Gwenola GOUESBETa,b, Gwenaël JANa, Patrick BOYAVAL a*
a Laboratoire de Recherches de Technologie Laitière, INRA, 65 rue de St-Brieuc,35042 Rennes Cedex, France
b Rhodia-Food, BP 10, Z.A. de Buxières, 86220 Dangé St-Romain, France
Abstract — Lactobacillus delbrueckii ssp.bulgaricusis a lactic acid bacterium widely used in thedairy food industry. Since the industrial processes are a succession of constraints, it is essential to under-stand the behaviour of L. bulgaricus when facing usual stresses. The influence of heat stress was inves-tigated on the viability of L. bulgaricuscells grown in a chemically defined medium. The suscepti-bility of cells to heat-shock was obvious only above 55 °C. We investigated the acquisition ofthermotolerance as a result of exposure to a moderate heat-shock, and the acquisition of a cross-stress-tolerance by exposure to a mild osmotic stress. When cells were submitted, before lethal tem-perature challenge (65 °C), to a heat pre-treatment at 50 °C or to a hyper-osmotic pre-treatment, theviability of cells increased. For the industrial strain RD 546, the addition of glycine betaine (GB) in0.4 mol.L–1 NaCl during the pre-treatment decreased the acquired thermotolerance, while GB aloneenhanced cell viability. The thermotolerance of the type strain was not influenced by GB. We demon-strated that the stress tolerance induced by a moderate heat-shock was dependent on protein syn-thesis, while the effect of GB on RD 546 thermotolerance was independent of such biosynthesis.Thermotolerance acquired in presence of GB depends on a strain-dependant mechanism that dif-fers from the mechanism involved after a moderate heat-shock.
Résumé — Stress thermique et thermotolérance chezLactobacillus delbrueckiissp. bulgaricus.Lactobacillus delbrueckiissp. bulgaricus est une bactérie lactique largement utilisée en industriealimentaire. Les procédés industriels étant une succession de contraintes, il est essentiel de connaîtrele comportement de L. bulgaricusface aux stress rencontrés. L’influence du stress thermique sur laviabilité de 2 souches de L. bulgaricus,cultivées en milieu chimiquement défini, a été étudiée. La via-bilité des cellules n’est affectée qu’au-delà de 55 °C. Les cellules acquièrent une thermotolérance vis-à-vis d’un choc thermique à 65 °C durant 10 min, c’est-à-dire une viabilité accrue, après expositionà un prétraitement thermique modéré à 50 °C ou un prétraitement hyperosmotique. Pour la
Lactobacillus delbrueckii ssp.bulgari-cus is a widely used lactic acid bacteriumin the dairy food industry, especially inyoghurt manufacturing. The knowledge ofphysiological adaptation by L. bulgaricusto different stresses is essential for the under-standing of bacterial behaviour when fac-ing dairy processes and during starter elab-oration. One of the industrial processes thatcauses a large loss of viability is bacterialconservation, by freezing, freeze-drying orby spray-drying. During these processes,bacteria are subjected to adverse conditions,one of the most encountered and drasticstresses being the heat stress. The best-described effects induced by high tempera-tures concern the protein denaturation [28],but membranes, nucleic acids and certainenzymes have been equally identified ascellular sites of heat injury [25]. Heat stressis also responsible for a disturbance of thetransmembrane proton gradient, leading toa decrease of the intracellular pH [21, 22,29]. Most of the studies about heat stress inGram-positive bacteria describe the syn-thesis of a protein family (HSP), a phe-nomenon attributed to a universal responseafter a heat-shock [11]. The HSP, mainlychaperones and proteases, are responsiblefor refolding or degrading. The expression ofthe corresponding genes is positively or neg-atively controlled at the transcriptional level.The positive regulation is based on the tran-
scription modulation by alternative sigmafactors, whereas the negative regulationrequires the intervention of repressor depen-dent mechanisms. For Bacillus subtilis, twomajor regulation systems are used for theheat-shock response [11, 18]. One implies arepressor encoded by hrcAacting on a con-served motif (CIRCE) in some thermoin-ducible genes promoters as found in groEand dnaKoperons. Nevertheless many heat-shock genes are positively controlled inB. subtilisby a general stress σ factor, σB.The σB factor is involved in the inductionof heat-shock response by different stim-uli [31].
