CHAPITRE 4 : Continuous production of mixed lactic starters containing probiotics using immobilized cell technology

Table des matières

Y. Doleyres, I. Fliss et C. Lacroix

Centre de recherche en sciences et technologie du lait STELA

Université Laval

Québec, Qc, G1K 7P4, Canada

Soumis dans "Biotechnology Progress"

La production d’une culture mixte composée de Lactococcus lactis subsp. lactis biovar. diacetylactis MD et de Bifidobacterium longum ATCC 15707 a été étudiée lors d’une fermentation continue de 17 jours avec cellules immobilisées conduite à différentes températures entre 32 et 37°C. Le système de fermentation à deux étages était composé d’un premier réacteur (R1) contenant des cellules des deux souches immobilisées séparément dans des billes de gel de κ-carraghénane et de gomme de caroube et d’un second réacteur (R2) opéré avec les cellules libres relarguées du premier réacteur. Le système a permis de produire une culture mixte concentrée avec un rapport de souches dont la composition dépendait de la température et du temps de fermentation. Une culture mixte stable (avec un ratio cellulaire de 22:1 L. diacetylactis / B. longum ) a été produite à 35°C dans l’effluent de R2, alors que la culture mixte était rapidement débalancée en faveur de B. longum à plus haute température (37°C) ou de L. diacetylactis à plus basse température (32°C). Une redistribution des souches dans les billes immobilisant à l’origine une culture pure de L. diacetylactis ou de B. longum a également été obervée. À la fin de la fermentation, le ratio cellulaire (7:1 L. diacetylactis / B. longum ) dans des échantillons de plusieurs billes (environ 0.5 g) était similaire à celui de billes individuelles. La détermination de la distribution spatiale des deux souches dans les billes de gel par immunofluorescence couplée à la microscopie confocale a montré que le contamination croisée était limitée à une couche périphérique des billes de 100 μm d’épaisseur. Les données de cette étude valident un précédent modèle de dynamique des populations et de relarguage cellulaire dans des billes de gel lors de fermentations avec cellules immobilisées.

The production of a mixed lactic culture containing Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707 was studied during a 17-day continuous immobilized-cell culture at different temperatures between 32 and 37°C. The two-stage fermentation system was composed of a first reactor (R1) containing cells of the two strains separately immobilized in κ-carrageenan/locust bean gum gel beads and a second reactor (R2) operated with free cells released from the first reactor. The system allowed continuous production of a concentrated mixed culture with a strain ratio whose composition depended on temperature and fermentation time. A stable mixed culture (with a 22:1 ratio of L. diacetylactis and B. longum ) was produced at 35°C in the effluent of R2, whereas the mixed culture was rapidly unbalanced in favour of B. longum at a higher temperature (37°C) or L. diacetylactis at a lower temperature (32°C). Strain redistribution in beads originally immobilizing pure cultures of L. diacetylactis or B. longum was observed. At the end of culture, the strain ratio (7:1 L. diacetylactis/B. longum ) in bulk bead samples was similar to that of individual beads. The determination of the spatial distribution of the two strains in gel beads by immunofluorescence and confocal laser-scanning microscopy showed that bead cross-contamination was limited to a 100 μm peripheral layer. Data from this study validate a previous model for population dynamics and cell release in gel beads during mixed immobilized-cell cultures.

Continuous fermentations with lactic acid bacteria immobilized in polysaccharide gel beads have major advantages over free-cell cultures for the production of biomass, including high productivity and stability, stable strain ratios in mixed cultures, prevention from washing-out, reduction of susceptibility to contamination and bacteriophage attack, enhancement of plasmid stability, and protection of the cells from shear forces in the stirred reactor (Champagne et al., 1994; Lamboley et al., 1997; 1999; 2001; Macedo et al., 1999).

Applications of the immobilized cell technology for the continuous inoculation prefermentation of milk in the production of fresh cheese (Sodini-Gallot et al., 1995; Sodini et al., 1997) or yogurt (Prévost et al., 1985), and for the production of mesophilic lactic starters (Lamboley et al., 1997; 1999; 2001) or probiotic bacteria (Doleyres et al., 2002a,b ; chapters 2, 3) lead to high process productivity and stability, due to the high immobilized cell concentration and cell release activity from beads to bulk medium. However, a complex redistribution of strains among gel beads initially immobilizing a pure culture was observed (Lamboley et al., 1997; 1999; 2001; Sodini et al., 1997). A theoretical model for cell release from cavities at the bead periphery has been recently proposed to explain this cross-contamination phenomenon (Lacroix et al., 1996).

