Fermentation technologies for the production of exopolysaccharide-synthesizing Lactobacillus rhamnosus concentrated cultures
Claude P. Champagne*
Financial support: This work was carried out within the Research Network on Lactic Acid Bacteria, supported by the Natural Sciences and Engineering Research Council of Canada, Agriculture and Agri-food Canada, Novalait Inc., The Dairy Farmers of Canada and Rosell-Lallemand Inc.
Keywords: alginate beads, fed-batch culture, temperature.
exopolysaccharide (EPS)-producing cultures such as Lactobacillus
rhamnosus RW-9595M present a challenge for the culture producers
because the high viscosity of the fermented growth medium makes it
difficult to recover the cells by centrifugation or filtration. This
study examined four approaches to reduce viscosity of the medium while
producing high cell densities: incubation temperature, extended incubation
in the stationary growth phase, production in alginate gel beads and
fed-batch fermentation technology. Automated spectrophotometry (AS)
was used to study the effects of temperature, pH and lactate level
on growth of the strain. In AS assays, there was no significant difference
in final maximal biomass production at temperatures ranging between
Exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) are of interest to the food industry because they can improve the texture of yoghurts (De Vuyst and Degeest, 1999; De Vuyst et al. 2001), decrease the risk of syneresis and improve yields in cheeses (Broadbent et al. 2001). In addition to their technological benefits, LAB EPS have been recognized to have antitumoral, antiulcer and blood cholesterol lowering activities, as well as the ability to enhance the immune system (Chabot et al. 2001; Ruas-Madiedo et al. 2002). Therefore EPS-producing strains are of commercial value for both their technological and putative probiotic properties.
In order to supply cultures directly to consumers or to the food industry, manufacturers must propagate the strains in appropriate media, recover the cells and stabilize cell concentrates by freezing or drying. With high EPS producing bacteria, such as Lactobacillus rhamnosus RW-9595M (Macedo et al. 2002; Bergmaier et al. 2003), cell recovery presents a challenge for manufacturers because viscosity of the fermented medium could hinder centrifugation or filtration operations. There is therefore a need to develop fermentation technologies which can generate high biomass yields, while keeping the viscosity at a level which would enable easy recovery of the cells from the fermented broth.
A balance between carbon and nitrogen sources is necessary to obtain a high EPS concentration (De Vuyst and Degeest, 1999), but other factors such as growth temperature, pH and agitation affect EPS production (Gamar-Nourani et al. 1998). High incubation temperatures have been shown to reduce EPS production by lactobacilli (Grobben et al. 1995; Mozzi et al. 1996b), but it is unknown if the viscosity reduction is sufficient to enable easy recovery of cells. Fed-batch fermentation with high carbohydrate levels was successful in enhancing EPS production (Cheirsilp et al. 2003) but is it unknown if a reverse strategy (i.e. fed-batch with low carbohydrate level) could be used to generate a fermented medium with high cell concentration and low-viscosity.
In addition to the traditional concentrated starter technology, immobilized cell technology (ICT) has been proposed for the production of bacterial concentrates (Doleyres and Lacroix, 2005). Although the production of concentrates in alginate beads has the advantage of eliminating the need for a concentration step, the EPS yields are sometimes lower (Champagne et al. 1993) and some strains are adversely affected by entrapment in alginate gels (Lamboley et al. 2003). No data are available on the ICT production of EPS-producing strains of L. rhamnosus.
The objective of the present study was to examine four methods for the production of high bacterial populations while controlling viscosity of the medium: high incubation temperatures, extended incubation on the stationary growth phase (SGF), fed-batch fermentations and biomass production in alginate beads.
cultures of L. rhamnosus RW-9595M were prepared by mixing a
fresh MRS-grown culture with sterile rehydrated skim milk (20% w/w)
and sterile glycerol (20% w/w) in a 1:2:2 ratio, and placing 1 ml
fraction in Cryovials (Nalge, Rochester, USA). The cell
suspensions were then frozen and kept at
Method 1 was based on the use of over-optimal growth temperatures,
it was necessary to ascertain the optimum growth temperature for this
particular strain. With respect to Method 4, the strategy required
prior knowledge of the amount of sugar needed to carry out an extended
fermentation. Thus, preliminary assays using automated spectrophotometry
(AS) were carried out to ascertain the conditions of pH, temperature
and maximum lactate concentration that were to be subsequently used
for fermentations. The AS assays were not conducted on the modified-MRS
(MMRS) or whey media used for fermentations because such media generate
biomass levels which are too high. Indeed, in AS assays it is recommended
to limit the growth of the culture to optical density (OD) values
under 1.0. Thus AS assays were conducted in the basal medium described
by Morishita et al. (1981) for lactobacilli. It
had the following composition per liter:
”l of sterile media were then dispensed in wells (Polystyrene sterile
96 well plates, Costar, NY,
assays with Method 1, the growth medium was selected to generate the
highest possible biomass and, hence, a high EPS level. This was done
to simulate industrial conditions which are designed to attain high
biomass, as well as to generate the highest possible viscosity in
order to test the value of our hypothesis. The medium used to evaluate
the effect of temperature on cell growth and viscosity was formulated
with ingredients of commercial sources recognized as enabling very
good growth of strain RW-9595M as well as high EPS levels (Macedo
et al. 2002). The whey permeate (WP) based medium was prepared
by first adding
cultures were conducted in three
Method 1, samples were taken at the beginning of the SGP. For Method
2, the incubation of the same media was simply extended at 42 or
with Method 3 and Method 4 were done simultaneously in order to compare biomass
levels of the three fermentation technologies (free, immobilized and
fed-batch). However, the WP medium could not be used for Method 4,
because it was necessary to have a low level of carbohydrate at the
beginning of the fermentation (low C/N ratio), which the WP media
did not enable. It was therefore necessary to use a synthetic medium
where the carbohydrate could be added separately from the other ingredients.
