中国优良酒类酒球菌的分离与鉴定

中国优良酒类酒球菌的分离与鉴定

ISOLATION AND IDENTIFICATION OF PREDOMINANT OENOCOCCUS OENI FROM CHINESE WINE

作者：李翠霞　　导师：王　华　李　华

西北农林科技大学　　微生物学2011届硕士

摘　要

为了分离筛选出适合葡萄酒酿造的优良酒类酒球菌（Oenococcus oeni，O. oeni）菌株，用ATB分离培养基从我国新疆、河北、甘肃葡萄酒产区酒精发酵结束的干红葡萄酒中进行酒类酒球菌的分离鉴定，从中筛选出适合中国葡萄酒酿造的优良菌株。主要研究结果如下：

1．　采用ATB分离培养基从新疆、河北、甘肃葡萄酒产区酒精发酵结束的干红葡萄酒中共分离得到49株苹果酸—乳酸细菌，通过对其进行培养特征、形态学特征、苹果酸分解实验、革兰氏染色实验、过氧化氢酶实验、生理生化实验鉴定，结果表明：分离菌株均为酒类酒球菌。

2．　通过对分离纯化的49株菌株进行单因子（pH、酒精、SO₂）耐受性实验，选出单因子抗性较好的14株菌进行复合因子（pH×酒精×SO₂)耐受性实验，结果表明：XJ-2a比对照菌株SD-2a有较强的适应性。XJ-2b、HB-1A、XJ-3a、HB-1B也与对照菌株SD-2a的生长量无明显差别，具有较强的酿酒适应性。

3．　用种属特异性PCR（species-specific PCR）对分离菌株进行分子生物学鉴定。结果表明：14株单因子抗性较好菌株均能扩增出大小为1025bp的唯一清晰条带，说明这14株菌均为酒类酒球菌。

4．　用16S rRNA序列同源性分析对分离菌株进行分子生物学验证。结果表明：14株单因子抗性较好菌株的测序结果与数据库中酒类酒球菌的同源性均达到了99%以上，进一步证明了这14株菌均为酒类酒球菌，并构建了相应的系统进化树。除此之外，通过对14株实验菌株与链球菌科中7个属中模式菌株的16S rRNA基因序列建立相似性矩阵，结果表明：14株实验菌株的基因序列与O. oeni的相似性达到100%，Leuconostoc mesenteroides与O. oeni亲源关系相对较近，相似性达到了83.5%。

5．　优良菌株O.oeni-XJ-2a生长曲线的测定。结果表明：O.oeni-XJ-2a比对照菌株O.oeni-SD-2a最大菌悬液的光密度值（OD₆₀₀）大0.267，有较好的生长特性。

本文通过形态学特征、生理生化鉴定、种属特异性PCR以及16S rRNA序列同源性分析，可将这14株菌鉴定为酒类酒球菌（O. oeni）。

关键词　酒类酒球菌　分离　鉴定　species-specific PCR　16S rRNA

Abstract

In order to screen Oenococcus oeni with good performance for the MLF,The excellent Oenococcus oeni for Chinese wine were isolated and identification from Xinjiang wine,Heibei wine,Gansu wine after Alcohol fermentation with ATB isolation medium. The main results of the experiment as follows:

1.　Forty-nine strains of malolactic bacteria were isolated and purified from Xinjiang wine,Heibei wine,Gansu wine after Alcohol fermentation with ATB isolation medium. According Cultivating characteristics,tomorphological,malic acid decomposition,catalase experiment and physiological biochemical characteristics,they were identified to be O. oeni.

2.　The 49 isolates were tested for their capacity of resistance to the individual stress condition of pH,ethanol%(V/V) and SO₂ content．According to the results，the fourteen selected strains’ growth under the combination of external stress physical conditions of ethanol (V/V)，pH and SO₂ (mg/L) were also tested，the results showed that,in selective strains,the resistance of XJ-2a is stronger than SD-2a,and XJ-2b,HB-1A,XJ-3a,HB-1B have higher applicability in wine producing.

3.　The analysis of species-specific PCR was used to make molecule identify. The result as follows: The fourteen better resistant strains of single factor all have only one clear strips with 1025bp,the results showed that these fourteen strains were O. oeni.

4.　The analysis of 16S rRNA PCR was used to make molecule identify. The results as follows: The results showed that the 16S rRNA gene sequence similarity of fourteen strains and model strain were all above 99 %,comparing with the type strains of O. oeni from GenBank database. It further proved that the fourteen strains were O. oeni,and the homologous phylogenetic tree had been established based on 16S rRNA sequence of O. oeni. Besides,The results of 16S rRNA gene sequences similarity matrix of model strains of 7 Genera in streptococcaceae showed that the similarity of the gene sequences of fourteen strains reaches 100%,and the relationship of Leuconostoc mesenteroides and O. oeni is the closest,the similarity reached 83.5%.

5.　Thedetermination of O.oeni-XJ-2a's growth curve. The results showed that the Optical Density (OD₆₀₀) of O.oeni-XJ-2a's biggest bacterium levitation liquid is more than O.oeni-SD-2a's only 0.267,in other words, O.oeni-XJ-2a have better growth characteristics.

In conclusion,according to tomorphological and physiological biochemical characteristics,which showed that these strains belong to O. oeni;Finally,the species-specific PCR and homology analysis of 16S rRNA sequences also support the results.

Key words　Oenococcus oeni　Isolation　Identification　Species-specific PCR　16S rRNA