Thermotolerance represents the abilityof treated cells to exhibit increased survivalafter a severe heat-shock [19]. The heat-inducible thermotolerance allows bacteria,after a non-lethal heat-shock, to tolerate asecond heat stress higher in intensity [2].Teixeira et al. [24] demonstrated that Lac-tobacillus bulgaricusNCFB1489 shows aninducible thermotolerance when pre-treatedat 52 °C before being stressed at 64 °C.
Cells’ survival to a determined stress canbe improved by pre-treatment of cells by amild shock (heterologous pre-treatment) likelow pH, moderate temperature, oxidativeagent, UV irradiation or moderate osmolar-ity [4, 8, 10, 20, 30]. The beneficial effect ofa mild osmotic pre-treatment on thermotol-erance reported for several bacteria has beenpoorly correlated to the known osmoregu-lation phenomena. On the other hand, the
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souche industrielle RD 546, l’addition de glycine bétaïne (GB) dans le milieu de prétraitementcontenant 0,4 mol.L–1 NaCl diminue la thermotolérance acquise, tandis que la glycine bétaïne seuleen tant que prétraitement avant le choc létal augmente la viabilité cellulaire. La thermotoléranceinduite par un prétraitement thermique modéré est dépendante de la synthèse protéique, tandis quel’effet de la GB sur la thermotolérance est indépendant de biosynthèse. La thermotolérance acquiseen présence de GB nécessite un mécanisme souche-dépendant distinct par rapport à celui impliquélors d’un prétraitement thermique.
Cells were harvested in exponential orstationary phase by centrifugation (16 000 g,10 min; Biofuge 15, Heraeus, Sepatech,3360 Osterode, Germany) at room temper-ature. The pellet was resuspended in MPLmedium at room temperature to reach anOD570of 1 unit, which corresponds to a pop-ulation of 2 × 108 CFU.mL–1. Then 200 µLaliquots were transferred into a water bath atthe challenge temperature for 10 min. Atintervals indicated, samples were immedi-ately diluted in a 1 g.L–1 peptone, 8.5 g.L–1
NaCl, pH 7.0 solution at room temperatureand poured in duplicate on MRSA plates.The viable cell numbers in these sampleswere determined after 24 h of an anaerobicincubation at 37 °C. The data is expressed asthe percent of colony-forming ability afterexposure to the challenge temperature, withthe colony-forming units at 0 min set to100%.
2.3. Adaptation conditions:pre-treatment
Pre-treatments were performed on expo-nential cells resuspended at OD570of 1 unitin MPL medium. Cells were exposed to agiven stress agent in non-lethal conditionsbefore exposure to a heat challenge. Differ-ent pre-treatments (adaptation conditions)were applied for 30 min. Cells were incu-bated at several temperatures (50 °C, on ice)or at room temperature in MPL media con-taining different mild stressing agents(0.4 mol.L–1 NaCl, 0.4 mol.L–1 NaCl plus10 mmol.L–1 GB, 0.6 mol.L–1 trehalose,0.66 mol.L–1 glycine betaine, 0.48 mol.L–1
sucrose or 0.64 mol.L–1 glycerol). Thensamples were submitted to the heat chal-lenge, 65 °C for 10 min. Viable cell numberwas determined on MRSA plates.
For experiments testing the effect of theinhibition of protein synthesis on ther-moadaptation, chloramphenicol (Cm) wasused at 10µg.mL–1. Exponential cells were
effect of the well-known osmoprotectantglycine betaine (GB) on bacterial growthunder high osmolarity has been widelydescribed in numerous bacteria [5]. Thegrowth of many osmotically stressed bacte-ria is protected by less than 1 mmol.L–1 GB.
