derived from the spontaneous reaction of ethanol with urea or citrulline in Chinese rice wine. Polymerase chain reaction–denaturing gradient gel electrophoresis showed that five species, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis and Lactobacillus coryniformis were the most abundant bacteria in the Chinese rice wine production process. Five strains belonging to these species can degrade arginine primarily in the exponential growth phase and accumulate citrulline in MRS‐Arg medium. In addition, an L. brevis strain was shown to be capable of assimilating citrulline, indicating the potential of this strain suggesting a potential route to reduce citrulline content and ethyl carbamate formation in Chinese rice wine fermentation. Copyright © 2018 The Institute of Brewing & Distilling     

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     

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。     

黄酒中氨基甲酸乙酯直接减除技术的研究

黄酒中含有微量氨基甲酸乙酯(Ethyl Carbamate),简称EC,直接降低黄酒中的EC含量,具有重要的现实意义。在各种吸附性材料中,优选得特异性功能树脂材料L2、L3,复配后以合适的添加量体积分数10%处理酒样,对黄酒中的EC能较有效减除,去除率在60%以上,基本达到EC限量要求,同时对酒体风味的保持较好。材料对EC的减除为吸热过程,实际应用中需保证作用温度大于20℃。面向中国黄酒中EC含量的直接降低,提供了一个实用、便捷的全新途径。     

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.     

HACCP体系在客家黄酒生产过程中的应用

HACCP，即危害分析与关键控制点体系，是一种规范操作和卫生标准的控制危害的预防体系。客家黄酒具有独特的风味和源远流长的酒文化，其生产过程是复杂的生化反应过程，存在着生物危害、化学危害、物理危害等。HACCP体系在客家黄酒应用中的关键控制点有：原料的质量、设备的清洗、发酵、炙酒、灌装及生产用水等，该体系具有响应时间快、确定失误准等优点，对生产和保证产品质量具有很好的指导。     

The overexpression of DUR1,2 and deletion of CAR1 in an industrial Saccharomyces cerevisiae strain and effects on nitrogen catabolite repression in Chinese rice wine production

Urea is acknowledged as the predominant precursor of ethyl carbamate (EC) in Chinese rice wine. During Chinese rice wine fermentation, urea accumulates owing to the nitrogen catabolite repression effect when preferred nitrogen sources are available. In previous research, two metabolically engineered strains were constructed with overexpression of DUR1,2 and deletion of CAR1 from an industrial Chinese rice wine yeast N85. The decreasing effect of urea and EC was demonstrated during small‐scale Chinese rice wine fermentations. The present study compared the urea utilization rate of the parental and metabolically engineered yeast strains, using a preferred and non‐preferred nitrogen source culture media, leading to alleviated urea accumulation and thus EC formation. The qRT‐PCR results showed that, in all of the culture media, DUR1,2 was overexpressed with the inserted strong promoter PGK1p. During pilot scale fermentations, the urea and EC content decreased with the engineered strains. These results confirmed that the engineered strains could resist the nitrogen catabolite repression effect. Copyright © 2016 The Institute of Brewing & Distilling     

黄酒微生物及其与风味形成关系的研究进展

黄酒是我国传统发酵食品,在其风味物质形成过程中,微生物起到了非常重要的作用。本研究综合分析了黄酒原料处理、麦曲、酒药以及酿造过程等方面对微生物群落结构,并探讨了微生物群落结构与黄酒风味形成的关系。其中,浸米过程中的微生物主要以乳酸菌为主,麦曲主要以霉菌为主,酒药主要以酵母菌为主。而在整个黄酒酿造过程中霉菌先减少后增多,并在后酵过程起到产酶糖化的作用;酵母先增加后减少,并在前主酵过程中起到产酒精增强黄酒风味的作用;细菌尤其是乳酸菌在浸米及酿造过程中起到降解大分子物质的作用从而促进黄酒呈香呈味,且对有机酸的产生即黄酒酸味起到积极作用,而其余各种细菌在黄酒不同的酿造阶段相互作用并对黄酒的风味起到积极作用。根据目前的研究现状,提出可利用多组学技术分析黄酒中主要产香微生物并通过后续的高通量筛选应用到黄酒发酵生产中,优化黄酒风味的形成过程。     

