CHAPITRE 3 : Quantitative determination of the spatial distribution of pure and mixed-strain immobilized cells in gel beads by immunofluorescence

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

Publié dans "Applied Microbiology and Biotechnology", 59  : 297-302, 2002.

Une nouvelle méthode basée sur l’utilisation d’anticorps polyclonaux et la microscopie confocale a été développée pour détecter et quantifier deux souches, Lactococcus lactis subsp. lactis biovar. diacetylactis MD et Bifidobacterium longum ATCC 15707, immobilisées séparément ou co-immobilisées dans des billes de gel. Ainsi, l’établissement d’un profil de concentration de biomasse pour chaque souche a été mesuré durant la colonisation des billes lors de fermentations successives en batch avec pH contrôlé. La croissance cellulaire s’est déroulée principalement dans une couche périphérique de billes d’une épaisseur de respectivement 200 et 300 μm pour L. diacetylactis and B. longum . Ces fermentations en batches répétés avec cellules immobilisées ont également permis de produire une culture mixte contenant une souche peu compétitive de bifidobactérie, grâce à la croissance des cellules immobilisées et à la forte libération cellulaire de la périphérie des billes dans le milieu de fermentation. Par ailleurs, aucun phénomène particulier de coopération ou d’inhibition entre les deux souches fut observé lors des fermentations en culture mixte.

A new method was developed to detect and quantify two strains, Lactococcus lactis subsp. lactis biovar. diacetylactis MD and Bifidobacterium longum ATCC 15707, immobilized separately and co-immobilized in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy. The establishment of biomass concentration profiles for each strain was measured during colonization of beads using successive pH-controlled batch fermentations. Growth occurred preferentially in 200 and 300 μm peripheral layers of the beads for L. diacetylactis and B. longum , respectively. Repeated-batch cultures with immobilized cells permitted the production of a mixed culture containing a non-competitive strain of bifidobacteria, as a result of immobilized cell growth and high cell-release activity from the beads. During co-immobilized fermentations, there were no apparent interactions of strains.

Fermented dairy products containing probiotics are increasingly gaining popularity with consumers because of their perceived importance in human health (Chandan, 1999). Due to their low competitivity for fermenting milk or other media when they are grown in mixed cultures with lactic acid bacteria, bifidobacteria are usually propagated in pure cultures, and then added in combination with the lactic starter used for milk fermentation (Tamime et al. 1995).

The immobilized cell technology with lactic acid bacteria in polysaccharide gel beads has been studied for the continuous production of mixed cultures of mesophilic lactic acid bacteria with controlled composition (Lamboley et al. 1997, 1999, 2001). The high immobilized cell concentration and cell release from the beads to the bulk medium lead to high process productivity and biological stability. However, a complex redistribution of strains among gel beads initially immobilizing a pure culture was observed. An immunofluorescent method involving double color labelling and confocal microscopy has been recently developed to specifically detect lactic acid bacteria and probiotic cells co-immobilized in gel beads (Prioult et al. 2000). This method could be used for studying the complex microbial dynamics for mixed cultures in gel beads. However, this would require the localization and quantification of microcolonies in beads.

The aim of the present research was therefore to develop a quantitative analysis for the detection of immobilized cells, using the immunological method described by Prioult et al. (2000). Bead colonization during successive batch fermentations with a model mixed culture containing Lactococcus lactis subsp. lactis biovar. diacetylactis as a competitive strain, and Bifidobacterium longum as a non-competitive probiotic strain, immobilized separately or co-immobilized, was analyzed with this new method for cell detection .

Bead colonization was first studied during single-strain repeated-batch fermentations. In L. diacetylactis cultures, immobilized cell concentration increased exponentially during the first 8-12 hours of the first batch fermentation to reach 5.4±1.3x109 CFU/g beads and only doubled to 1.1±0.2x1010 CFU/g at the end of the third culture (32 h; Figure 3.2a). Viable-cell counts in the broth at the end of batch cultures were very similar, and remained low between 5.7±0.2x107 CFU/ml and 1.7±0.1x108 CFU/ml. The total cell concentration in beads measured by ELISA was two- to threefold higher than viable-cell counts, at the beginning of the stationary phase of the first batch culture (12 h). Confocal microscopy images and analysis of spatial biomass distribution showed that L. diacetylactis growth was almost nil at the bead centre but mainly occurred in a 200 μm peripheral layer of the beads (Figure 3.3a). Cell concentration estimated by reference to the ELISA test reached 5.0x109 cells/g at the bead centre at the end of the first batch culture (14.5 h) and did not increase thereafter. However cell concentration in the peripheral layer increased during the three successive batch cultures and reached 2.7x1010 cells/g near the bead surface at the end of the third batch (31.5 h). Increasing the number of beads analyzed from 1 to 5 produced smoother profiles but did not change cell concentrations as a function of bead depth (Figure 3.4).