We describe in this study the physiolog-ical effect of heat stress and the inductionof a thermotolerance by pre-exposure to dif-ferent mild stresses on L. bulgaricuscells.Cells harvested during stationary phase havethe higher thermotolerance. If cells are pre-treated at 50 °C or with 0.4 mol.L–1 NaCl, anacquired thermotolerance was observed. Wetested the incidence of a mild osmotic pre-treatment coupled with the presence of GB.An effect on viability after a heat challengewas observed for only one strain. Relation-ships between osmotic and heat stress inL. bulgaricusare discussed.
2. MATERIALS AND METHODS
2.1. Bacterial strains and media
The Lactobacillus delbrueckiissp. bul-garicus type strain ATTC11842 and thestrain RD 546 isolated from industrialyoghurt were used. The rich medium wasMRS (Difco Laboratories, Detroit, Michi-gan, USA) [6], and defined medium was theminimal medium MPL [16] with 5 g.L–1
D-glucose as carbon source. Liquid cultureswere performed at 37 °C without stirring,MRS agar (MRSA) plates were incubatedanaerobically (Anaerocult® A, Merck, 64271Darmstadt, Germany) at 37 °C. Cells fromsingle colonies were inoculated into MRSmedium and grown for 16 h at 37 °C. Theywere then subcultured at 1:50 (v/v) dilutioninto MPL medium supplemented with5 g.L–1 D-glucose and incubated for 10 hat 37 °C without stirring. This MPL pre-culture was used to inoculate MPL work-ing cultures. Growth of cell cultures wasdetermined by OD570measurements (modelDU 7400 spectrophotometer, Beckman,93220 Gagny, France).
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Figure 2.Effect of heat pre-treatment on L. bul-garicusthermotolerance. Exponential cells werecentrifuged, resuspended in MPL medium andincubated 10 min at 65 °C without pre-treatment(■), or after an incubation at 50 °C for 30 min(h), or after 30 min on ice ( ). Each point is theaverage of at least five independent experiments.Standard deviation is shown by bars.
G. Gouesbet et al.
resuspended at OD570 of 1 unit in MPLmedium containing 10µg.mL–1 Cm andwere incubated at room temperature for20 min before pre-treatment and challengeat 65 °C for 10 min.
3. RESULTS
3.1. Effect of heat-shockon the survival of Lactobacillusdelbrueckiissp. bulgaricus
The survival of the two L. bulgaricusstrains, the ATTC11842 and RD 546 strains,from cultures grown to exponential phase,was tested at increasing temperatures. Asshown in Figure 1, viability of cells was notaltered for heat challenge between 37 °C to55 °C. The strains became heat sensitive fora challenge temperature above 55 °C. Thepercentage of survivors dropped sharply fora 10 min heat-shock at temperatures above
55 °C. A temperature of 65 °C for 10 minwas then chosen as the fixed challenge con-dition. Compared with exponential-phasecells, stationary-phase cells of L. bulgari-cuswere significantly more resistant to heatat 65 °C for 10 min (results not shown). Inan attempt to study thermoadaptation in ahomogenous context, log phase cells (OD570below 0.6) were used for the measurementof the thermotolerance in subsequent exper-iments.
3.2. Induction of a thermotoleranceby a heat pre-treatment
In this study, we investigated the adaptiveheat-shock response of L. bulgaricusstrains.The non-lethal temperature selected for thepre-treatment was 50 °C as L. bulgaricusstrains were not affected by a heat challengeat this temperature (Fig. 1).
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Figure 1. Effect of increasing temperatures onL. bulgaricussurvival in MPL medium. Expo-nential cells were centrifuged, resuspended inMPL medium and incubated 10 min at indicatedtemperature. ATTC11842 (n); RD 546 (●). Eachpoint is the average of at least five independentexperiments. Standard deviation is shown bybars.
L. bulgaricus and heat stress
sucrose, showed a weak protection towardsheat stress compared to the NaCl effect(Fig. 3). The thermotolerance was increasedcompared to the control by a three-fold anda four-fold factor for trehalose and sucrose,respectively. For the RD 546 strain, theeffect of 0.66 mol.L–1 trehalose was com-parable to a pre-treatment in 0.4 mol.L–1
NaCl, while 0.48 mol.L–1 sucrose was lessefficient on thermoprotection. However, thepre-treatment in sucrose did give rise to atwo log increase in RD 546 survival com-pared to the control.
