酒酒球菌產(chǎn)生物胺和氨基甲酸乙酯相關(guān)基因檢測及影響其產(chǎn)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：趙艷卓　　導師：劉樹文

西北農(nóng)林科技大學　　發(fā)酵工程2011屆碩士

摘　要

氨基甲酸乙酯（Ethyl Carbamate，EC）和生物胺（Biogenic Amines，BAs）廣泛存在于發(fā)酵食品中，葡萄酒中亦有存在。氨基甲酸乙酯是一種致癌物質(zhì)，過量的生物胺會引起過敏反應，二者存在會影響葡萄酒安全性。國內(nèi)外學者對二者在發(fā)酵食品，包括葡萄酒中的產(chǎn)生機理、影響因素、檢測方法，以及產(chǎn)生菌株的檢測等進行了研究。隨著科學技術(shù)的發(fā)展，用分子生物學方法對產(chǎn)生氨基甲酸乙酯和生物胺的菌株進行鑒定，篩選不具有產(chǎn)生氨基甲酸乙酯和生物胺相關(guān)基因的菌株進行葡萄酒釀造，可以提高葡萄酒的質(zhì)量和安全性。

本研究對實驗室從中國主要葡萄與葡萄酒產(chǎn)區(qū)篩選保存的50株酒酒球菌產(chǎn)氨基甲酸乙酯和生物胺的相關(guān)基因——精氨酸脫亞胺酶（arcA）、鳥氨酸轉(zhuǎn)氨甲酰酶（arcB）、氨甲酰激酶（arcC）、組氨酸脫羧酶（hdc）、鳥氨酸脫羧酶（odc）、酪氨酸脫羧酶（tdc）基因進行了檢測，并研究了乙醇、SO₂、精氨酸、pH四因素對氨基甲酸乙酯產(chǎn)生的影響。主要結(jié)果如下：

1.　產(chǎn)氨基甲酸乙酯相關(guān)基因的檢測

采用引物arcAF/arcAR、arcBF/arcBR和CK5’/CK3’分別擴增酒酒球菌的arcA、arcB和arcC基因。結(jié)果顯示，供試的50株酒酒球菌均擴增出單一明亮的目的條帶，測序后在基因庫中進行比對確定是arcA、arcB和arcC基因。說明本研究所用的50株酒酒球菌均具有arcA、arcB和arcC基因，全部具有產(chǎn)生氨基甲酸乙酯的潛力。

PCR擴增體系為標準PCR體系（20μL）。反應條件為：初始變性溫度95℃，時間5min；循環(huán)數(shù)30；變性溫度95℃，時間30s；復性時間30s；延伸溫度72℃，時間30s；最終延伸溫度72℃，時間5min。

2.　產(chǎn)生物胺相關(guān)基因的檢測

本研究中合成了6對引物：引物CL1mod/JV17HC和PHDC1/PHDC2擴增組氨酸脫羧酶基因（hdc），引物P1-rev/P2-for和TD2/TD5擴增酪氨酸脫羧酶基因（tdc），引物3/16和odcf/odcr擴增鳥氨酸脫羧酶基因（odc）。對這6對引物在不同退火溫度下擴增效果進行比較，選擇引物CL1mod/JV17HC、P1-rev/P2-for、3/16進行hdc、tdc和odc基因的檢測。結(jié)果顯示，供試的50株供試酒酒球菌均不具有hdc、tdc和odc基因。

PCR擴增體系為標準PCR體系（20μL）。反應條件為：初始變性溫度95℃，時間5min；循環(huán)數(shù)30；變性溫度95℃，時間30s；復性時間30s；延伸溫度72℃，時間30s；最終延伸溫度72℃，時間5min。

3.　影響氨基甲酸乙酯產(chǎn)生因素的研究

本文采用固液萃取法結(jié)合GC-MS方法研究了酒精度、精氨酸、pH和SO₂這四種因素對模擬酒中氨基甲酸乙酯形成的影響。結(jié)果顯示，提高乙醇含量可以促進氨基甲酸乙酯的形成；低濃度的精氨酸會促進氨基甲酸乙酯的形成，高濃度則抑制其形成；在實驗pH和SO₂水平下，氨基甲酸乙酯含量變化不明顯。

關(guān)鍵詞　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