To help cells to better resist the stressful conditions associated with the freeze-drying process during starter production, we investigated the effect of various osmotic conditions on growth, survival and acidification activity of Lactobacillus buchneri R1102, after freeze-drying and during storage for 3 months at 25°C.
Methods and Results
High survival rates during freeze-drying, but not during storage, were obtained when 0·1 mol l−1 KCl was added at the beginning of fermentation, without any change in membrane properties and betaine accumulation. This condition made it possible to maintain a high acidification rate throughout the process. In contrast, the addition of 0·6 mol l−1 KCl concentrations at the beginning of fermentation led to a high survival rate during storage that was related to high intracellular betaine levels, low membrane fluidity and high cycC19:0 concentrations. However, these modifications induced the degradation of acidification activity during storage. When a moderate stress was applied by combining 0·1 mol l−1 KCl at the beginning and 0·6 mol l−1 KCl at the end of fermentation, betaine accumulated in the cells without any membrane alteration, allowing them to maintain high acidification activity and survival rate during storage.
Specific osmotic conditions during fermentation induced intracellular betaine accumulation and modifications of membrane character-istics, thus affecting stress resistance of Lact. buchneri R1102. A slight osmotic stress made it possible to maintain a high acidification activity, whereas a high osmotic stress at the end of fermentation led to the preservation of cell survival during freeze-dried storage.
Significance and Impact of the Study
This study revealed that the survival and preservation of acidification activity of freeze-dried Lact. buchneri R1102 during starter production can be improved by using appropriate osmotic conditions.
Freeze-drying is frequently used to preserve the viability and technological properties of lactic acid and probiotic bacteria at the industrial scale (Béal et al. 2001a). This process makes it possible to obtain stable products at chilling temperature, thus reducing the costs of storage and transportation (Carvalho et al. 2004). Nevertheless, viability and activity losses occur during the freeze-drying process, as well as during long-term storage (Béal et al. 2001a; Zamora et al. 2006; Coulibaly et al. 2010).
Pre-adaptation of lactic acid bacteria consisting of submitting the cells to a moderate stress before the true stress is an efficient approach to improve their resistance to freeze-drying and long-term storage. It has been demonstrated that conducting the fermentation of Lactobacillus rhamnosus at a suboptimal pH (Saarela et al. 2009) or applying heat shock or cold shock treatments to Lactococcus lactis (Broadbent and Lin 1999) before freeze-drying helped to improve the stability of the bacteria. The positive effect of a mild heat stress on Lactobacillus coryniformis was confirmed by Schoug et al. (2008). The positive effect on resistance to drying of an osmotic shock (0·6 mol l−1 KCl) applied at the mid-log phase of Lact. rhamnosus was observed by Prasad et al. (2003) and was confirmed with other lactobacilli and bifidobacteria after air-drying (De Angelis and Gobbetti 2004; Champagne et al. 2012). Survival of Lactobacillus delbrueckii subsp. lactis to freeze-drying was improved up to 200-fold by applying an osmotic shock with 0·6 mol l−1 NaCl to growing cells (Koch et al. 2007).
This phenomenon is ascribed to different physiological changes. Cells are first able to increase the synthesis of ABC transport-related proteins (Prasad et al. 2003), which allows the internalization of osmoprotectant organic compounds known as ‘compatible solutes’ (Obis et al. 1999), once they are added to the culture medium. The osmoregulated ABC-transport system of Lact. lactis was shown to sense osmotic stress via changes in the physical state of the membrane, membrane properties and protein–lipid interactions (Van der Heide and Poolman 2000). These compatible solutes help balance the osmotic pressure to maintain the turgor pressure necessary for cell elongation and preserve the protein functions inside the cells (Romeo et al. 2001). Secondly, osmotic shocks act on the fatty acid composition of the membrane of Lact. lactis by changing the cycC19:0 content (Guillot et al. 2000). They also increase the degree of saturation of membrane fatty acids of Lactobacillus bulgaricus, thus modifying cellular permeability (Tymczyszyn et al. 2005). Finally, stress proteins (GroEL, DnaK), glycolytic enzymes, phosphocarrier protein HPr and ABC transport-related proteins are upper-synthesized in Lact. rhamnosus as a response to hypertonic conditions (Prasad et al. 2003). These authors also reported higher levels of carbohydrates in the cytoplasm after osmotic shock, which was confirmed by Tymczyszyn et al. (2005).
Despite these physiological changes, application of osmotic stress results in lower growth rates and biomass yields (Koch et al. 2007), which limits its use in industrial plants. Consequently, there is a need for further studies about the effect of osmotic stress on bacterial resistance to freeze-drying, while maintaining high performance during fermentation and storage. In addition, no information is available on starter production of Lactobacillus buchneri. This species ensures the heterolactic fermentation process and increases the aerobic stability of the silage (Hu et al. 2009; Reich and Kung 2010).
