A Study Of Acid Tolerance Of Lactobacillus Plantarum ZDY 2013 And Expression Of Glutamate Dehydrogenase Gene

Lactobacillus plantarum as an important kind of lactobacillus, which was often found in fermend vegetable and fruits, has gain more attention in fields of food, silage ang healthcare. As one of probiotics in human gastrointestinal tract, L. plantarum has to survive from extreme acidic condition or digestion of various enzymes through the whole gastrointestinal tract to play a beneficial function. Thus the acid resistance ability of bacteria is generally accepted as one of the best criteria for selecting potential probiotics.In this dissertation, L. plantarum ZDY 2013 was used as an initial strain,with L. plantarum ATCC 8014 as a control, the physiological responses and metabolic regulations during acid stress were investigated by using microbial physiology technology. Furthermore, the expression vector of glutamate dehydrogenase(GDH) gene from L. plantarum in Escherichia coli was construted to discove the effect of GDEon the acid resistence of the cells. The main results were described as follows:1. The different stress responses between L. plantarum ZDY 2013 and L. plantarum ATCC 8014 during acid stress were compared on the level of micro-environment and cell membrane. Transmission electron microscopy analysis showed that L. plantarum ZDY 2013 maintained a more intact cell membrane during acid stress, which may contribute to decrease the effuse of intracellular materials and influx of deleterious materials, thus alleviating the damage induced by acid stress. Acid stress resulted in the pHi decrease of cells, but compared with L. plantarum ATCC 8014(pHi 5.28), L. plantarum ZDY 2013(pHi 5.66) exhibited higher pHi. And the activity of Na+, K+-ATPase and intracellular ATP pool of L. plantarum ZDY 2013 were higher 35% and 41%, respectively, when compared with L. plantarum ATCC 8014. Moreover, the content of intracellular arginine, glutamate, alanine and histidine were detected higher in L. plantarum ZDY 2013 than that in L. plantarum ATCC 8014 during acid stress. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 were increased by 1.2- and 1.3- fold, respectively, when compared with L. plantarum ATCC 8014 in acid stress.2. Glutamate dehydrogenase gene was amplified by PCR from the genomic DNA of L. plantarum ZDY 2013, and then inserted into the expression plasmid pET-32a(+). The recombinant plasmid was transformed into E. coli BL21(DE3) and the protein was induced with IPTG. The host bacteria containing recombinant plasmid was grown and induced with IPTG. The combined protein was purified using Ni-NTA affinity chromatography and the glutamate dehydrogenase activity was determined. At the same time, the survival rate of cells under acid stress was determined, and the results showed it was increased by 1.4-fold in the host bacteria containing recombinant plasmid at pH 4.5.