In B. longum cultures , viable-cell counts in beads increased during the first two batch cultures to a very high value of 9.6±1.0x1010 CFU/g after 26 h culture (Figure 3.2b). Viable-cell counts in the broth continuously increased with the number of batch cultures, from 8.2±0.6x108 CFU/ml at the end of the first batch to 2.5±0.2x109 CFU/ml for the third batch. As observed with L. diacetylactis , B. longum cell concentration determined by ELISA was similar to viable cell counts in beads during the exponential growth of the first batch culture but was higher during the stationary phase, with a maximal ratio of 3.2 after 14.5 h culture. Growth occurred mainly in a 300 μm peripheral layer but did not stop at the bead centre during the experiment. Cell concentration at the bead centre and periphery reached 8.0x1010 and 1.9x1011 cells/g at the end of the third culture (Figure 3.3b).

Microbial dynamics in beads and broth was then studied during fermentations with co-immobilized cells to observe possible interactions between the two strains. Figure 3.5 shows the strain concentrations in beads and broth during repeated-batch fermentations carried out at pH=6.2 with an inoculum composition of 9:1 B. longum / L. diacetylactis . Viable-cell counts of L. diacetylactis in beads increased until 9-12 h of culture during the first batch and remained constant during the subsequent batches at 2.1±0.1x1010 CFU/g. For B. longum , viable-cell counts in beads increased during repeated-batch fermentations up to 9.9±0.1x1010 CFU/g at the end of the fourth batch (26 h). Lactococci free-cell counts in the broth at the end of the batch cultures did not change from the first to the fourth batch, averaging 2.2±0.7x108 CFU/ml. On the other hand, B. longum viable-cell counts at the end of batch cultures progressively increased from 1.1±0.1x108 to 1.3±0.1x109 CFU/ml from the first to the fourth batch culture. L. diacetylactis and B. longum concentrations in beads determined by ELISA were four- to fivefold higher than the viable-cell counts after an initial culture period (Figure 3.5). L. diacetylactis concentration at the bead centre reached 1.2x1010 cells/g at the end of the second batch culture (21 h) and did not change thereafter (Figure 3.6a). Growth was concentrated in a 200-250 μm peripheral layer and peaked at 5.3x1010 cells/g near the bead surface (80 μm) at the end of the experiment. B. longum cell concentration near the bead centre and the bead surface (60-120 μm) increased gradually during the repeated cultures, to maxima of approximately 7.3x1010 and 1.5x1011 cells/g (Figure 3.6b).

A second experiment with co-immobilized cells was conducted under the same conditions except for pH control (5.8) and strain ratio (1:1) in the inoculum used for bead preparation, to tentatively favor the growth of L. diacetylactis and decrease the dominant behaviour of B. longum observed in the first experiment. Very similar data were obtained for both co-immobilized culture experiments, for both immobilized and free-cell concentrations and biomass profiles in gel beads (data not shown).

This study is the first to report the specific and quantitative analysis of the spatial biomass distribution of L. diacetylactis and B. longum , immobilized separately or co-immobilized in gel beads, using ELISA and an immunofluorescent method involving double colour labelling and confocal microscopy. Viable-cell counts determined on Petri dishes can only be correlated to the ELISA test and confocal images when no loss of viability is postulated since polyclonal antibodies detect both live and dead biomass and when the antigen-antibody reaction is not altered for immobilized cells compared with free cells used to calibrate the ELISA test. Clearly, this was not the case during fermentations, during which the ELISA test measured a much higher cell concentration than plate counts (ratio from 2 to 5) after an initial growth period (Figures 3.2 and 3.5). Increasing the number of beads analyzed (from 1 to 5) to determine biomass profiles smoothed the curves but did not change the concentration profiles, indicating a high uniformity of colonization and biomass profiles among beads (Figure 3.4).

Quantitative determination of the spatial distribution of the two strains in beads showed that, after an initial colonization, cell growth occurred preferentially in a 200 or 300 μm layer near the bead surface for L. diacetylactis and B. longum , respectively, for both separately and co-immobilized cells (Figures 3.3 and 3.6). Conditions are more favourable for cell growth close to the gel bead surface due to diffusional limitations for both substrates and inhibitory products, in this case lactic and acetic acids (Arnaud et al. 1992; Lamboley et al. 1997), which result in a sharp pH gradient in beads (Masson et al. 1994). In addition, a cell concentration decrease can be observed close to the bead surface, which is explained by the high cell-release activity from the bead to the broth medium (Figures 3.3 and 3.6). The thicker growth layer and the higher total and viable-cell concentrations for immobilized B. longum than for L. diacetylactis indicate that this culture is more resistant to lactic and acetic acid local concentrations in beads, for the culture conditions used in this study. Using electron microscopy, Arnaud and Lacroix (1991) reported that immobilized cells of Lactobacillus casei were concentrated in a 400 μm peripheral layer. Using sequential dilution of alginate beads, Prévost and Diviès (1988) observed that 43 and 90% of Streptococcus salivarius subsp. thermophilus immobilized cells were localized in a peripheral layer of 130 and 410 μm.