On the basis of these considerations, this work aims at studying the influence of various osmotic conditions on the growth, survival and acidification activity of Lact. buchneri R1102 after freeze-drying and storage of the starters for 3 months at 25°C. To explain the behaviour of the cells as a consequence of osmotic stress, intracellular betaine accumulation, membrane fatty acid composition and membrane fluidity were determined. These biological characteristics were linked to the resistance of the bacteria to freeze-drying and storage using a statistical approach.
Materials and methods
Bacterial strain and media
Frozen aliquots of Lact. buchneri R1102 (Lallemand SAS, Blagnac, France) were stored at −80°C in MRS broth (Biokar Diagnostics, Beauvais, France) supplemented with glycerol (28%). They were subcultured twice in MRS broth at 37°C for 24 h. Inoculation was carried out at an initial concentration of 2 × 107 CFU ml−1. For starter production, the culture medium was composed of MRS (Biokar, Beauvais, France) to which 50 g l−1 glucose (Merck, Darmstadt, Germany) and 2 mmol l−1 betaine (Aldrich, Steinheim, Germany) were added. It was sterilized in the fermentor at 115°C for 15 min.
Fermentation and cooling
Cultures were performed in a 3·6-l fermenter (Labfors 4, Infors, Massy, France) with 2·0 l final working volume. Fermentations were performed at 37°C with pH controlled at 6·2 by adding 16% NH4OH solution. The fermenter was flushed with air (1·2 vvm) and stirred at 120 rpm. Conductivity measurements were used to characterize fermentation kinetics and to stop the fermentations. Different osmotic conditions were carried out by adding 0·1 mol l−1 or 0·6 mol l−1 KCl at the beginning or at the end of fermentation, as detailed in the ‘Experimental design’ section and in Table 1.
Table 1. KCl concentrations and corresponding values of aw measured at the beginning and at the end of the fermentations of Lactobacillus buchneri R1102
KCl at the beginning of fermentation (M)
KCl at the end of fermentation (M)
Mean of two independent experiments.
Values are the means of at least two measurements (± standard deviation).
When a conductivity variation of 21·8 ± 2·8 mS cm−1 was obtained, cells were harvested and concentrated by centrifugation (13 000 g, 15 min, 8°C). Concentrated cells were washed twice in sterile physiological water (NaCl, 9 g l−1) and aliquots of 1 g were frozen at −80°C before determination of membrane fatty acids and intracellular betaine concentrations, and analysis of membrane fluidity. Concentrated cells were cryoprotected with a protective medium containing 8% sucrose (w/w) and 5% sodium glutamate (w/w). The mixtures were frozen at −45°C before lyophilization in the freeze-drier (Alpha 1-4 LSC, Christ, Fisher Scientific, Illkirch, France). Freeze-drying was performed for 42 h at 10 Pa for sublimation, and at 20°C and 3 Pa for secondary drying. Freeze-dried samples were stored for 3 months at 25°C before analysis. One gram of freeze-dried starter culture was rehydrated in 100 ml peptone water for 10 min at 25°C before use. All these treatments corresponded to a concentration factor of 51 ± 11 between rehydrated cells and cells obtained at the end of fermentation.
Measurement of water activity
The water activity of fermentation media was determined before fermentation (initial aw) and at the end of fermentation (final aw). It was measured at 25°C using a LabMaster-aw water activity meter (Novasina, Precisa, Poissy, France).
Quantification of betaine accumulation
Before analysis of the intracellular betaine concentration, protein concentration was measured using the colorimetric method (Bradford 1976) with bovine serum albumin as a standard. Cells were then submitted to a hypo-osmotic shock in order to allow them to release the accumulated betaine in the medium (Obis et al. 1999). Washed cell pellets were resuspended in a solution containing 3 mmol l−1 HCl (20 min, 25°C). Cells were removed by centrifugation (20 000 g, 5 min, 20°C), and the betaine concentration of cell-free extracts was determined by HPLC measurements (Waters Corp., Milford, MA). A prepurification of the sample was performed by micro-solid-phase extraction on C18 micro tips (OMIX, C18 100 μl, Varian, Inc., Palo Alto, CA). Betaine was recovered in the nonbound fraction, which was loaded onto a strong cation exchanger column (Chrompack 1000, 50 × 4·6 mm, Varian, Inc). Isocratic elution was performed in 4 mmol l−1 HCl solution at 40°C, at a flow rate of 0·4 ml min−1. Eluted compounds were detected by UV detection at 195 nm (Photo Diode Array Detector 2996, Waters Corp). Quantification of betaine was obtained by external standard calibration.
Cultivability was measured by plate counts on MRS agar (AES-Chemunex, Combourg, France). The plates were incubated for 48 h at 37°C under aerobic conditions. The results were the average values of at least three counts (in CFU ml−1).
