Two-Dimensional Electrophoresis Study of Lactobacillus delbrueckii subsp. bulgaricus Thermotolerance

Abstract

The response of Lactobacillus delbrueckii subsp. bulgaricus cells to heat stress was studied by use of a chemically defined medium. Two-dimensional electrophoresis (2-DE) analysis was used to correlate the kinetics of heat shock protein (HSP) induction with cell recovery from heat injury. We demonstrated that enhanced viability, observed after 10 min at 65°C, resulted from the overexpression of HSP and from mechanisms not linked to protein synthesis. In order to analyze the thermoadaptation mechanisms involved, thermoresistant variants were selected. These variants showed enhanced constitutive tolerance toward heat shock. However, contrary to the wild-type strain, these variants were poorly protected after osmotic or heat pretreatments. This result suggests that above a certain threshold, cells reach a maximum level of protection that cannot be easily exceeded. A comparison of protein patterns showed that the variants were able to induce more rapidly their adaptive mechanisms than the original strain. In particular, the variants were able to express constitutively more HSP, leading to the higher level of thermoprotection observed. This is the first report of the study by 2-DE of the heat stress response in L. delbrueckii subsp. bulgaricus.Lactobacillus delbrueckii subsp. bulgaricus is one of the most important industrial lactic acid bacteria. This fact is mainly due to its use in yogurt and fermented milk for human consumption. However, the price of current commercial preparations restricts its use, mainly because of the high susceptibility of L. delbrueckii subsp. bulgaricus to preservation processes. For example, this species would be a very interesting probiotic (40) if the production cost of these sensitive bacteria could be decreased substantially. This goal could be achieved by increasing cell survival or by using a less deleterious production process. Direct spray drying of vegetative microbial cells is emerging (3) as an efficient and inexpensive preservation method with a high output and may lower this cost. Knowledge of the resistance of these bacteria to heat stress would be helpful for mastering cell viability during this dehydration treatment. Teixeira et al. (35) studied L. delbrueckii subsp. bulgaricus thermotolerance and clearly showed the successful application of such a preservation method to concentrated suspensions of these bacteria (34,35). When cells were submitted to a heat pretreatment at 50°C or to a hyperosmotic pretreatment before lethal temperature challenge (65°C), the viability of the cells increased (13). Nevertheless, little is known of potential methods for enhancing cell survival and mechanisms used by the cell to protect itself from heat stress or to repair damage. Two-dimensional electrophoresis (2-DE) of proteins is currently one of the highest-resolution analytical techniques available for the study of protein expression patterns (the promising genome-wide DNA arrays are not yet available for L. delbrueckii subsp. bulgaricus). New technological developments have improved r...

Proteomic Analysis of Global Changes in Protein Expression during Bile Salt Exposure of Bifidobacterium longum NCIMB 8809

“…DnaK and GroEL are induced by bile in Enterococcus faecalis (12,15), by salt stress in Lactococcus lactis (26), by heat stress in Bacillus cereus and Lactobacillus delbrueckii subsp. bulgaricus (17,42), and by acid stress in L. lactis (21).…”

Heat and Osmotic Stress Responses of Probiotic Lactobacillus rhamnosus HN001 (DR20) in Relation to Viability after Drying

“…There are only a few reports describing the physiological stress responses in lactic acid bacteria (LAB), particularly Lactobacillus species (5,22,32,46,48). Further, there is a paucity of information describing the physiological mechanisms underlying the stress-induced improvement in the survival of LAB in relation to industrial processing and storage conditions.…”

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes

“…The resistance to heat stress is higher when the cells were previously exposed and adapted to this type of stress in the stationary phase, otherwise, when pre-adapted in exponential phase, the cells are more sensitive. In particular, the storage stability of the culture that was heat shocked after stationary phase was superior to that of culture heat shocked after log phase [34,23,30].…”

“…In every strain tested the involvement of Heat Shock Proteins (HSPs such as DnaK, GroEL and GroES during the heat stress was clear) [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. The role of these stress proteins is complex; in fact, the bind substrate proteins in a transient non-covalent manner prevent premature folding and promote the attainment to the correct state in vivo.…”

“…have been studied for Lactobacillus delbrueckii subsp. bulgaricus [23] and Lactobacillus paracasei [24,25], Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus helveticus [26], Lactobacillus collinoides [27] , Lactobacillus sakei [28], Lactobacillus johnsonii [29], Lactobacillus rhamnosus [30], Lactobacillus plantarum [31][32][33] and Lactobacillus salivarius [34]. The heat resistance of LAB is a complex process involving proteins with different roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing and control of ribosomal functions [31].…”