To validate this hypothesis and identify factors of cross-contamination, an immunofluorescent method involving double-color labelling and confocal microscopy has been developed to specifically detect cells of one strain of lactic acid bacteria ( Lactococcus lactis subsp. lactis biovar. diacetylactis ) and one probiotic ( Bifidobacterium longum ) co-immobilized in gel beads (Prioult et al., 2000). Recently, a quantitative analysis for the localization and quantification of microcolonies in beads was developed with the same method (Doleyres et al., 2002a; chapter 3). Repeated-batch cultures with immobilized cells also allowed the production of a mixed culture containing lactic acid bacteria and a non-competitive strain of bifidobacteria.

The objective of the present work was to demonstrate and quantify the cross-contamination in polysaccharide gel beads during continuous production of a model probiotic lactic starter composed of two strains, Bifidobacterium longum ATCC 15707 and Lactococcus lactis subsp. lactis biovar. diacetylactis MD, immobilized separately in gel beads, using a recently developed quantitative immunofluorescence method (Doleyres et al., 2002a; chapter 3). Moreover, the effect of temperature on the production of a balanced mixed culture of lactic acid bacteria and non-competitive cells of bifidobacteria in the fermentation broth was also studied during a 17-day continuous immobilized-cell fermentation.

Batch fermentations were first carried out for bead colonization for 16 h in a stirred tank bioreactor (Bioflo model C30, New Brunswick Scientific Co., Edison, NJ, USA) containing 400 ml supplemented MRS medium and 100 ml of B. longum or L. diacetylactis beads. The bioreactor was controlled at 37°C and purged with CO2 to ensure anaerobic conditions, with mixing set at 120 rpm. The pH was controlled at 6.0 by addition of 6 N NH4OH. Colonized beads were then stored overnight in citrate buffer (0.1 M, pH=5.4) containing KCl 0.3 M before starting the continuous immobilized-cell culture.

A two-stage fermentation system was used for the continuous fermentation. The first reactor (R1) was a flat-bottomed custom-built glass bioreactor (R1) containing 20 ml of each of the two types of colonized gel beads, for a total culture volume of 120 ml. The second reactor of 600 ml (R2, Bioflo model C30, New Brunswick Scientific Co.) was operated in series and inoculated with free cells released from beads in R1. The reactors were adapted for continuous culture with immobilized cells, as described previously (Norton et al., 1994), and CO2 was injected in the headspace of the two reactors to maintain anaerobic conditions during cultures. Flow rate of supplemented MRS medium was set at 240 ml/h during the 17-day culture and pH was controlled at 6.0 by addition of 6 N NH4OH. Temperature in both reactors was first set at 37°C, then decreased to 32°C at day 6, and finally increased to 35°C at day 13 until the end of the experiment. Broth samples were taken daily from the two reactors for cell enumeration and HPLC analysis, whereas bead samples were taken every 3 days for cell enumeration and confocal laser-scanning microscopy analysis.

Bead preparation and confocal microscopy observation using specific fluorescent polyclonal antibodies for the two strains, and quantitative determination of the spatial distribution of cells from the two strains in beads were performed as previously described (Prioult et al., 2002; Doleyres et al., 2002a; chapter 3). Briefly, the biomass volumetric fraction (percentage of space occupation) for each strain as a function of the gel bead radius was derived from the position and surface area of the colonies in the labelled sections. For this determination, bead sections were divided into 2.5 μm thickness annular regions and the number and fraction (%) of fluorescent pixels containing biomass were determined in each region as a function of radius. Each pixel could contain cells from none, one, or the two strains. The sum of biomass occupation for the two strains could therefore be 100% or more in certain parts of the beads. Five beads were used to determine biomass profiles in beads originally immobilizing B. longum or L. diacetylactis after 15 days continuous culture.