Acidification activity measurements
The Cinac system (Ysebaert, Frépillon, France) was used to quantify the loss of acidification activity of Lact. buchneri R1102 after freeze-drying and after 3 months of storage at 25°C (Fonseca et al. 2000). Acidification was performed in triplicate in MRS broth (Biokar Diagnostics) at 37°C. To standardize the measurements, one hundred microlitres of fresh culture diluted to 1/4 or of rehydrated cell suspensions diluted to 1/50 were introduced into 150-ml Erlenmeyer flasks containing 120 ml MRS broth. From a technological perspective, this measurement allows discrimination of the samples on the ground of their global acidification activity that depends upon both physiological state of the cells and cell concentration of the samples. For each sample, the time necessary to reach pH 5·5 (tpH5·5, in min) was used to characterize the acidification activity of the bacterial suspensions. The higher the tpH5·5 was, the longer the latency phase was and, consequently, the lower the acidification activity was.
Fatty acid analysis
The relative fatty acid composition of bacterial membranes was determined by gas chromatography as described by Béal et al. (2001b). Cell pellets were washed three times in 0·05 mol l−1 Tris buffer, pH 8·8. Methylation and extraction were simultaneously performed at 4°C by adding 1·5 ml sodium methoxide (1 mol l−1 in methanol) (Sigma-Aldrich, Saint-Quentin-Fallavier, France), shaking for 2 min and adding 1 ml hexane (Sigma-Aldrich). After the addition of undecanoic acid methyl ester (0·1 mg ml−1 in hexane; Sigma-Aldrich) as the internal standard, and decantation for 5 min, the upper phase was removed and stored at −80°C in an airtight glass bottle until analysis. The analyses were performed on a gas chromatograph (HP 6890, Hewlett Packard, Avondale, PA) equipped with a capillary column packed with 70% cyanopropyl polysilphenylene-siloxane (BPX 70, 60 m*0·25 mm; SGE, Ringwood, Vic., Australia). Helium was used as the carrier gas (1·2 ml min−1) and the injection volume was 2 μl. Injection was carried out splitless for 2 min. The oven temperature was raised from 65 to 230°C at 5°C min−1 and maintained for 10 min at 230°C. Injection and detection temperatures were 230°C. The fatty acid methyl esters were identified by using a mass selective detector (Agilent 5973, Hewlett Packard) at a scan rate of 273 scan s−1. The electron impact energy was set at 70 eV and data were collected in the range of 30–400 atomic mass units. The mass spectra were compared with the NBS75K and WILEY 275.L data banks (Hewlett Packard). The identity of the fatty acid methyl esters was confirmed by comparing their retention times with those of known standards (BAME and FAME, Supelco, Bellefonte, PA). Results were expressed as relative percentages of each fatty acid, which were calculated as the ratio of the surface area of the considered peak to the total area of all peaks. These values led to the determination of the ratios of unsaturated-to-saturated fatty acids (U/S) and cyclic-to-saturated fatty acids (C/S). Analy-ses were carried out in quadruplicate using two inde-pendent extractions, from one culture sample for each osmotic condition, except for the reference fermentation whose extractions were duplicated from two independent cultures samples.
Determination of membrane fluidity
Fluorescence anisotropy was assessed to characterize the membrane fluidity of the cells using a spectrofluoro-meter (Fluorolog-3, Horiba Jobin Yvon, Longjumeau, France) in a T format (Cao-Hoang et al. 2008). Cell pellets were washed three times (5000 g, 5 min, 25°C) in 50 mmol l−1 MES buffer (2-[N-morpholino] ethanesulfonic acid)-KOH, 10 mmol l−1 glucose, pH 5). They were resuspended in the same buffer to reach an optical density of 0·6 at 600 nm. Fluorescence anisotropy was measured by using hydrophobic 1,6-diphenyl-1,3,5-hexatriene (DPH) (Molecular Probes, Eugene, OR). Two milliliters of cell suspension was added with 4 μl of 1·5 mmol l−1 DPH solution (in tetrahydrofuran; Sigma-Aldrich), and measurements were performed in the dark at 37°C for at least 10 min. Excitation and emission wavelengths were set at 340 and 431 nm, respectively, and the steady-state anisotropy (r) was calculated according to Lakowicz (1988). Calculated anisotropy values were the means of at least five assays.
Different osmotic conditions were studied by adding KCl to the fermentation medium at concentrations of 0·1 or 0·6 mol l−1. They were applied either at the beginning of fermentation, i.e., throughout the culture, or at the end of fermentation for 45 min, as summarized in Table 1. To evaluate the variability, the reference fermentation without the addition of KCl was carried out in duplicate. Each fermentation condition made it possible to quantify three types of physiological responses of Lact. buchneri R1102: the growth kinetics that were indirectly evaluated by NH4OH consumption, and the survival and the loss of acidification activity of the starters, which were measured after freeze-drying and during three months of storage at 25°C. The membrane fatty acid composition and the membrane fluidity were determined at the moment the cells were recovered to explain the physiological responses observed under the different osmotic conditions.
The comparisons of bacterial counts, acidification activities, betaine contents and membrane fatty acid compositions were achieved by using analysis of variance with Statbox™ v6.7 (Grimmersoft, Neuilly-sur-Seine, France). Multiple comparison tests were carried out according to Newman-Keuls tests. When P values were lower than 0·05, differences were considered as being statistically significant. Because measurement conditions differed between the end of fermentation, on the one hand, and after freeze-drying and rehydration, on the other, multiple comparison tests were carried out separately on these two sets of measurements.
