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。     

Analysis of flavour compounds in beer with extruded sorghum as an adjunct using headspace solid‐phase micro‐extraction and gas chromatography–mass spectrometry

Sorghum is a widely used adjunct that is used in the production of beer and increasingly affects beer flavour as the amount added increases. The aim of this work was to establish a simple, solvent‐free technique, without derivatization, to analyse flavours and typical volatile compounds present in extruded and unextruded sorghum beer, and to compare the flavour differences of the two types of beer. Headspace solid‐phase micro‐extraction gas chromatography–mass spectrometry was used to determine the flavours in the two beers and eight typical volatile compounds were quantified using GC. Forty‐five flavour compounds were identified and quantified in extruded white sorghum beer, while 31 flavour compounds were identified in unextruded white sorghum beer. Extruded or unextruded white sorghum can be used to produce ale beer, but the primary flavour content in the extruded white sorghum beer was higher than in the unextruded white sorghum beer. Copyright © 2016 The Institute of Brewing & Distilling     

气相色谱-串联质谱法测定黄酒和酱油中的氨基甲酸乙酯

目的 建立气相色谱-串联质谱法(GC-MS/MS)测定黄酒和酱油中氨基甲酸乙酯的分析方法。 方法 样 品添加同位素内标氨基甲酸乙酯-d5 后, 直接上样到填装好 Extrelut TM NT 有机硅藻土的柱中进行基质固相分散 萃取, 先用正己烷淋洗除杂, 再用乙酸乙酯:乙醚(1:9, v/v)的混合溶剂洗脱氨基甲酸乙酯, 洗脱液经浓缩后采用 GC-MS/MS 多反应监测(MRM)模式测定。结果 方法检出限和线性范围分别为 2 ?g/kg 和 5~1000 ?g/kg (R＞ 0.999)。在 50、200、500 ?g/kg 三个添加水平下, 黄酒基体中氨基甲酸乙酯的加标回收率为 97.4%~98.8%, RSD 为 4.0%~8.4%。向酱油基体中添加 10、50、200 ?g/kg 三个浓度水平, 氨基甲酸乙酯的加标回收率为 96.2%~104.0%, RSD 为 5.3%~10.0%。结论 该方法定量准确、操作简单、灵敏度高、定性确证可靠, 适用于 黄酒和酱油中氨基甲酸乙酯的测定。     

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。     

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.     

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。     

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。     

Effects of sterilization temperature on the concentration of ethyl carbamate and other quality traits in Chinese rice wine

Ethyl carbamate (EC), which is present in Chinese rice wine, has a large potential for carcinogenicity and genotoxicity. EC is produced during the process of rice wine fermentation and storage. High concentrations of precursors, as well as high temperatures, will significantly accelerate the formation of EC. The present work aims to reduce EC formation by optimizing the production process, especially the boiling procedure. With various boiling sterilization temperatures, EC accumulated to different concentrations but the lower the temperature, the less EC was formed. To preserve the quality traits of Chinese rice wine, including biological and non‐biological stability, as well as the sugar component, an 80°C boiling temperature is suggested. The present study provides direction for process optimization, which combined with improved production technology and metabolic engineered yeast strains, can reduce the content of EC in Chinese rice wine. Copyright © 2014 The Institute of Brewing & Distilling     

黄酒酵母的改良及对产物中尿素和氨基甲酸乙酯含量的影响

氨基甲酸乙酯（EC)是一种潜在致癌物,在黄酒发酵过程中尿素是它的前体物质。该研究通过紫外诱变和基因过表达筛选获得改良黄酒酵母菌种,并对黄酒产品的理化指标进行检测可知,与出发菌株相比,改造菌株的发酵性能和黄酒的出酒率、酒精度、总糖、总酸、氨基酸态氮和β-苯乙醇没有明显的差异,而诱变菌株JF501-A62发酵产物尿素含量降低了67%,EC含量降低了59%;基因过表达菌株JF501-B5发酵产物尿素含量降低了88%,EC含量降低了63%。两者均有很好的发酵性能,并取得了较好地降低产品中尿素含量、进而降低氨基甲酸乙酯含量的效果。与紫外诱变相比,基因过表达的改良方法获得了尿素含量更低的菌株,并贮存6个月之后产品中的EC含量更低。     