This study also showed that immobilized cell technology allowed the production of a model mixed-lactic starter containing a competitive strain ( L. diacetylactis ) and a non-competitive strain of bifidobacteria. Immobilized cell technology had already been used for the efficient and stable continuous production of mixed-strain mesophilic lactic starters of controlled composition and activity in fermented medium, as a result of high immobilized cell counts and cell-release activity from gel beads (Lamboley et al. 1997, 1999, 2001). Fermentation conditions, such as temperature (37°C) and medium composition, were thus chosen to promote growth of B. longum . With these conditions, bead colonization during repeated pH-controlled batch fermentation was complete at the end of the first batch culture for both strains, but a much higher viable-cell concentration in beads was obtained for B. longum (1.0x1011 CFU/g) than for L. diacetylactis (1.1x1010 CFU/g) (Figure 3.2). Similar concentrations in gel beads were reported during continuous fermentations with B. longum (Camelin et al. 1993), but much higher concentrations (1.2x1011 CFU/g) were obtained with lactococci (Lamboley et al. 1997). As a result of immobilized cell growth and cell release, a much higher concentration of B. longum (1.7±1.1x109 CFU/ml) was produced in the broth at the end of batch cultures compared with L. diacetylactis (1.1±0.5x108 CFU/ml) (Figure 3.2).

During the production of mixed cultures of non-isogenic lactococci, two strains exhibiting a competitive relationship in free-cell cultures were found to be cooperative during fermentations carried out with immobilized cells (Audet et al. 1995). However this phenomenon was not observed in the present study. L. diacetylactis strain was apparently not influenced by the presence of the other strain during mixed co-immobilized fermentations (Figures 3.2a and 3.5). However B. longum maximal concentration in beads and broth was reached later in mixed cultures compared with pure cultures (end of fourth and first batch, respectively), with no effect on maximal ELISA and viable cell counts (Figures 3.2b and 3.5). At the end of the fourth batch fermentation, a mixed free-cell culture containing 2.3x108 CFU/ml L. diacetylactis and 1.3x109 CFU/ml B. longum was produced in the broth (Figure 3.5). B. longum counts could be further increased with the number of batch cultures, and by starting a continuous culture. In the previous chapter, a cell production of 4.9x109 CFU/ml was measured in the broth for the same strain in immobilized cell continuous cultures. On the other hand, L. diacetylactis cell counts in the broth did not change from the end of the first to the fourth batch cultures, indicating the unfavourable conditions for this strain in mixed cultures.

To promote L. diacetylactis growth in mixed cultures, a new set of experiments was performed with pH decreased from 6.2 to 5.8 and L. diacetylactis concentration increased approximately tenfold in the inoculum used for bead preparation. These changes gave very little effect on the growth and fermentation of both strains, suggesting that culture conditions, such as lower fermentation temperatures, should be changed to promote the growth of L. diacetylactis and balance the mixed cultures.

The present study shows that cell immobilization in polysaccharide gel beads can be used to produce a mixed-lactic starter containing a non-competitive strain of bifidobacteria. Viable-cell counts in the broth during repeated pH-controlled batch fermentations were related to biomass concentrations of each strain in the beads. Moreover, a new method was tested to specifically localize and quantify biomass profiles in gel beads, using specific polyclonal antibodies and confocal laser-scanning microscopy observations. This method will be used to study the complex microbial phenomena that occur in beads during continuous production of mixed-strain lactic starters using immobilized cell technology (chapter 4).

B3

B2

B1

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Figure 3. 4 : Effects of bead numbers on biomass concentration in L. diacetylactis beads.

Figure 3. 5 : Total biomass concentration determined by ELISA (●,○, log cells/g) and viable cell counts in beads (■,□, log CFU/g) and broth (♦,◊, log CFU/ml) during repeated pH-controlled (pH=6.2) batch fermentations (B1 to B4) with co-immobilized B. longum (dark symbols) and L. diacetylactis (open symbols) (ratio 9:1 in the inoculum). The arrows indicate medium changes after 12, 21 and 23.5 h.

Figure 3. 6 : L. diacetylactis (a) and B. longum (b) concentration profiles in beads during repeated pH-controlled batch fermentations (B1 to B4) with co-immobilized strains. The medium was changed after 12, 21 and 23.5 h.