Principal component analysis (Statbox™ v6.7) was carried out to explore links between variables such as membrane fatty acid relative contents, membrane fluidity, loss of acidification activity and cell survival during freeze-drying and storage, as well as similarities between starter samples produced under different osmotic conditions.
Influence of osmotic conditions on fermentation kinetics
Fermentation kinetics of Lact. buchneri R1102 cultivated under different osmotic conditions were compared according to the variations of electrical conductivity in the culture medium. Electrical conductivity is the consequence of the total concentration of ammonium lactate and KCl in the culture medium. It therefore differed at the beginning of the different fermentation conditions. The initial conductivity was significantly higher in the culture media prepared with 0·1 mol l−1 KCl (21·1 mS cm−1) and 0·6 mol l−1 KCl (78 mS cm−1) than without the addition of KCl (6·9 ± 0·9 mS cm−1). Figure 1 shows the variations in conductivity during the fermentations of Lact. buchneri R1102, which were calculated on the basis of differences with initial conductivity.
By considering the standard deviation calculated from the two control fermentations, no significant difference was observed between conditions C, T0.1i, T0.6f and T0.1i0.6f. This was confirmed by calculating the time necessary to achieve a conductivity variation of 15 mS cm−1, which was equal to 11·8 ± 0·1 h for the two control cultures, and to 12·3 ± 0·7 h for conditions T0.1i, T0.6f and T0.1i0.6f. The statistical comparison of means provided a P value of 0·218, indicating that the difference was not significant. In contrast, adding 0·6 mol l−1 KCl at the beginning of fermentation (trial T0.6i) strongly affected the fermentation kinetics as 19·3 h were necessary to reach 15 mS cm−1 instead of 11·8 h. In addition to conductivity variations during growth, the addition of 0·6 mol l−1 KCl at the end of fermentation (T0.6f and T0.1i0.6f) led to a strong increase in final conductivity that reached 102 ± 2 mS cm−1. These values were close to the final value observed at the end of the fermentation trial T0.6i (100 mS), which was conducted at high initial conductivity. Consequently, the addition of 0·1 mol l−1 KCl in the culture medium did not affect the fermentation kinetics, whereas supplementation with 0·6 mol l−1 KCl induced strong osmotic stress.
Influence of osmotic conditions on intracellular betaine accumulation
High osmotic conditions allowed exogenous betaine to accumulate in intracellular medium. Figure 2 shows that betaine accumulation differed according to the osmotic conditions. A similar concentration of accumulated betaine (53·5 ± 6·9 nmol mg−1) was detected in cells grown without KCl and in the presence of 0·1 mol l−1 KCl during growth (trials C and T0.1i). In contrast, when 0·6 mol l−1 KCl was added, either at the beginning or at the end of fermentation (T0.6i, T0.6f and T0.1i0.6f), intracellular accumulated betaine was 1·9–2·5 times higher than that of the previous trials (P <1%). However, sample T0.1i0.6f displayed lower intracellular betaine content (101·2 ± 5·8 nmol mg−1) compared to starter T0.6f (136·3 ± 1·3 nmol mg−1) (P <1%). This result indicated that a strong osmotic shock was necessary to allow high betaine accumulation, in contrast to a more progressive shock.
Effect of osmotic conditions on membrane fatty acid composition and fluidity
The relative concentrations of membrane fatty acids of Lact. buchneri R1102 were determined by considering the cells recovered at the end of the fermentation process, including osmotic stress, just before stabilization. On the basis of gas chromatography analysis, a total of seven main different fatty acids representing more than 99% of the total fatty acid content were observed in the membrane of Lact. buchneri R1102, regardless of the experimental conditions. The relative percentages of these fatty acids were calculated (Table 2). Three fatty acids represented 87–88% of total fatty acid composition: palmitic acid (C16:0), oleic acid (C18:1) and lactobacillic acid (cycC19:0). Saturated fatty acid content differed slightly among the samples (42·6–45·4%), whereas unsaturated (31·9–39·6%) and cyclic (15·8–25·3%) fatty acid concentrations displayed greater changes.
Table 2. Relative membrane fatty acid composition and fluorescence anisotropy of Lactobacillus buchneri R1102 membranes, as a function of osmotic conditions
C: Control without the addition of KCl; T0.1i: 0·1 mol l−1 KCl at the beginning of the fermentation; T0.6i: 0·6 mol l−1 KCl at the beginning of the fermentation; T0.6f: 0·6 mol l−1 KCl at the end of the fermentation; T0.1i0.6f: 0·1 mol l−1 KCl at the beginning of the fermentation and 0·6 mol l−1 KCl at the end of the fermentation.
Values are the means of four measurements (± standard deviation).
