产γ－氨基丁酸屎肠球菌的筛选及γ－氨基丁酸的定量

摘要

本试验旨在利用分子生物学方法鉴定分离得到1株产γ－氨基丁酸（ GABA）屎肠球菌，并定量测定所产GABA的量。从泡菜、酸奶、土壤、新鲜牛奶样品中筛选出一目标菌株F6，进行形态学特征与革兰氏染色鉴定；再扩增菌株F6的16S rDNA基因，然后测定该基因序列和构建系统发育树；同时采用高效液相色谱（ HPLC）法定量测定菌株F6发酵液中的GABA含量。结果表明：菌株F6菌落大而光滑、圆形、直径1～2 mm、边缘整齐、乳白色；在分离培养基上菌落周围形成透明圈，使分离培养基呈黄色；在MRS固体培养基上菌落不透明，周围有透明圈。革兰氏染色鉴定菌株F6为阳性菌。分子生物学鉴定分析显示，菌株 F6的16S rDNA 基因序列与GenBank数据库中屎肠球菌（ Enterococcus faecium）的相似性大于99％。 HPLC法测定得到GA⁃BA标准曲线线性方程为Y＝7080733．1395X－4511．6927（R2＝0．9994），通过方程得出菌株F6发酵液中的GABA含量为7．1 g／L，保留时间为7～10 min。结果提示，本试验筛选得到1株高产GABA的屎肠球菌F6。%This study was aimed to isolate and identify an Enterococcus faecium by molecular biological meth⁃ods which produced γ⁃aminobutyric acid ( GABA) , moreover, the amount of GABA was also quantitatively determined. One target strain of Enterococcus faecium F6 was screened out from the samples of pickles, yo⁃gurt, soil and fresh milk, and was identified according the morphological characters and Gram stain methods. After the 16S rDNA gene of bacterial strain F6 was amplified, the gene sequence and phylogenetic tree were analyzed. Meanwhile, the GABA content in the fermented solution of bacterial strain F6 was quantitatively de⁃termined by high performance liquid chromatography ( HPLC) method. The results showed that the bacterial strain F6 took a large, smooth, round and a neat edge of shape with a diameter of 1 to 2 mm and in a milky white color. A transparent circle was formed around the colony on the separating medium which changed to be yellow. On the MRS solid medium, the colony was opaque with a transparent circle around. Gram stain was used to identify F6 as a positive strain. The 16S rDNA gene sequence of bacterial strain F6 was more than 99%similarity to that of Enterococcus faecium in GenBank showed by molecular biological analysis. HPLC method was used to draw out the GABA standard curve linear equation as follows:Y=7 080 733.139 5X-4 511.692 7 ( R2=0.999 4) , and the content of 7.1 g/L of GABA with 7 to 10 minutes of retention time was determined in the fermented solution of F6 according to linear equation. The results indicate that a high yield GABA of En⁃terococcus faecium F6 is finally obtained.