酒酒球菌产生物胺和氨基甲酸乙酯相关基因检测及影响其产生因素的研究
DETECTION OF GENES RELATED TO BIOGENIC AMINES AND ETHYL CARBAMATE IN OENOCOCCUS OENI AND STUDY OF INFLUENCING
FACTORS ON ETHYL CARBAMATE PRODUCTION
作者：赵艳卓 导师：刘树文
西北农林科技大学 发酵工程2011届硕士
摘 要
氨基甲酸乙酯（Ethyl Carbamate，EC）和生物胺（Biogenic Amines，BAs）广泛存在于发酵食品中，葡萄酒中亦有存在。氨基甲酸乙酯是一种致癌物质，过量的生物胺会引起过敏反应，二者存在会影响葡萄酒安全性。国内外学者对二者在发酵食品，包括葡萄酒中的产生机理、影响因素、检测方法，以及产生菌株的检测等进行了研究。随着科学技术的发展，用分子生物学方法对产生氨基甲酸乙酯和生物胺的菌株进行鉴定，筛选不具有产生氨基甲酸乙酯和生物胺相关基因的菌株进行葡萄酒酿造，可以提高葡萄酒的质量和安全性。
本研究对实验室从中国主要葡萄与葡萄酒产区筛选保存的50株酒酒球菌产氨基甲酸乙酯和生物胺的相关基因——精氨酸脱亚胺酶（arcA）、鸟氨酸转氨甲酰酶（arcB）、氨甲酰激酶（arcC）、组氨酸脱羧酶（hdc）、鸟氨酸脱羧酶（odc）、酪氨酸脱羧酶（tdc）基因进行了检测，并研究了乙醇、SO2、精氨酸、pH四因素对氨基甲酸乙酯产生的影响。主要结果如下：
1. 产氨基甲酸乙酯相关基因的检测
采用引物arcAF/arcAR、arcBF/arcBR和CK5’/CK3’分别扩增酒酒球菌的arcA、arcB和arcC基因。结果显示，供试的50株酒酒球菌均扩增出单一明亮的目的条带，测序后在基因库中进行比对确定是arcA、arcB和arcC基因。说明本研究所用的50株酒酒球菌均具有arcA、arcB和arcC基因，全部具有产生氨基甲酸乙酯的潜力。
PCR扩增体系为标准PCR体系（20μL）。反应条件为：初始变性温度95℃，时间5min；循环数30；变性温度95℃，时间30s；复性时间30s；延伸温度72℃，时间30s；最终延伸温度72℃，时间5min。
2. 产生物胺相关基因的检测
本研究中合成了6对引物：引物CL1mod/JV17HC和PHDC1/PHDC2扩增组氨酸脱羧酶基因（hdc），引物P1-rev/P2-for和TD2/TD5扩增酪氨酸脱羧酶基因（tdc），引物3/16和odcf/odcr扩增鸟氨酸脱羧酶基因（odc）。对这6对引物在不同退火温度下扩增效果进行比较，选择引物CL1mod/JV17HC、P1-rev/P2-for、3/16进行hdc、tdc和odc基因的检测。结果显示，供试的50株供试酒酒球菌均不具有hdc、tdc和odc基因。
PCR扩增体系为标准PCR体系（20μL）。反应条件为：初始变性温度95℃，时间5min；循环数30；变性温度95℃，时间30s；复性时间30s；延伸温度72℃，时间30s；最终延伸温度72℃，时间5min。
3. 影响氨基甲酸乙酯产生因素的研究
本文采用固液萃取法结合GC-MS方法研究了酒精度、精氨酸、pH和SO2这四种因素对模拟酒中氨基甲酸乙酯形成的影响。结果显示，提高乙醇含量可以促进氨基甲酸乙酯的形成；低浓度的精氨酸会促进氨基甲酸乙酯的形成，高浓度则抑制其形成；在实验pH和SO2水平下，氨基甲酸乙酯含量变化不明显。
关键词 酒酒球菌 生物胺 氨基甲酸乙酯 基因 PCR
Abstract
Ethyl Carbamate (EC) and biogenic amines (BAs) are widely found in fermented foods including wine. They decrease the safety of wine,for EC is a carcinogen and excessive BAs cause allergic reaction. So,the producing mechanism,influencing factors and testing methods,also the detection of EC- and BA-producing bacterium in fermented foods were studied. With the development of science and technology,molecular biological techniques play an important role in the identification of EC- and BA-producing bacterium. Screening and utilizing malolactic bacterium strains that do not possess genes related to EC and BA producing in wine brewing can improve the quality and safety.
50Oenococcus oeni (O. oeni) strains selected from China’s major grape and wine region were detected for the genes related to EC and BA: arginine deiminase (arcA),ornithine transcarbamylase (arcB),carbamate kinase (arcC),histidine decarboxylase (hdc),ornithine decarboxylase (odc),tyrosine decarboxylase (tdc),in addition,the effect of ethanol,SO2,arginine and pH on EC forming was observed. The main results are listed as follows:
1. Testing of genes related to ethyl carbamate
Gene arcA,arcB and arcC in O. oeni were amplified by primer sets arcAF/arcAR,arcBF/arcBR and CK5'/CK3',respectively. The results showed that 50 O. oeni strains possess the arcA,arcB and arcC gene after sequencing the amplified single bright band and comparison with the gene sequence in Genebank,indicating all the strains has the potential to produce EC.
The PCR amplification system (20μL) was standard. The reaction conditions were: initial denaturation temperature,95℃,5min;30 cycles,denaturation temperature,95℃,30s;annealing time 30s;extension temperature,72℃,30s;the final extension temperature 72℃,5min.
2. Testing of genes relevant to biogenic amines
Six pairs of primer sets were synthesized in the experiment: Primer sets CL1mod/JV17HC and PHDC1/PHDC2 for hdc gene test,primers sets P1-rev/P2-for and TD2/TD5 for tdc gene,primers sets 3/16 and odcf/odcr for odc gene. After comparing the amplified effects of the six pairs of primers at different annealing temperature,primers CL1mod/JV17HC,P1-rev/P2-for,3/16 were chosen for hdc gene,tdc gene and odc gene detection. The results showed that all the 50 O. oeni strains do not have histidine decarboxylase gene,tyrosine decarboxylase gene and ornithine decarboxylase gene.
The PCR amplification system (20μL) is standard. The reaction conditions were: initial denaturation temperature,95℃,5min;30 cycles,denaturation temperature,95℃,30s;annealing time 30s;extension temperature,72℃,30s;the final extension temperature 72℃,5min.
3. Effect of influencing factors on EC forming
The effect of alcohol,arginine,pH and SO2 on EC production was studied in the model wine via solid-liquid extraction and GC-MS method. The results showed that increased alcohol content promoted the formation of EC,also at the low concentration of arginine,but the formation was inhibited at high concentration of arginine;while EC yield did not change significantly at the experimental pH and SO2 levels.
Key words Oenococcus oeni Biogenic amine Ethyl carbamate Gene PCR
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