As seen in Table 2, significant differences between the mean values of these variables according to osmotic conditions were evaluated using analysis of variance (anova). On the basis of these analyses, the relative contents in C12:0, C14:0 and C16:1 did not significantly change according to osmotic conditions (P >5%). The C16:0 relative content was significantly lower (P <5%) for sample T6·0i. The C18:1 (P <0·1%) and cycC19:0 (P <1%) relative concentrations were lower and higher for samples T0.6i and T0.6f, respectively. Sample T0.1i0.6f displayed an intermediate composition between these two samples and the reference ones. Finally, the C18:0 content was slightly but significantly higher (P <0·1%) in lactobacilli grown in KCl containing medium than in the control condition. These results showed that the addition of a low KCl concentration (0·1 mol l−1) in the medium did not affect the membrane fatty acid composition, except for a slight increase in the percentage of C18:0. On the contrary, when 0·6 mol l−1 KCl was added to the culture medium either at the beginning (T0.6i) or at the end of fermentation (T0.6f, T0.1i0.6f), the C18:1 percentage decreased and the cycC19:1 relative concentration increased. This phenomenon was less obvious when 0·1 mol l−1 KCl was first added to the medium (T0.1i0.6f). Finally, a significant decrease in the C16:0 proportion was observed when 0·6 mol l−1 KCl was added at the beginning of fermentation.
Membrane fluidity of Lact. buchneri R1102, which was indirectly characterized by fluorescence anisotropy, varied according to osmotic conditions. As can be seen in Table 2, the values of anisotropy were not significantly different for samples C, T0.1i, T0.6f and T0.1i0.6f. On the contrary, fluorescence anisotropy was significantly higher when considering sample T0.6i that was obtained by adding a high KCl concentration (0·6 mol l−1) at the beginning of fermentation. On the basis of these results, it was concluded that membrane fluidity was affected only when KCl was added at the beginning of fermentation and at a high concentration. On the contrary, when it was added at the end of fermentation or at a lower concentration, membrane fluidity was not affected.
Influence of osmotic conditions on bacterial concentrations and survival
To assess the effect of osmotic stress on the survival of Lact. buchneri R1102 after freeze-drying and during storage, bacterial concentrations were determined by cultivability measurements at the end of fermentation, after freeze-drying and after 3 months of storage at 25°C. Statistical comparison of means was carried out by separately considering the values obtained before freeze-drying and those obtained after, in order to better highlight the differences. As seen in Table 3, the cultivability of Lact. buchneri R1102 at the end of fermentation (1·7 ± 0·3 109 CFU ml−1) was not significantly different among the four experiments conducted with low initial KCl concentrations (0 and 0·1 mol l−1 KCl). In contrast, the cultivability of the fermentation trial performed with 0·6 mol l−1 KCl (T0.6i) was 4·2 times lower than that of the previous ones.
Table 3. CFU counts measured at the end of the fermentation (Xtr), after freeze-drying (Xt0) and after three months (Xt3) of storage at 25°C of Lactobacillus buchneri R1102 starters produced under different osmotic conditions
Cell cultivability (CFU ml−1)
Mean of two independent experiments.
C: Control without the addition of KCl; T0.1i: 0·1 mol l−1 KCl at the beginning of the fermentation; T0.6i: 0·6 mol l−1 KCl at the beginning of the fermentation; T0.6f: 0·6 mol l−1 KCl at the end of the fermentation; T0.1i0.6f: 0·1 mol l−1 KCl at the beginning of the fermentation and 0·6 mol l−1 KCl at the end of the fermentation.
Values are the means of three independent measurements (± standard deviation).
Multiple comparison tests were carried out separately for samples at the end of the fermentation (a–b) and samples after freeze-drying and storage (c–f). Superscript letters show difference at P <0·05.
After freeze-drying, the bacterial concentration increased as a consequence of centrifugation, freezing and dehydration. As can be seen in Table 3, it reached 7·0 ± 0·9 1010 CFU g−1 for five experiments, but was 3·5 times lower for experiment T0.6i. On the basis of these results, the survival during freeze-drying was calculated as the ratio between the CFU counts obtained after freeze-drying to those obtained at the end of fermentation. This ratio was equal to 69% for sample T0.1i and to 49 ± 6% for the four other experiments. This result indicates that a moderate osmotic stress slightly improved the survival of Lact. buchneri R1102 during freeze-drying.
During storage of lyophilized starters at 25°C for 3 months, the CFU counts slightly decreased, regardless of the osmotic conditions. The final CFU counts that distinguished experiment T0.6i remained 3·3 times lower than those of the other ones. The stability of the starters during storage was calculated as the ratio between the CFU counts obtained after 3 months of storage at 25°C to those obtained after freeze-drying. This ratio was equal to 65 ± 2% for fermentations done without KCl during culture (C and T0.6f) or with low KCl concentrations (T0.1i) added at the beginning of fermentation, whereas it was equal to 87 ± 2% for the experiments conducted with high KCl concentrations added at the beginning (T0.6i) or at the end of fermentation with an addition of 0·1 mol l−1 KCl at the beginning of fermentation (T0.1i0.6f). These results showed that high osmotic stress during culture led to a better survival of Lact. buchneri R1102 during storage at 25°C of freeze-dried starters.