Half beads were also stained with fluorescent dyes SYTO9 and propidium iodide for observation of total and dead biomass, respectively (LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes Inc., Eugene, OR, USA). Confocal micrographs were made at an excitation wavelength of 488 nm with a FITC emission filter for SYTO9 and at an excitation wavelength of 543 nm for propidium iodide.

B. longum and L. diacetylactis counts in beads and effluents of R1 and R2 varied with fermentation time and temperature (Figure 4.1). B. longum counts in beads increased during the first 5 days at 37°C to reach 1.5±0.1x1010 CFU/g at day 5, but decreased to 5.5±1.3x109 CFU/g at day 11, after the temperature was changed to 32°C at day 6. A stable B. longum concentration was further observed in beads when temperature was set at 35°C, averaging 9.6±1.6x109 CFU/g from day 14 to day 17. The same tendency was observed for B. longum cell counts in the effluents of R1 and R2. Due to high bead colonization and favourable temperature (37°C) for B. longum growth, cell counts in the effluents of R1 and R2 increased from day 2 to day 6 from 4.3±0.1x108 to 1.1±0.2x109 CFU/ml and from 3.4±0.1x108 to 6.9±0.1x108 CFU/ml, respectively. Then bifidobacteria cell counts decreased when the temperature was changed to 32°C, to 1.9±0.1 and 1.8±0.1x108 CFU/ml in R1 and R2 at day 13, respectively, and remained stable from day 14 to day 17 at 35°C, averaging 3.7±1.1 and 3.4±0.9x108 CFU/ml in R1 and R2, respectively.

L. diacetylactis counts in beads varied in an opposite direction compared to B. longum counts with temperature. A significant decrease was observed with fermentation time at 37°C to a low concentration in beads of 2.8±0.4x109 CFU/g at day 5, followed by a large increase when temperature was set at 32°C, to reach a very high concentration of 6.4±0.6x1010 CFU/g at day 11. Then, L. diacetylactis counts in beads decreased slightly at 35°C to 3.7±0.4x1010 CFU/g at day 17. Even though L. diacetylactis cell counts in the effluents of R1 and to a lesser extent in R2 increased during the first 5 days at 37°C, concentrations remained low at 5.4±1.2x107 and 1.7±0.1x108 CFU/ml in R1 and R2 at day 6, respectively. However, as observed in beads, cell counts in the fermented broth from R1 and R2 greatly increased when temperature was decreased to 32°C to reach very high values of 2.3±0.3 and 6.0±0.3x109 CFU/ml at day 13, respectively. The temperature change to 35°C at day 13 induced an approximately twofold decrease of L. diacetylactis counts in R1 that reached 1.2±0.2x109 CFU/ml at day 17, whereas counts in R2 remained stable from day 14 until the end of the study, averaging 7.0±1.1x109 CFU/ml.

Lactic and acetic acid concentrations in the effluents of R1 and R2 during the 17-day continuous culture are reported in Figure 4.2. Organic acid concentrations were constant in R1 and R2 from day 2 to day 6 at 37°C averaging 7.0±0.5 and 9.0±0.4 g/l for acetic acid, and 5.2±0.9 and 8.5±0.7 g/l for lactic acid, respectively. After a temperature decrease to 32°C, the acetic acid concentration decreased to 4.1±0.5 and 4.6±0.7 g/l in R1 and R2 at day 13, respectively, whereas lactic acid concentrations increased after a 2-day delay to 9.2±1.7 and 24.5±2.4 g/l in R1 and R2 at day 13, respectively. No change in lactic and acetic acid concentrations in the effluents of the two bioreactors was further observed during the following days (day 14 to day 17) carried out at 35°C compared with that at 32°C.