高温流化法糊化在黄酒生产中的应用

采用“高温流化”和“蒸煮”两种不同方法处理大米 ,并进行酿造试验。通过对两种不同方法处理后大米成份的差异、发酵过程中失重变化趋势和酿造酒的常规理化指标的对比分析认为 :高温流化法糊化技术完全适用于我国传统黄酒生产中 ,可有效降低酿造酒中氨基氮含量 ;另外 ,通过对同样采用高温流化法糊化技术生产的“松竹梅”清酒的对比分析认为 ,中国黄酒和日本清酒在酿造工艺方面有很大的差异     

黄酒生产中氨基甲酸乙酯的监测与控制

氨基甲酸乙酯（EC）是一种基因致癌物,黄酒中含量较高。应用SPE结合GC/MS方法对广东黄酒生产过程中产生的氨基甲酸乙酯进行全程监测分析,在水和糖化液里未能检出EC,米酒、发酵液、煎酒液和成品中EC平均含量分别为8.2μg/kg、7.9μg/kg、21.6μg/kg、52.7μg/kg,其中70%的EC是在煎酒和贮存过程中产生。需加强对黄酒生产工艺和贮存条件的管理和改进,以减少EC的产生,确保广东地产黄酒的竞争性及安全性。     

浅析绍兴黄酒生产中的美拉德反应

针对绍兴黄酒中的美拉德反应,介绍了美拉德反应的机理和影响因素,认为黄酒生产中发生美拉德反应的反应底物有还原糖和氨基酸,在黄酒生产中发生美拉德反应的条件是水分、温度和pH值,在黄酒制曲过程、发酵过程、煎酒过程和贮存过程中都会产生美拉德反应,同时讨论了黄酒中美拉德反应的利与弊.     

黄酒生产过程中生物胺的形成及变化

采用高效液相色谱的方法分析某企业春、秋和冬酿黄酒生产过程中的总生物胺及色胺、组胺、苯乙胺、酪胺、尸胺、腐胺、精胺、亚精胺8种生物胺含量,探讨了黄酒原料及整个发酵生产过程中生物胺形成和分解机制。结果表明,冬酿黄酒中主要的生物胺是腐胺、酪胺和色胺,在整个生产过程中呈现先降低后升高的趋势,其中后酵生物胺含量最高,为186.7 mg/L;春酿、秋酿和冬酿黄酒中前酵阶段生物胺含量差异不明显（P＞0.05）,后酵阶段秋酿黄酒和冬酿黄酒生物胺含量显著高于春酿黄酒（P＜0.05）,煎酒阶段冬酿黄酒生物胺含量显著高于春酿黄酒和秋酿黄酒（P＜0.05）;冬酿和秋酿黄酒中酪胺含量分别为72.11 mg/kg和30.26 mg/kg,以及高含量的腐胺和色胺,具有潜在的食品安全隐患。     

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。     

Study on the changing concentration of ethyl carbamate in yellow rice wine during production and storage by gas chromatography/mass spectrometry

Ethyl carbamate (EC) is a carcinogen that existed wildly in yellow rice wine. Raw materials, semi- and finished wines were tested by gas chromatography/mass spectrometry as soon as being collected from factories. EC was not detected in rice and wheat starter, while it was detected after fermentation, sterilization and storage. Results indicate that EC is mainly generated from the processes of wine boiling, jar-filling and storage. As a result, production technology is suggested to be improved so as to reduce the concentration of the EC. Furthermore, yellow rice wine is advised to be stored at 4?°C and consumers are proposed to choose fresh wine.     

