中国黄酒中氨基甲酸乙酯控制策略及机制的研究
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摘要
“民以食为天，食以安为先。”发酵饮料酒中存在微量氨基甲酸乙酯（EC），对人体健康有潜在不利影响。各酿造酒中，黄酒的EC问题较为突出。在分析探讨各种EC含量控制策略过程中，以黄酒适用肠杆菌（Enterobacter sp.）R-SYB082尿素降解酶，形成从源头上控制中国黄酒中EC的有效措施，同时结合材料科学与技术，形成采用功能性材料进行黄酒中EC直接去除的技术，首次建立了对底物尿素和产物EC含量的集成控制策略，使中国黄酒中EC达到限量要求，对丰富中国黄酒的酿造科学和安全性理论与技术有重要的意义和价值。主要结果如下：
（1）针对目前黄酒中EC传统检测方法存在的耗时、不够稳定、需有机溶剂等问题，基于顶空-固相微萃取-气质联用技术（HS-SPME-GC-MS），建立了自动化、快速简便测定黄酒中EC的方法。以氨基甲酸丙酯（nPC）为内标，添加NaCl至0.39g mL-1酒样，采用聚丙烯酸酯（PA）萃取头70℃下萃取45min。此法解决了黄酒中复杂化合物易形成干扰的问题，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的平均总需时，从国际标准SPE（固相萃取）法的2h以上缩短到只要57min，相对标准偏差小于2.89％，检测限低至1.19μg L-1，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。
（2）针对Enterobacter sp. R-SYB082尿素降解酶活力低的问题，通过优化发酵策略，包括优化初糖浓度为40g L-1、通入CO2提高反应器的厌氧水平等，Enterobacter sp.R-SYB082的尿素降解酶活从1100U L-1优化提高到2303U L-1，并放大至30L规模获得相似酶活。此Enterobacter sp. R-SYB082酶具有好的稳定性，且通过自然冷藏及CO2气体介入成功获得激活。该酶在黄酒中的尿素去除率（66.47%），比广泛使用的日本商业脲酶（去除率58.79%）高。该酶对黄酒中底物尿素的有效降解，从源头上实现了对EC的控制。
（3）开展了对Enterobacter sp. R-SYB082尿素降解酶克隆表达的研究。通过基因比较发现，Enterobacter sp. R-SYB082酶是一种酸性脲酶同功酶——脲基乙醇酸酰胺水解酶（UAH）而非脲酶，该酶能实现黄酒中待去除底物尿素及产物EC的联合控制，具有很好的应用特性与工业开发前景。首次报道了脲酶缺陷型菌株以此酶介导的全新氮同化代谢途径。并成功构建了具有自主知识产权的工程菌BL21-pET28-allA，获得该酶的活性表达。
（4）基于定向去除技术和分子间作用原理，从各种吸附性材料中优选得特异性功能材料L，以直接去除黄酒中EC。并对去除机制进行研究发现：其强碱性［-N+R3］基团对EC的离子交换作用，是EC定向减除的主要动力。经定向修饰和复配后，再经酒样的特殊工艺微调处理，其能选择性地有效减除黄酒中的EC。此减除为吸热过程，实际应用中温度需高于20℃效果更佳。应用L材料动态处理的较合适速度为0.25BV h-1，对于EC含量在300μg L-1以内的黄酒，能降至50μg L-1以下，达到限量要求；且处理后酒体的风味与原酒接近。
配合Enterobacter sp. R-SYB082尿素降解酶对尿素的降解，和功能性材料对EC的终端定向减除，可以实现中国黄酒中尿素含量降至10mg L-1以内，以及EC含量在300μg L-1以内的黄酒降至50μg L-1以下，两者综合，首次建立了从底物到产物对黄酒中EC含量的集成控制策略。为中国黄酒中EC含量的降低，提供了实用、便捷的新途径，具有保障食品安全、利于应对国外技术壁垒的特殊含义。也为其他饮料酒中EC的控制，提供了一条新思路。
“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.
（1）针对目前黄酒中EC传统检测方法存在的耗时、不够稳定、需有机溶剂等问题，基于顶空-固相微萃取-气质联用技术（HS-SPME-GC-MS），建立了自动化、快速简便测定黄酒中EC的方法。以氨基甲酸丙酯（nPC）为内标，添加NaCl至0.39g mL-1酒样，采用聚丙烯酸酯（PA）萃取头70℃下萃取45min。此法解决了黄酒中复杂化合物易形成干扰的问题，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的平均总需时，从国际标准SPE（固相萃取）法的2h以上缩短到只要57min，相对标准偏差小于2.89％，检测限低至1.19μg L-1，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。
（2）针对Enterobacter sp. R-SYB082尿素降解酶活力低的问题，通过优化发酵策略，包括优化初糖浓度为40g L-1、通入CO2提高反应器的厌氧水平等，Enterobacter sp.R-SYB082的尿素降解酶活从1100U L-1优化提高到2303U L-1，并放大至30L规模获得相似酶活。此Enterobacter sp. R-SYB082酶具有好的稳定性，且通过自然冷藏及CO2气体介入成功获得激活。该酶在黄酒中的尿素去除率（66.47%），比广泛使用的日本商业脲酶（去除率58.79%）高。该酶对黄酒中底物尿素的有效降解，从源头上实现了对EC的控制。
（3）开展了对Enterobacter sp. R-SYB082尿素降解酶克隆表达的研究。通过基因比较发现，Enterobacter sp. R-SYB082酶是一种酸性脲酶同功酶——脲基乙醇酸酰胺水解酶（UAH）而非脲酶，该酶能实现黄酒中待去除底物尿素及产物EC的联合控制，具有很好的应用特性与工业开发前景。首次报道了脲酶缺陷型菌株以此酶介导的全新氮同化代谢途径。并成功构建了具有自主知识产权的工程菌BL21-pET28-allA，获得该酶的活性表达。
（4）基于定向去除技术和分子间作用原理，从各种吸附性材料中优选得特异性功能材料L，以直接去除黄酒中EC。并对去除机制进行研究发现：其强碱性［-N+R3］基团对EC的离子交换作用，是EC定向减除的主要动力。经定向修饰和复配后，再经酒样的特殊工艺微调处理，其能选择性地有效减除黄酒中的EC。此减除为吸热过程，实际应用中温度需高于20℃效果更佳。应用L材料动态处理的较合适速度为0.25BV h-1，对于EC含量在300μg L-1以内的黄酒，能降至50μg L-1以下，达到限量要求；且处理后酒体的风味与原酒接近。
配合Enterobacter sp. R-SYB082尿素降解酶对尿素的降解，和功能性材料对EC的终端定向减除，可以实现中国黄酒中尿素含量降至10mg L-1以内，以及EC含量在300μg L-1以内的黄酒降至50μg L-1以下，两者综合，首次建立了从底物到产物对黄酒中EC含量的集成控制策略。为中国黄酒中EC含量的降低，提供了实用、便捷的新途径，具有保障食品安全、利于应对国外技术壁垒的特殊含义。也为其他饮料酒中EC的控制，提供了一条新思路。
“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.
引文
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