Figure 4.3 shows sugar concentrations in the effluents of R1 and R2 during the continuous culture. Lactose concentration decreased during the first 5 days at 37°C in both R1 and R2 to 16.0 and 1.0 g/l at day 6, respectively. Lactose concentration increased during the first 2 days at 32°C to 29.7 and 23.2 g/l at day 8 in R1 and R2, but then decreased with time at the same temperature in R1 to 23.1 g/l at day 13, and to a greater extent in R2 to a low value of 8.0 g/l. Lactose concentration remained constant during the following 4 days at 35°C in R1 (23.5±0.6 g/l) and R2 (4.1±0.4 g/l). Glucose and galactose concentrations increased during the first 4 days at 37°C to 7.6 and 7.1 g/l and 12.6 and 12.9 g/l for R1 and R2 at day 6, respectively. After decreasing the temperature to 32°C, glucose and galactose concentrations in R1 and R2 dropped rapidly to low values of 2.3 and 3.8 g/l for glucose and 3.2 and 5.5 g/l for galactose at day 7, respectively, and then remained constant until the end of the 17-day culture at 32°C or 35°C.

B. longum and L. diacetylactis viable cell counts were determined in individual beads to observe cross-contamination occurring in beads originally immobilizing a pure culture of one of the two strains, as a function of fermentation time (Table 4.1). At day 2, beads immobilizing the two different strains were distinct with no ( L. diacetylactis beads) or a low ( B. longum beads) cross-contamination by the other strain. B. longum concentration in L. diacetylactis beads increased rapidly to approximately 1/7 of total cell concentration in these beads at day 8, but did not change thereafter. In contrast, L. diacetylactis contamination was slower in beads originally immobilizing B. longum but was more intense after 11 days, with a high L. diacetylactis / B. longum ratio of 7±3, which did not change for longer fermentation times. Consequently, beads originally immobilizing a pure culture of B. longum or L. diacetylactis could not be distinguished from day 11 to the end of the experiment, as the strain ratios determined by viable cell enumeration became identical for the two types of gel beads.

Using specific fluorescent polyclonal antibodies for the two strains and confocal laser-scanning microscopy, contamination by the other strain was detected after 14 days at the periphery of both types of beads (Figure 4.4). The peripheral layer of approximately 100 μm contained a mixed culture of B. longum and L. diacetylactis cells occupying a very high fraction of B. longum bead volume (60-65% and 90-95% for each strain, respectively) (Figure 4.5a). After 14 days, colonization of B. longum beads was constant in the core of the beads, with an occupation of approximately 30% (Figure 4.5a). There was also approximately a 150-μm thick intermediate layer, between the external mixed-strain layer and the core of the beads, where the fraction of bead volume occupied by biomass was very low, equal to approximately 10%. Biomass profiles in L. diacetylactis beads exhibited the same tendencies after 14 days continuous culture (Figure 4.5b), with a 100-μm peripheral layer containing a mixed culture of the two strains, with a volume occupation of 60% L. diacetylactis and 45% B. longum . As observed for B. longum beads, there was approximately a 150-μm thick layer between the external layer and the core of the beads where the fraction of bead volume occupied by biomass was very low at 10%. Moving deeper in the beads toward the centre, the occupation ratio increased to 30% but then progressively decreased to approximately 10% at the bead centre (Figure 4.5b). Using a two-color fluorescence assay of bacterial viability (LIVE/DEAD BacLight Bacterial Viability Kit), the intermediate layer containing low biomass concentration in beads, detected with polyclonal antibodies, appeared to contain a large concentration of dead biomass, unlike the periphery and the core of the beads which contained live biomass (Figure 4.6).