Determination of ethyl carbamate in Chinese yellow rice wine using high‐performance liquid chromatography with fluorescence detection

Ethyl carbamate (EC) is a naturally occurring component in the fermented products, especially the Chinese yellow rice wine. EC has been found showing a potential for carcinogenity and causing food safety problems. This study is to investigate the application of the existing HPLC‐FLD method to the analysis of EC in Chinese yellow rice wine, and to validate it for EC analysis with high through‐out and applicability in Chinese yellow rice wine sample. The parameters examined in this study were fully evaluated. Results indicated that good linearity was obtained with a correlation coefficient exceeding 0.9990, the limit of detection and quantification was 73.2 and 243.9 μg L^?1, respectively. Recoveries ranged between 98.30% and 101.30%, and the precision of this method was lower than 5% (RSD). The method conducted in this work was successfully applied to determine EC concentration in Chinese yellow rice wine samples from different origins. The negative correlation between EC formation and urea level in yellow rice wine samples is evaluated with the developed HPLC‐FLD method.     

黄酒中氨基甲酸乙酯的分析与控制

氨基甲酸乙酯是一种存在于黄酒中的致癌物质。本文采用气质联用法检测了广东市场上42种黄酒样品中氨基甲酸乙酯含量,28个样品中EC含量超过30μg/kg唱,占66．67％,其中7个样品中EC含量超过了100μg/kg（日本清酒中EC的限量标准）,最高的高达1210μg/kg。研究了脲酶对黄酒处理的效果,结果发现,一定条件下添加脲酶能够去除酒里的尿素,破坏产生氨基甲酸乙酯的主要途径,从而控制氨基甲酸乙酯含量不再剧增。并在单因素试验结果的基础上,进行正交实验,得出脲酶最佳的处理条件为酶浓度150mg／L、处理温度为25℃,pH值为3．5,处理时间10d。该条件下进行处理,可使酒中氨基甲酸乙酯含量降低45．83％。     

应用粳米淋饭酒母、苦荞麦粉喂浆开发清爽营养荞麦黄酒工艺

根据苦荞麦的营养成分和特征,结合酶法低温蒸煮新技术,在传统黄酒酿造工艺的基础上,采用粳米饭搭窝、酒药培养糖化制成淋饭酒母,然后在酵母扩配和发酵过程中将粳米喂饭改为苦荞麦粉喂浆,在糖化发酵剂上又融合了白药、红曲、麦曲之长,增加了酒醪中微量成分和代谢产物含量,并在后道工序中引入勾兑调味技术和冷冻吸附工艺,试制开发出具有清醇、爽适、营养、保健的苦荞黄酒。     

Effect of ‘wheat Qu’ addition on the formation of ethyl carbamate in Chinese rice wine with enzymatic extrusion liquefaction pretreatment

Ethyl carbamate (EC), which has been confirmed as probably carcinogenic to humans, can be formed during the alcoholic fermentation of Chinese rice wine. In this study, EC and two possible precursors (urea and arginine) were analysed in wines fermented from rice processed by enzymatic extrusion liquefaction pretreatment, with different wheat Qu content gradients from 6 to 18% (dry basis, d.b.). The EC concentration decreased 22.5% with the addition of wheat Qu reduced to 6% for the enzymatic extrusion‐processed rice wine (EE). Moreover, although the arginine was generated 6.33–8.39 times higher in the enzymatic extruded rice, instead of the traditional treated rice at the same wheat Qu level, the corresponding level of urea formed from arginine was not high in the EE. In addition, rice treated by extrusion combined with enzymatic hydrolysis was found to be more utilizable and fermentable, and the lost quality of the final traditional rice wine with the low wheat Qu level (6–14%) could be improved using enzymatically extruded rice as the fermenting material. Therefore, it may be feasible to inhibit EC accumulation in Chinese rice wine by wheat Qu control, combined with enzymatic extrusion pretreatment for rice. Copyright © 2015 The Institute of Brewing & Distilling     

Ethyl carbamate formation regulated by ornithine transcarbamylase and urea metabolism in the processing of Chinese yellow rice wine

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 utilisation 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 citrulline 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 association (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.