Influence of osmotic conditions on acidification activity
Acidification activity measurements were used to compare the ability of Lact. buchneri R1102 to restart acidification after growth under different osmotic conditions. Acidification activity was assessed as the time necessary to reach pH 5·5 (tpH5·5, in min) after freeze-drying and after 3 months of storage at 25°C. Statistical comparison of means was carried out by separately considering the values obtained before freeze-drying and those obtained after, in order to better highlight the differences.
As can be seen on Fig. 3, acidification activity of starters obtained at the end of fermentation, after freeze-drying and after 3 months of storage at 25°C, differed under the different osmotic conditions. Starters produced in the control conditions or in the presence of 0·1 mol l−1 KCl could not be distinguished on the basis of their acidification activity, either at the end of fermentation (421 ± 18 min), after freeze-drying (542 ± 18 min) or after 3 months of storage (559 ± 8 min). In contrast, samples obtained after the addition of 0·6 mol l−1 KCl, either at the end (T0.6f and T0.1i0.6f) or at the beginning (T0.6i) of fermentation, displayed lower acidification activity, as shown by the elevated tpH5·5 values that were comprised between 500 and 710 min after fermentation, between 610 and 840 min after freeze-drying, and between 625 and 1280 min after 3 months of storage. These results pointed out that a strong osmotic stress led to a degradation of the acidification activity, whereas a moderate osmotic stress helped to maintain this functionality.
Figure 3 shows that tpH5·5 increased at each step of the production, but differently according to the osmotic conditions. The loss of acidification activity during the stabilization process, including concentration, freezing and freeze-drying, was determined as the difference between the tpH5·5 measured at the beginning of storage and at the end of fermentation. It was equal to 120 ± 16 min for samples T0.1i, T0.6i, T0.6f and T0.1i0.6f, compared to 177 ± 22 min for the two control samples. The loss in acidification activity during storage was quantified by calculating the difference between acidification activities measured after 3 months at 25°C and at the beginning of storage. It was equal to 60 min and 440 min for experiments T0.6f and T0.6i, respectively. These values were clearly higher than those obtained with the other samples (15 ± 9 min). This result indicates that acidification activity significantly decreased during the storage of these two samples, which were submitted to a strong osmotic stress, whereas it remained stable under the other osmotic conditions. Finally, combining 0·1 mol l−1 KCl at the beginning of the fermentation and 0·6 mol l−1 KCl at the end of the fermentation (T0.1i0.6f) limited the degradation of the acidification potential of the starter during storage (10 min).
Principal component analysis between membrane characteristics and stability of the starters during freeze-drying and storage
Two principal component analyses (PCA) were performed to establish a link between technological and biological characteristics and to explain the behaviour of the bacteria submitted to different osmotic conditions. These analyses were established to describe the relationship between the main characteristics of the cell membranes (anisotropy and percentages of C16:0, C18:0, C18:1, cycC19:0 fatty acids) and cell survival (Fig. 4a), or the loss of acidification activity (Fig. 4b) of Lact. buchneri R1102 during freeze-drying and storage, as a function of osmotic conditions and intracellular betaine accumulation.
As can be seen in Fig. 4a, the first two dimensions accounted for 78% of the data variance. The F1 axis (49% of the total variation) distinguished the starters according to C18:1 and cycC19:0 relative contents, betaine concentration (Bet) and membrane anisotropy (Aniso). The F2 axis (29% of the total variation) differentiated the starters according to the percentages of C16:0 and C18:0, and to cell survival during freeze-drying and storage (FdSurv and StoSurv). As seen on Fig. 4b, the first two dimensions accounted for 89% of the data variance. The F1 axis (57% of the total variation) distinguished the starters according to C18:1 and cycC19:0 relative contents, betaine concentration (Bet), membrane anisotropy (Aniso) and loss of acidification activity during 3 months of storage (StoLoss). The F2 axis (32% of the total variation) differentiated the starters according to the percentage of C16:0 and C18:0 and to the loss of acidification activity (FdLoss) during freeze-drying.
Using both of the principal component analyses, the first two axes made it possible to separate the five starters into three similar groups. The first group included the starter T0.6i that was located in the lower right side of the graph. It was characterized by a high loss of acidification activity and a high survival rate during storage, which were associated with high membrane anisotropy, together with high intracellular betaine and cycC19:0 contents, but low C16:0 and C18:1 relative concentrations. The second group contained the reference samples (C1 and C2) and was located in the lower left part of the graph. It showed a high loss of acidification activity during freeze-drying and a low survival rate during freeze-drying, which were linked to low C18:0, cycC19:0 and intracellular betaine contents. The third group, located in the upper part of the graph, was composed of three starters (T0.1i, T0.1i0.6f and T0.6f) that displayed a slight loss of acidification activity during freeze-drying, a high survival rate during freeze-drying (mainly for the T0.1i sample) and a low survival rate during storage. These characteristics were associated with high C16:0 and C18:0 contents but different betaine contents.
