Acid Tolerance Of Lactobaillus Brevis Ncl912 And Its Differentially Expressed Protein Under Acid Strese

Lactic acid bacterial (LAB) are important food microorganisms that have been used in food production for thousands years. LAB can improve food nutritions, flavors, textures and preservation, and are also of value in maintaining and promoting human health. Lactobacilli are Gram-positive rods belonging to the group of lactic acid bacteria. In recent years, the industrial applications of Lactobacilli as starters, producers, and probiotics have greatly increased. However, the growth of Lactobacilli is characterized by the production of organic acids, which accumulate and lead to a reduction of pH in its growth environment. As the probiotics, these bacteria also encounter a transient acidic environment in the stomach after consumption. Acid stress greatly affects the growth and bioactivities of Lactobacilli. Therefore, they must possess the acid tolerance ability and be able to induce a series of acid stress responses to survive and grow in acidic environments prior to performing their health benefits. It would greatly drive the appilication of lactic acid bacterial in food industry when the acid tolerance ability of Lactobacilli could be enhanced. The proteomics studies on lactic acid bacterial under acid stress may not only provide a better understanding the changes of the protein expressed in acid stress response, but comprehending the correlative genes and proteins under stress which will obtain the further insight into acid stress response mechanism. The results are as follows:1. The growth and survival of Lactobacillus brevis NCL912, a high y-aminobutyric acid (GABA)-yielding bacterium isolated from fermented vegetables, were investigated at different acidic pH levels. The results show that L. brevis NCL912 grows at pH 3.0-6.4, and the optimal growth pH is 5.0. L. brevis NCL912 could be survive for 4-6 h at pH 2.0.2. The growth, survival, GAB A yield and glutamate decarboxylase activity of L. brevis NCL912 in the media with or without the addition of sodium L-glutamate were compared. The strain attained higher cell concentrations in the media with L-MSG than that without L-MSG in the same growth period. The addition of sodium L-glutamate significantly enhances acid resistance and glutamate decarboxylase activity (p<0.05). Sodium L-glutamate is an important factor for the growth and survival of L. brevis NCL912 at different acidic pHs. It was implied that the acid stress response of L. brevis NCL912 was related to glutamate decarboxylase-GABA antiporter system. 3. Acid stress responses involve a very comprehensive network system of genes and proteins. Therefore, the differentially expressed proteins of L. brevis NCL912 under acid stress were investigated at the global proteomic level by using the two-dimensional gel electrophoresis and mass spectrum. The results showed that 25 proteins were differentially expressed under acid stress. Among them, eight protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, of which 7 were up-regulated and 1 was down-regulated. The function of down-regulated protein was unknown and the putative functions of up-regulated proteins were categorized as stress response proteins, DNA repair, protein synthesis and glycolysis. Real-time flurescent quantitative PCR was used to further validate these differentially expressed proteins at the mRNA level and a positive correlation between the content of the proteins and their mRNA levels was found.4. The differential protein expression of L. brevis NCL912 in the media with sodiun L-glutamate under acid condition were investegated by 2-dimensional gel electrophoresis and mass spectrometry. Real-time flurescent quantitative PCR was used to estimate the expression of selected proteins at the mRNA level. The results showed that 26 proteins were differentially expressed under acid stress. Among them, eleven protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, of which 10 were up-regulated and 1 was under-shifted. According to the putative functions, the up-regulated proteins were categorized as stress response proteins and the other related to protein synthesis and glycolysis which were related to acid stress induction. Another cluster of cell membrane formation-related proteins and cell communication-related proteins might be induced by sodium L-glutamate. Real-time flurescent quantitative PCR data revealed a positive correlation between the content of the proteins and their mRNA levels.5. The proteins and their functions of L. brevis NCL912 in the media with or without sodium L-glutamate were compared and analyzed. The analysis of bioinformatics showed that L. brevis NCL912 might launch the common acid resistance systems such as the protein synthesis, glycometabolism and stress proteins to protect the cell against acid stress which is irrelevant to the sodium L-glutamate. However, the exogenous amino acid (sodium L-glutamate) might also induce the proteins which were involved in the cell membrane formation and signal transporter. It was implied that the acid resistance-responsive system is a very complex network system which is not only related to the exogenous amino acid, but also the expression of the cell proteins.