Regulation Mechanism Of Glutathione On Stress Resistance Of Lactic Acid Bacteria

Glutathione (GSH) is an important non-protein sulfhydryl compound with multiple functions such as anti-oxidation, scavenging free radicals, detoxification and enhancing immunity. So far, it has been widely used as a key functional factor in food industry. In this study, Lactobacillus sanfranciscensis DSM20451T and Lactococcus lactis ssp. cremoris SK11 which are important starters in the fermentation of food industry and Lactococcus lactis ssp. Lactic NZ9000 which is a valuable host bacteria were chosen to investigate the physiological regulation of GSH on lactic acid bacteria under cold and acid stresses. Furthermore, this paper presents the physiological state of lactic acid bacteria during cold and acid treatments and the effect of GSH on the physiological activity and structural morphology of cells. The possible mechanisms of the protective role of GSH were also investigated. The main results of this dissertation were described as follows:1. L. sanfranciscensis DSM20451T is incapable to biosynthesize GSH but capable to uptake it from the ambient, and the intracellular GSH concentration enhanced with the increasing amounts and time of exogenous addition. When the addition of GSH in MRS was 4.8 mM, the intracellular GSH concentration was 20.03±0.07 (nmol·mg protein-1), and the uptake of GSH was close to be saturated then.2. L. sanfranciscensis DSM20451T cells withdrawn from various phases of preculture showed different cold resistance, and the survival of later-stationary cells (36 h in preculture) was higher than others. Meanwhile, the protective role of GSH was most significant at this phase, the survival of L. sanfranciscensis DSM20451T(GSH+) cells precultured to this phase was 8.43 and 6.40 fold higher than (GSH-) cells, respectively after a cold treatment at 4°C or -20°C for 30 d. After exposed at 4°C or -20°C for 15 d, the lag phase of L. sanfranciscensis DSM20451T(GSH+) cells in reculture broth shortened 13.5 h and 12 h than that of (GSH-) cells, respectively.3. GSH does not disturb the growth and metabolism of L. sanfranciscensis cells under normal conditions. However, it prevents the activity decrease of certain key enzymes in metabolism such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase(PK) and lactate dehydrogenase (LDH) under cold stress. Meanwhile, the intracellular ATP level of L. sanfranciscensis DSM20451T(GSH+) cells was significant higher than (GSH-) cells, after a cold treatment at -20°C for 20 d, the ATP concentration of L. sanfranciscensis DSM20451T(GSH+) and (GSH-) cells was 6.4% and 1.9% of their initial values, respectively. In addition, as a vital factor for cells, the intracellular pH (pHi) of L. sanfranciscensis DSM20451T(GSH-) cells decreased remarkably, but the pHi of L. sanfranciscensis DSM20451T(GSH+) cells with same cold treatment was higher than the initial. The results above showed that the regulation of GSH on cell metabolic activity, energy level and intracellular pH homeostasis play important role in improving the cold resistance of cells.When recultured after cold treatment, the activities of key metabolic enzymes in L. sanfranciscensis DSM20451T (GSH-) cells did not change during the first 18 h of lag phase, while the PK and GAPDH activities of (GSH+) cells increased 3.17- and 1.66-fold. Meanwhile, the intracellular GSH concentration of L. sanfranciscensis DSM20451T(GSH+) cells recovered during the reculture process, and reached 78.8% of the value before cold treatment 18 h later. The relationship between the change of intracellular GSH and enzyme activities suggested that the protection of GSH to these enzymes may related to"S-glutathiolation".4. Under cold stress, GSH remained the cell membrane of L. sanfranciscensis DSM20451T intact and smooth, and increased the proportion of unsaturated fatty acids in membrane. The ratio of unsaturated and saturated fatty acids (U/S) of L. sanfranciscensis DSM20451T(GSH+) and (GSH-) cells was similar before cold treatment, while the difference increased under cold stress and was most significant with a treatment at 4°C for 14 d, and the U/S of L. sanfranciscensis DSM20451T(GSH-) cells was only 0.41 fold of (GSH+) cells then.5. The protection of GSH on cell membrane against cryodamage is concerned with preventing membrane fatty acids from oxidation and protecting certain key enzymes on membrane. The redox status of L. sanfranciscensis DSM20451T cells changed significantly during the cold treatment: SOD activity increased initially but started to decease 7 d later. When exposed to cold conditions, [NAD+]/[NADH] decreased and the intracellular sulfhydryl level decreased continuously. The changes of these indexes suggested that the oxidative stress was triggered off during the cold treatment. In this process, GSH prevented the oxidation of membrane unsaturated fatty acids such as linoleic acid, thus maintained the fluidity of cell membrane. Furthermore, different from the sharp decline of Na+,K+-ATPase activity of L. sanfranciscensis DSM20451T(GSH-) cells, the (GSH+) cells sustained 75.8% and 82.1% of initial activity after a treatment at 4°C and -20°C for 30 d, respectively, indicating that GSH protects the Na+,K+-ATPase which regulates the membrane permeability and proton gradient.6. With the 2-DE electrophoresis and flight mass spectrometry technology, differences of protein spots before and after cold treatment were observed, and no significant effect of GSH on cell physiological status was found under normal conditions. The total and different protein spots of L. sanfranciscensis DSM20451T (GSH+) and (GSH-) cells were more than those before cold treatment, suggesting that the protein expression changed under cold stress. Further study revealed that GSH regulated proteins participated in metabolism (e. g. beta-phosphoglucomutase and phosphotransacetylase), stress adaption (e. g. UspA family, beta-phosphoglucomutase and phosphotransacetylase), anti-oxidation (e. g. glutathione peroxidase) and DNA replication, protein transcription and translation, thus improved the cold resistance of L. sanfranciscensis DSM20451T cells.7. GSH protects L. sanfranciscensis DSM20451T, Lactococcus lactis ssp. cremoris SK11 and Lactococcus lactis ssp. Lactic NZ9000 derivation against acid stress, and the most significant protection appeared after cells were precultured to later-stationary phase. The survival of L. sanfranciscensis DSM20451T(GSH+) cells was 6.14 fold of (GSH-) cells when treated at pH4.0 for 10 h. After exposed at pH2.5 for 30 min and at pH4.0 for 10 h, the survival of SK11(GSH+) cells was 17.2- and 15.9-fold higher than SK11(GSH-). Similarly, the protective role of GSH in NZ9000 derivation was also remarkable, the survival of NZ9000(pNZ3203) was 15 fold higher than the control NZ9000(pNZ8148) when treated at pH2.5 for 30 min, and 18 fold higher than NZ9000(pNZ8148) at pH4.0 for 10 h. The mechanism that GSH improves the acid resistance of cells was performed mainly by preventing the pHi decline and protecting the activity of GAPDH. In addition, the ways how exogenous and intracellular biosynthesized GSH work were different. Under acid stress, GSH in SK11(GSH+) cells consumed sharply and remained only 7.6% at pH4.0 for 4 h, seemed like a"suicide"mold. However, the GSH concentration in NZ9000(pNZ3203) changed slightly, and it seemed that GSH can protect cells against acid stress with its low consumption.