This study is the first to report the continuous production of a mixed culture containing lactic acid bacteria and bifidobacteria immobilized in gel beads. The fermentation process, with two reactors in series continuously fed with MRS medium, allowed continuous production of a concentrated mixed culture of the two strains. Temperature appeared to be a crucial parameter for the strain ratio in the mixed culture (Figure 4.1). A high temperature close to 37°C favoured the growth of B. longum (maximal concentration of 8.3±0.1x108 CFU/ml in the effluent of R2 at day 5), whereas a low temperature of 32°C promoted L. diacetylactis growth (maximal concentration of 7.5±0.1x109 CFU/ml in R2 at day 12). However, a stable ratio of strain concentrations was obtained in the effluent of R2 at 35°C for 4 days and until the end of the experiment, with a high concentration of both strains in the effluent (3.4±0.9x108 and 7.0±1.1x109 CFU/ml for B. longum and L. diacetylactis , respectively). During a previous study on repeated pH-controlled batch fermentations carried out at 37°C with co-immobilized cells of B. longum and L. diacetylactis , a mixed free-cell culture containing 2.3x108 CFU/ml L. diacetylactis and 1.3x109 CFU/ml B. longum was produced in the broth at the end of the fourth batch culture (Doleyres et al., 2002a; chapter 3). These results are close to cell counts determined in the effluent of R1 when temperature was initially set at 37°C (day 2 to day 6). High biological stability and cell volumetric productivities were also obtained during long-term continuous fermentations (exceeding 50 days) of a supplemented whey permeate medium using immobilized cells to produce mixed-strain mesophilic lactic starters, and no strain became dominant nor was eliminated from the reactor (Lamboley et al., 1997; 1999; 2001). Data in our study show that immobilized cell technology and a culture parameter such as temperature can be used to control the strain ratio of a mixed culture of lactic acid bacteria and less competitive strains like bifidobacteria, and that biological stability can be obtained for at least 4 days at 35°C. Lamboley et al. (1997) also showed that fermentation parameters (temperature, dilution rate and pH) can be used to efficiently control ratios of three lactococci in the effluent of a continuous immobilized-cell bioreactor. However, changing the bead ratios in the inoculum of the immobilized-cell bioreactor had only a limited effect on the composition of the culture produced in the effluent (Lamboley et al., 2001).

During the entire 17-day continuous fermentation, L. diacetylactis viable cell counts were higher (up to fivefold) in the effluent of R2 than in that of R1 (except for day 4), whereas B. longum viable free-cell counts were always lower in R2 than in R1. The low competitivity of B. longum free cells in R2 can be mainly explained by the high lactic acid concentration in R2 (maximum of 25.5 g/l at day 14 compared with 9.8 g/l in R1), which inhibited B. longum growth in R2. Acetic acid concentration measured in R1 and R2 were very similar, particularly at 32 and 35°C (Figure 4.2). However, the synergic inhibitory effect of lactic and acetic acids on this strain (Desjardins et al., 1990) could also explain the absence of growth of B. longum in R2. Consequently, lactic acid production in R2 was almost totally due to L. diacetylactis activity.

Lactose hydrolysis was high in R1 when fermentation was carried out at 37°C (residual lactose concentration of 16.0 g/l in R1 at day 6), indicating a high enzymatic activity of bifidobacteria which resulted in significant glucose and galactose accumulations in the fermentation medium. However, lactose concentration remained higher at 32-35°C at 22-30 g/l than at 37°C, and glucose and galactose concentrations did not exceed 5 g/l, indicating a high utilization of monosaccharides by L. diacetylactis . This preference for B. longum to metabolize lactose before glucose was previously demonstrated during free-cell batch fermentations of a medium containing the two sugars (Doleyres et al., 2002b; chapter 2).

The use of a second reactor R2 in series with the first immobilized cell reactor R1, with a higher volume to increase biomass concentration in the effluent, may not be a good strategy for promoting the growth of bifidobacteria in mixed cultures containing other competitive strains of lactic acid bacteria, like L. diacetylactis . However, the inhibitory conditions in R2 could also increase cell tolerance to various stresses, such as freeze-drying or oxygen, that cells generally encounter following their production (Reilly and Gilliland, 1999).

The development of a mixed culture of the two strains in beads originally immobilizing a pure culture as a function of fermentation time was observed and quantified with selective enumeration methods and the use of immunofluorescence and confocal laser-scanning microscopy. Strain redistribution lead to identical strain ratios (7±3/1 L. diacetylactis/B. longum ) after 11 days in beads originally immobilizing L. diacetylactis or B. longum (Table 4.1). This phenomenon of strain redistribution among gel beads initially immobilizing a pure culture has been previously observed during long-term continuous cultures of mesophilic lactic acid bacteria by plating individual beads on Petri dishes (Lamboley et al., 1997; 1999; 2001, Sodini et al., 1997). To explain this massive cross-contamination of gel beads, a theoretical model for contamination was proposed (Lacroix et al., 1996). In this model, cell release from the gel cavities results from high pressure due to cell expansion, collisions and shearing forces in the bioreactor. Afterwards, the walls of the emptied cavities made of viscoelastic polysaccharide gel material could close again, entrapping a sample of the surrounding bulk medium. Recolonization of the cavities occurs as a result of the activity of both the original bead culture and the contaminating flora.