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     

Chemical profiling of volatile compounds of various home‐made fruit spirits using headspace solid‐phase microextraction

Fruit spirits are typical alcoholic beverages in the Moravian region of the Czech Republic. The aim of this work was to find differences in chemical composition among volatile profiles of spirits produced from various kinds of fruits. Twenty‐four samples of seven kinds of fruit spirits (plum, apple, pear, cherry, mirabelle, apricot and raspberry spirits) were analysed for examination of their volatile profiles. The most abundant family of compounds was esters, particularly ethyl esters. Sesquiterpenes proved to be a significant group of volatile substances by observing the differences between pome and stone fruit spirits [by relative higher content of (E ,E )‐α‐farnesene and by presence of α‐zingiberene and (E )‐α‐bisabolene in volatile profiles of pome fruit spirits]. Only in stone fruit spirits were propyl decanoate and ethyl salicylate found. Some other compounds were observed as being characteristic for individual kinds of analysed fruit spirits, e.g. γ‐decalactone for apricot spirits, (E )‐β‐farnesene for apple spirits, (Z )‐9‐tetradecen‐1‐ol for mirabelle spirits or some apocarotenoids for raspberry spirits. This work could potentially be the basis for checking the fruit origin of these distillates, or a partial manual as to how to differentiate individual kinds of pome‐fruit spirits (pear and apple spirits) or stone‐fruit spirits (plum, mirabelle, apricot spirits). Copyright © 2017 The Institute of Brewing & Distilling     

黄酒中氨基甲酸乙酯形成机理及控制方法研究进展

氨基甲酸乙酯是一种存在于黄酒中的致癌物质.该文主要从氨基甲酸乙酯的形成机理,酿造工艺对其形成影响和控制方法等方面进行综述,同时对未来研究方向进行了展望.旨在为企业更好地控制黄酒中的氨基甲酸乙酯含量提供参考.     

多维气相/气质联用法测定葡萄酒中的氨基甲酸乙酯

建立了多维气相/气质联用（MDGC/GCMS）法测定葡萄酒中氨基甲酸乙酯的检测方法。采用硅藻土固相萃取柱对葡萄酒中氨基甲酸乙酯（EC）进行提取净化,利用MDGC/GCMS对葡萄酒中EC进行检测。结果表明,葡萄酒中EC与干扰物均得到很好分离,EC在0～1 000 μg/L范围内线性关系良好（R=0.999 3）,检出限（S/N=3）为1.0 μg/L,加标回收率为82.97%～86.82%,相对标准偏差（RSD）为2.29%;方法的精密度和重复性试验RSD分别为2.42%和2.88%。表明该方法准确度高、重复性和精密度良好,适用于葡萄酒中的氨基甲酸乙酯的检测。     

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     

红葡萄酒中氨基甲酸乙酯检测方法的优化

该研究使用高效液相色谱-荧光检测法（HPLC-FLD）检测红葡萄酒中氨基甲酸乙酯（EC）,并进一步优化检测方法。结果表明,最佳检测条件为吸附时间10 min、正己烷作为淋洗溶剂、乙酸乙酯与乙醚（5∶95,V/V）作为洗脱溶剂,荧光反应时间5 min。在最佳的实验条件下对氨基甲酸乙酯进行检测,在50～200 μg/L范围内时,线性关系良好,该方法的平均加标回收率为88.5%～92.8%,精密度实验结果相对标准偏差（RSD）为2.9%～6.9%,检出限为5 μg/L,说明该检测方法具有较好的精密度和准确度,易于掌握,可用于葡萄酒中氨基甲酸乙酯的测定,具有良好的应用前景。