These results show that thermoprotec-tion of L. bulgaricuscan be achieved byionic and non-ionic pre-treatments, thatinduce a moderate increase of osmolarity.Then we tested the effect of a permeant
Figure 2 shows the influence of heat pre-treatment on L. bulgaricussurvival after aheat-shock challenge at 65 °C for 10 min.30 min incubation at 50 °C resulted inincreased viability of tested strains.ATTC11842 showed a ten-fold increase ofthermotolerance compared to viability undernon-adapted conditions. The RD 546 strain,extremely heat sensitive without pre-treat-ment, exhibited a three-log increase of via-bility.
A common lab. practice is to keep bac-terial samples on ice until experimentation,as cooled samples are thought to stay at theoriginal physiological state. The influence ofice incubation as a pre-treatment onATTC11842 thermotolerance after a heatchallenge was minor, but resulted in a reduc-tion of cells’ viability (Fig. 2). The effectwas obvious for the strain RD 546 as thepre-treatment at reduced temperature causeda survival reduction of 1 log after heat chal-lenge. The effect of ice incubation onviability of the control cells (unstressedcells) was weak, but deleterious, for theATTC11842 strain. For the strain RD 546,the ice pre-treatment led to a loss of 30%of viability of control cells in absence ofchallenge (results not shown).
3.3. Effect of osmotic pre-treatmenton thermotolerance
We tested the effect of several osmotica,used at concentrations generating thesame osmotic strength as 0.4 mol.L–1 NaCl(1 050 mOs.kg–1 H2O), on L. bulgaricusthermotolerance (Fig. 3). After pre-treat-ment of 30 min in 0.4 mol.L–1 NaCl, cellswere more resistant to a heat challenge(65 °C, 10 min). The effect of this salt pre-treatment was then compared to the effect ofnon-electrolyte osmotica (0.66 mol.L–1 tre-halose, 0.48 mol.L–1 sucrose) in order todifferentiate salt response from osmoticresponse. The type strain ATTC11842,when pre-treated for 30 min in presence of0.66 mol.L–1 trehalose or 0.48 mol.L–1
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Figure 3. Effect of osmotic pre-treatment onL. bulgaricus survival submitted to a challenge at65 °C. Exponential cells were centrifuged, resus-pended in MPL medium and incubated 10 min at65 °C without pre-treatment (■), or after incu-bation in 0.4 mol.L–1 NaCl (h), in 0.66 mol.L–1
trehalose ( ), in 0.48 mol.L–1 sucrose ( ) orin 0.64 mol.L–1 glycerol ( ). Each point is theaverage of at least five independent experiments.Standard deviation is shown by bars.
G. Gouesbet et al.
solute, glycerol on L. bulgaricusgrowth.The solute used at 0.64 mol.L–1 inducedonly a slight decrease in growth rate andwas not used as a carbon source (results notshown). Treatment in 0.64 mol.L–1 glycerolon L. bulgaricusthermotolerance prior to aheat challenge at 65 °C (Fig. 3) had no effecton RD 546 and ATTC11842 viability.
3.4. Effect of glycine betaineon L. bulgaricusthermotolerance
Effects of the osmoprotectant, GB, onthermotolerance was tested at 10 mmol.L–1
added to 0.4 mol.L–1 NaCl and alone at
0.6 mol.L–1 (concentration for which itdevelops an osmolarity equivalent to0.4 mol.L–1 NaCl) (Fig. 4). For the RD 546strain, GB partially reversed the thermotol-erance induced by the medium osmolarity.On the other hand, the solute had no influ-ence on ATTC11842 NaCl-induced ther-motolerance. When the osmoprotectant wasused as an osmoticum at 0.6 mol.L–1, itinduced thermoprotection for RD 546, butwas inefficient on ATTC11842 thermotol-erance. Thus, the universal osmoprotectantGB clearly triggers different responses inthe two strains of L. bulgaricus.