Relationships between growth of Lact. buchneri R1102, osmotic conditions and betaine accumulation
Bacterial growth of Lact. buchneri R1102 was affected by osmotic conditions. Longer culture time and lower final bacterial concentration were achieved when 0·6 mol l−1 KCl was added at the beginning of the fermentation (T0.6i). This extended time interval could be ascribed to the high initial conductivity (78 mS cm−1) and to the lower water activity value in this medium (Table 1). At low aw, less free water is available for bacterial growth, thus leading to a reduced specific growth rate and final biomass concentration. At lower KCl concentrations, water activity was not significantly affected, which explained why the growth curves were not disturbed. This result is in agreement with that of Champagne et al. (2012) obtained with Lact. rhamnosus. Nevertheless, the difference observed between condition T0.6i and the others remained slight because fermentation time was only 3 h longer and cell concentration was four times lower than that of the reference conditions. This result could be explained by the partial relief of KCl inhibition by betaine that accumulated in the intracellular medium as a result of strong osmolality (Schoug-Bergenholtz et al. 2012). A stimulatory effect of betaine on growth was observed with Lact. lactis submitted to an osmotic stress with NaCl (Guillot et al. 2000); moreover, some authors have previously associated the ability to grow in high osmolality medium with a stronger betaine transport activity (O'Callaghan and Condon 2000; Obis et al. 2001). This observation is consistent with the various levels of intracellular betaine accumulated by Lact. buchneri R1102 obtained under different osmotic conditions.
Relationships between membrane characteristics of Lact. buchneri R1102, osmotic conditions and betaine accumulation
Osmotic pressure in the culture medium strongly influenced the fatty acid composition of the bacterial membrane and, to a lesser extent, its fluidity. A significant increase in fluorescent anisotropy of DPH was observed when the cells were exposed to hypertonic conditions, for example, when a high KCl concentration (0·6 mol l−1) was added at the beginning of the fermentation (T0.6i). Similar observations have been reported in Bacillus subtilis cells cultured under hypersaline conditions (Lopez et al. 2000), in Escherichia coli (Beney et al. 2004) and Saccharomyces cerevisiae (Laroche et al. 2001) cells submitted to high osmotic pressure, but never in lactic acid bacteria.
The fatty acid relative content of the membrane of Lact. buchneri R1102 was also affected by the osmotic conditions. The largest variation was observed when the cells were exposed to high osmotic conditions at the beginning of fermentation (T0.6i) or at the end of fermentation (T0.6f). High osmolality mainly led to a cyclization of C18:1 into cycC19:0, thus reducing the unsaturated-to-saturated ratio (0·76 ± 0·01 instead of 0·86 ± 0·04 for the other conditions) and enhancing the cyclic-to-saturated ratio (0·53 ± 0·06 compared to 0·39 ± 0·02 for the other conditions). In condition T0.6i, a reduction of saturated fatty acid content in the membranes was also demonstrated. Such high relative cyclopropane fatty acid (CFA) content has been associated with stimulation of CFA synthase by the addition of glycine-betaine (Monteolivasanchez et al. 1993). These results agree with those of Guillot et al. (2000) and Tymczyszyn et al. (2005), who reported a decrease in the unsaturated-to-saturated ratio in cell membranes of Lact. lactis and Lact. bulgaricus grown in high osmolal medium. Moreover, an increase in cycC19:0 together with a decrease in C18:1 was demonstrated in Lact. lactis cells in the presence of NaCl (Guillot et al. 2000). In contrast, a decrease in the relative content of cycC19:0 was revealed in cells of Lact. bulgaricus grown in high osmolal MRS to which polyethylene glycol was added (Tymczyszyn et al. 2005). These differences indicated that the cell response at the membrane level was dependent on the nature of the stressing agent (salt or sugar) and on the bacterial species.
On the basis of these results and the principal component analyses (Fig. 4), a relationship between membrane fatty acid composition and fluorescence anisotropy of DPH, for example, membrane fluidity level, could be established. High anisotropy, for example, low membrane fluidity, was achieved in membranes characterized by high cycC19:0 and low C18:1, as observed in the membrane of the T0.6i starter. Low C16:0 relative content also affected anisotropy, but to a lesser extent. Some authors have described simultaneous changes in membrane fatty acid composition and fluidity properties (Tymczyszyn et al. 2005; Loffhagen et al. 2007; Mykytczuk et al. 2007), whereas no statistical relationship had been established between them.
In addition, our results revealed that fatty acid composition, membrane fluidity and intracellular betaine concentration were related (Fig. 4). In fact, higher intracellular betaine content was observed in more rigid membranes that contained higher cycC19:0 and lower C18:1, as observed for cells grown under high osmotic conditions for a long time (T0.6i). This relationship can be explained by two complementary mechanisms. Firstly, the lower membrane fluidity made it possible to retain intracellular osmocompatible solutes by reducing exchanges between the intracellular and extracellular compartments. Secondly, changes in membrane properties could modulate the transport of betaine, as previously described in Lact. lactis cells submitted to NaCl osmotic stress (Guillot et al. 2000; Prasad et al. 2003). This activation was attributed to the regulation of the activity of membrane-embedded proteins as a function of membrane properties (Obis et al. 1999).