With the use of confocal microscopy and the determination of the spatial distribution of the two strains in gel beads by immunofluorescence, our study validated this model. Indeed, contamination by free cells from the surrounding medium was limited to a peripheral layer of approximately 100 μm thickness due to diffusion limitations of cells in the gel. B. longum bead contamination by L. diacetylactis cells after 11 days was higher than L. diacetylactis bead contamination by B. longum cells, as a result of the medium containing more lactococci than bifidobacteria cells. However, contamination by the other strain was detected earlier in L. diacetylactis beads than in B. longum beads due to the fact that the optimal temperature for B. longum growth (37°C) was applied during the first days of fermentation (day 1 to day 6). In addition, the production of acetic acid by immobilized B. longum might have created local inhibitory conditions for L. diacetylactis growth in B. longum beads, which delayed its implantation and detection. The subsequent modification of temperature set point to lower values of 32 and 35°C increased competitivity of L. diacetylactis and produced a strain ratio greatly in favour of L. diacetylactis in both types of gel beads. The fact that this ratio was identical in both types of beads demonstrates that most if not all viable cells in beads were located in the peripheral layer and that the core of the beads contained mainly dead cells after 11 days continuous culture. However, confocal laser scanning microscopy and specific cell marking with fluorescent polyclonal antibodies did not permit to distinguish live and dead cells (Figure 4.4).

A biomass gradient was observed after 14 days continuous culture in the core (0-700 μm) of L. diacetylactis beads contrary to beads originally immobilizing B. longum that showed a flat profile (Figure 4.5). This was previously observed and explained by the fact that immobilized cells of B. longum are more resistant to lactic and acetic acid local concentrations in beads (Doleyres et al., 2002a; chapter 3). In this study, a cell concentration decrease was observed at the extreme periphery of the beads, which can be explained by the high cell-release activity from the beads to the medium. Our study showed that contamination of beads occurred in the high-cell density peripheral layer. A low biomass concentration layer was also observed for both types of beads, between the highly colonized peripheral layer and the core of the beads with intermediate colonization (Figure 4.5). A high concentration of dead cells was observed in this part of the beads using the LIVE/DEAD bacterial viability kit (Figure 4.6). Consequently, the polyclonal antibodies apparently marked only live biomass, due to modifications in cell wall structure following cell death. This cell mortality could be tentatively explained by initial cell growth during bead colonization with favourable conditions, followed by strong inhibition due to the high cell activity in the bead periphery and to the production of steep organic acid concentrations and pH gradients within the beads (Arnaud et al., 1991; 1992; Masson et al., 1994). In comparison, cells in the core of the beads grew with more inhibiting conditions and adapted progressively to these stress conditions in the course of bead colonization, and remained active during the 17 days of culture.

This work has demonstrated that cell immobilization in polysaccharide gel beads can be used to continuously and stably produce a mixed lactic culture containing a non-competitive strain of bifidobacteria. The use of confocal laser-scanning microscopy and fluorescent polyclonal antibodies also allowed the demonstration of the microbial dynamics of immobilized cells during mixed-strain fermentations and the cross-contamination by the other strains of mixed cultures that occurs in gel beads initially immobilizing a pure culture. Chapter 5 will assess the changes in physiological characteristics and compare tolerance to different stresses of cells produced during continuous immobilized-cell fermentations with that of cells produced during conventional free-cell batch fermentations.

Table 4. 1 : Change of the strain ratio in individual beads originally immobilizing a pure culture of B. longum or L. diacetylactis during continuous pH-controlled fermentation with immobilized cells at different temperatures 1.

1 Continuous culture was carried out at 37°C until day 6, 32°C between day 6 and 13, and 37°C afterward.

2 After 11 days culture, beads for the two strains could not be distinguished.

Figure 4. 3 : Lactose (□,■), glucose (∆,▲), and galactose (○,●) concentrations in effluents of R1 (dark symbols) and R2 (open symbols) determined by HPLC analysis during the continuous pH-controlled fermentation with immobilized cells of L. diacetylactis and B. longum .