3.5. Influence of protein synthesison thermotolerance
We investigated the involvement of pro-tein neosynthesis on the thermotoleranceresponse of strain RD 546; this strain exhib-ited the greater response. Addition of chlo-ramphenicol (10 µg.mL–1) caused a growtharrest by protein synthesis inhibition. Cellswere incubated in the presence of chloram-phenicol 20 min before pre-treatment andheat challenge. When chloramphenicol-treated RD 546 cells were submitted to amild heat pre-treatment before the heat chal-lenge, the acquired thermotolerance wasabolished (Fig. 5). However when chlo-ramphenicol treated cells were pre-treatedby 10 mmol.L–1GB for 30 min, the acquiredthermotolerance observed after the heat chal-lenge is preserved, allowing cells to reachthe same viability as measured in proteinsynthesis-permissive conditions.
4. DISCUSSION
This study investigated the heat-shockresponse and cross-protection in two strainsof L. bulgaricus. We used a chemicallydefined medium for culture and challengemedia. Previous studies used the richmedium MRS. Such a medium contains lotsof nutriments and solutes, of which the
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Figure 4. Effect of glycine betaine on L. bul-garicussurvival after a heat challenge. Expo-nential cells were centrifuged, resuspended inMPL medium and incubated 10 min at 65 °Cwithout pre-treatment (■), or after incubation in0.4 mol.L–1 NaCl (h), in 0.4 mol.L–1 NaCl +10 mmol.L–1 GB ( ), in 0.6 mol.L–1 GB ( )or in 10 mmol.L–1 GB ( ). Each point is theaverage of at least five independent experiments.Standard deviation is shown by bars.
L. bulgaricus and heat stress
This effect of growth phase on cell resis-tance has been reported for several micro-organisms after environmental stressesincluding high temperature, oxidising agents,osmotic shock and acid pH [10, 12, 13, 17].To minimise thermotolerance of L. bulgar-icusrelated to growth stage, all experimentswere subsequently performed on exponen-tial-phase cells.
A heat pre-treatment of L. bulgaricuscells grown in MRS has been reported toimprove the resistance of cells submitted toa lethal temperature challenge [24]. The heat-inducible thermotolerance is a widespreadphenomenon already described in severalmicro-organisms [19]. The enhancedcapability of bacteria to survive after expo-sure to a lethal temperature has been asso-ciated with the production of heat-shockproteins [15]. Indeed, we demonstrated inthis study that acquired thermotolerance inL. bulgaricusdisappeared when protein syn-thesis was blocked. We also showed thatincubation on ice can be deleterious for cellsurvival. Our results indicate that puttingcultures on ice could be a cause of artefact inexperiments.
Adaptation of cells by a moderate saltpre-treatment is known to increase the resis-tance of cells to a sudden exposure to hightemperature challenge [7, 8, 26], althoughthe mechanism by which this occurs isunclear. The thermotolerance was stimu-lated by the osmotic pressure for the twoL. bulgaricusstrains, with a slighter pro-tective effect for the sucrose in the case ofthe RD 546 strain. It has been demonstratedthat sugar and salt did not induce the samecellular response. Indeed, Glaasker et al. [9]have shown that sucrose-stressed and NaCl-stressed Lactobacillus plantarumcells didnot accumulate the same compounds inresponse to osmotic stress. Sugar-stressedcells contain sugar and sugar-derived com-pounds while salt-stressed cells do not. Inthe case of L. bulgaricusstrains, the decreasein turgor pressure produced by the additionof sucrose or NaCl should be compared interms of cytoplasmic accumulation.
effects on cell adaptation response areunknown. A chemically defined mediumallowed us to determine conditions and totest the real effect of added solutes on ther-motolerance. The use of the MPL mediumexplained, at least partly, the fluctuationsobserved between published results andours.
We observed a beneficial effect of sta-tionary phase on thermotolerance of cellsgrown in chemically defined medium.Teixeira et al. [24] have already demon-strated that Lactobacillus bulgaricusNCFB1489, when grown in the rich mediumMRS, shows a better thermotolerance instationary phase than in exponential phase.