Relationships between membrane fatty acid composition and fluidity of Lact. buchneri R1102 and survival during freeze-drying and storage, as a function of osmotic conditions and betaine accumulation
Different levels of cell survival were obtained during freeze-drying and storage, according to various osmotic conditions and subsequent intracellular betaine accumulation. As these differences could be partly explained by cell membrane characteristics, principal component analysis made it possible to characterize the relationship between the membrane properties of Lact. buchneri R1102 and cell survival during freeze-drying and storage, as a function of osmotic conditions and intracellular betaine accumulation (Fig. 4a).
On the basis of this analysis, lower survival rates of Lact. buchneri R1102 cells during freeze-drying and storage at 25°C were obtained when their membranes had a low relative C18:0 content, as in the case of the reference samples. In contrast, higher survival rates after freeze-drying were obtained for cells submitted to a moderate stress with 0·1 mol l−1 KCl (T0.1i). This result was related to an increase in C18:0 content together with the preservation of membrane fatty acid composition and fluidity similar to the reference starters (Table 2). Indeed, these biological properties allowed the permeation of water across the cell membrane to occur during osmotic stress, thus lowering its deleterious effect (Tymczyszyn et al. 2005).
Higher survival rates during storage were obtained for starters T0.1i0.6f and T0.6i, compared to the other conditions. This improvement could be partly attributed to high intracellular betaine accumulation for these samples (Fig. 2), and to high cycC19:0 relative concentration for T0.6i (Table 2), which were linked to low membrane fluidity. Some authors have linked the resistance of different lactic acid bacteria to various stresses to the high cycC19:0 content in their membranes (Annous et al. 1999; Wang et al. 2005). Sample T0.6f was close to sample T0.6i when considering cycC19:0, C18:1 and intracellular betaine content, but it displayed lower survival rates during storage. This specific behaviour of sample T0.6f was explained by the strong but short stress at the end of fermentation. This suggests that applying an osmotic stress for a long period of time may allow the cells to adapt themselves to the stress, which was not possible during a short stress, as observed with sample T0.6f. Judging from this observation, another mechanism may be involved, such as changes in protein synthesis (Prasad et al. 2003).
Relatiovnships between membrane fatty acid composition and fluidity of Lact. buchneri R1102 and loss of acidification activity during freeze-drying and storage, as a function of osmotic conditions and betaine accumulation
As a consequence of various osmotic conditions and subsequent betaine accumulation, different levels of acidification activity loss were achieved during freeze-drying and storage. The differences at the physiological level were explained on the basis of principal component analysis, which associated membrane fluidity and fatty acid composition of Lact. buchneri R1102 cells with their loss of acidification activity during freeze-drying and storage (Fig. 4b).
As shown by these analyses, high loss of acidification activity during freeze-drying but low loss of acidification activity during storage at 25°C was achieved when the cell membranes had a low relative C18:0 content, as in the case of the reference samples compared to all the other ones.
When Lact. buchneri R1102 was cultivated in the presence of low KCl concentrations (T0.1i and T0.1i0.6f), the starters were able to maintain good acidification activity after freeze-drying (T0.1i) or during storage (T0.1i0.6f). This low KCl concentration did not modify the membrane fatty acid contents, except for C18:0 concentration, or the membrane fluidity, compared to the reference samples. Consequently, it could be hypothesized that the activity of proteins located inside the membrane was less or not significantly altered by this slight osmotic stress.
In contrast, when cell membranes contained low C16:0 and C18:1, but high cycC19:0 relative concentrations that were linked to high anisotropy and low fluidity, a high loss of acidification activity was observed during storage, as shown by sample T0.6i and, to a lesser extent, by starter T0.6f. The decrease in residual activity after drying was also achieved for Lactobacillus plantarum cells grown with 1 mol l−1 NaCl (Linders et al. 1997). These characteristics could first be linked to ionic perturbations that occurred under high osmotic conditions and, second, to the lower unsaturated-to-saturated fatty acid ratio in these cell membranes, as previously demonstrated in S. thermophilus (Béal et al. 2001b). In addition, the accumulation of high betaine concentrations observed after a strong osmotic stress was either not involved or not sufficiently implicated to counteract these deleterious effects. Finally, our results revealed that strong osmotic stress degraded the metabolic activity of Lact. buchneri R1102, whereas low osmotic stress during fermentation allowed the cells to adapt themselves, thus helping them to maintain their acidification activity.
In conclusion, it was demonstrated that moderate osmotic stresses enhanced the stability of Lact. buchneri R1102 starters, either during their stabilization process or their storage, in terms of cell survival and acidification activity. Maintaining a high acidification activity required the application of a low osmotic stress, whereas a high survival rate during freeze-dried storage was favoured by a high osmotic stress at the end of fermentation. These results were associated with modifications at the membrane level in terms of fatty acid composition and with intracellular betaine accumulation. As protein synthesis may also be modified following osmotic stress, a complementary study is required to characterize these biological responses at the proteomic and the transcriptomic levels. Additionally, as biological mechanisms may differ according to the strain used, our results may be confirmed by including other strains of lactic acid bacteria.