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Figure 5. Effect of blocked protein synthesis onL. bulgaricusRD 546 acquired thermotolerance.Exponential cells were centrifuged, resuspendedin MPL medium and incubated 20 min at roomtemperature in presence of 10 µg.mL–1 chlo-ramphenicol and then submitted to a heat chal-lenge of 10 min at 65 °C without pre-treatment(■), after an incubation at 50 °C for 30 min (h),after an incubation at 50 °C for 30 min in pres-ence of 10 mmol.L–1 GB ( ), after incubationin 10 mmol.L–1 GB ( ). Each point is the aver-age of at least five independent experiments.Standard deviation is shown by bars.
G. Gouesbet et al.
Owing to its diffusion behaviour, theglycerol does not modify the intracellularpressure, it limits the cell volume decreaseusually observed during an osmotic shock[23]. As behaviour of the two L. bulgaricusstrains was not modified in presence of glyc-erol in response to heat-shock, we concludedthat the acquired thermotolerance induced byosmotica was a consequence of the increasedosmolarity rather than a reduction of wateractivity.
GB had an influence on thermoprotec-tion only for the RD 546 strain, while, whenadded in presence of NaCl, GB induced adecrease of the acquired thermotolerance.In Salmonella typhimurium,addition of1 mmol.L–1 GB reversed completely theability of NaCl to enhance viability at hightemperature [8]. From these authors, thethermotolerance can be quantified by twotypes of data: growth rate or survival. Theydemonstrated that these two phenomena arecontrolled by two different regulatory mech-anisms. Indeed, while GB acted on NaCl-induced thermotolerance, the osmoprotectantdid not have any influence on growth rate inthe presence of NaCl at high temperature.In conclusion, GB can act independently ondifferent mechanism(s) in cells. Its effecton thermoprotection in an osmotic contextcan be explained through its osmoprotec-tive properties by which GB decrease thedeleterious effect of osmotic stress and sodecrease the osmotic signal, inducing a cel-lular response of thermoprotection. Caldaset al. [3] have demonstrated that thermo-protection of Escherichia coliis enhanced byGB when cells were stressed at 42 °C.Although a physiological overlap betweenosmotolerance and thermotolerance has beendemonstrated [14, 27], the effect of theosmoprotectant is not clear. From Caldaset al.’s [3] hypothesis, GB could act as achemical chaperone at an intracellular con-centration as low as 50 mmol.L–1. GB alsoacts as a thermoprotectant in plants, indeedthe tolerance of Arabidopsisto high tem-peratures is enhanced by increasing the syn-thesis of GB [1]. It thus appears that GB isinvolved in thermoprotection through a
mechanism controlling survival of cells fac-ing heat stress, although no mechanism ofaction has so far been proposed. Here wedemonstrate that the effect of GB on ther-motolerance may not require protein syn-thesis. Moreover our results show that theeffect of GB on thermoprotection is strain-dependant, suggesting that an extensiveand comparative study of ATTC11842 andRD 546 strains would give us some cluesabout the implicated mechanism.
To summarise, mild starvation, or a read-ily performed heat pre-treatment are sim-ple conditions that can be used to obtainbetter survival of L. bulgaricuscells uponheat-shock, as occurs during industrial pro-cesses. Osmotic pre-treatment is also effi-cient but the presence of salt in the mediumcould be a problem during the preservationprocess and/or subsequent cell uses. As thisspecies is rather delicate, such pre-treat-ments could be used during starter elabora-tion to improve survival rate. We demon-strated in this study that thermotoleranceacquired after a mild heat-shock dependson protein synthesis. The characterisationof the implicated proteins is in progressusing whole cell protein 2-D electrophore-sis separation and mass spectrometry anal-ysis. Identification of the mechanismsinvolved in thermoprotection will provideuseful information leading to a better controlof bacterial behaviour when facing dairyprocesses and during starter elaboration.
ACKNOWLEDGEMENTS
This work was supported by grants from theMinistère de l’Éducation Nationale, del’Enseignement Supérieur et de la Recherche inthe programme Aliment Demain. We thankE. Maguin for providing the MPL medium com-position.
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