A MMRS was thus formulated for its carbohydrate-defined composition
as opposed to the WP-based medium where the lactose content cannot
be varied without changing the other solids content. The MMRS was
prepared as follows (per litre of medium):
inoculation procedure was carried out in order to compare this treatment
with batch fermentations performed with alginate beads, where the
same volume of fresh culture was entrapped in
alginate beads, fermentations inoculated with
fed-batch fermentations, the MMRS base (
fermentations (batch or fed batch) were carried out on
viable population in the medium (free cells) was determined by plating
appropriate dilutions of the cultures on MRS agar, and incubating
and products were analysed by HPLC using a Waters
(Mississauga, Canada) system coupled to Millennium software. Samples
were centrifuged and filtered on a HVLP 0.22 : membrane prior separation
on a Aminex HPX 87H column (BioRad,
Mississauga, Canada) heated at
media were centrifuged at
ml of broth of culture or supernatant were introduced in a capillary
Ubbelohde 1B unit placed into a water bath with a control temperature
Data are the average of three independent assays. CFU values were converted into their corresponding Log10 numbers and ANOVA were then carried out using the Student-Newman-Keuls procedure with Instat 3.0 software (GraphPad, San Diego, USA). The α = 0.05 level was used to ascertain the statistical significance of the differences. Paired t tests were used to evaluate the effect of extended incubations on viability drops at each incubation temperature (Method 2). Standard Error of the Means (SEM) values are presented in brackets in the tables.
Many studies show that EPS production in lactobacilli is strongly linked to biomass levels (De Vuyst et al. 1998; Torino et al. 2000). Although viscosity does not always correlate with EPS production (Shihata and Shah, 2002; Ruas-Madiedo et al. 2005) for L. rhamnosus RW-9595M, there is a correlation between EPS concentration in the medium and CRV values (Macedo et al. 2002). Then, by keeping the biomass yield of a culture at a low level, one could achieve the goal of having a low-viscosity fermented medium. However, from a starter manufacturer point of view, high biomass productions are desirable, which eventually lead to high medium viscosity. Attempts were thus made to find fermentation parameters which would enable high biomass as well as low EPS production.
AS evaluates growth from continuous OD readings, and media must be specially formulated for these assays in order to limit the growth in an OD range in which OD and biomass are linearly correlated. AS enabled the evaluation of the combined effects of lactate concentration, pH and temperature on the growth of L. rhamnosus RW-9595M.
on OD max data from media at pH 6.0 with no added lactate (0% lactate,
Figure 1) revealed that there was no significant
(P > 0.05) effect of incubation temperatures between 34 and
for ” max differed from that of OD max. At pH 6.0 and without added
lactate (0% lactate, Figure 2), the ANOVA revealed
that the highest growth rates (” max values) were obtained between
pH of the media were adjusted below 6.0, the ANOVA analysis of ”
max data in media with no added lactate revealed significantly lower
values (data not shown). These data served to select a pH value of
6.0 for subsequent biomass productions in fermenters. These results
are in line with those of Mozzi et al. (
Lactate concentrations higher than 4% substantially decreased the growth of L. rhamnosus RW-9595M, and negligible growth was noted in media adjusted at pH 6.0 having 6% lactate (Figure 1 and Figure 2). These AS results thus enabled the selection of 6% of glucose for the fed-batch assays (Method 4), in order to generate the 4% lactate level judged to be critically detrimental to biomass and growth rate levels. Indeed, preliminary data with this strain (not shown) suggested a conversion level of glucose to lactate of approximately 75% (Table 2).
Under pH control, lactate accumulation can generate growth inhibition (Cachon and Diviès, 1993), but the inhibitory level varies between strains, species, pH and temperature (Houtsma et al. 1996). The AS data was in line with the literature, showing lactobacilli to be more tolerant to lactate than are lactococci (Stieber et al. 1977; Cachon and Diviès, 1993).
pH-controlled batch productions in the whey-based medium, fermentation
times to reach the SGP were longer for temperatures below
of the media was 2-fold higher at
retrieval of cells was not possible for any of the cultures when using
a centrifugation force of
Since it was reported that EPS production (Pham et al. 2000) and CRV (Macedo et al. 2002) of L. rhamnosus cultures were at its highest at the beginning of the SGP and dropped steadily thereafter, an attempt was made to see if this phenomenon could be used to enhance recovery of the cells by centrifugation. Unfortunately, the CRV of the fermented medium was not significantly reduced after 5 hrs extended incubation in the SGP (Table 1). It was suggested that hydrolases activated by specific growth conditions caused degradation of the EPS (Pham et al. 2000), but our experimental conditions do not seem to have generated this EPS hydrolysis.
An apparent drop in viable counts of approximately 20% was noted during prolonged fermentation periods, but paired t tests on population differences are not shown to be significant when total populations are calculated (multiplication of the CFU vales by the average number of cells per chain).
In the ICT system, populations per g of beads in the MMRS were 10 times higher than for the free-cell control batch fermentations (Table 2). However, when the total yield of the bioreactor is considered, the total population from the beads represented only 40% of that obtained in free cells bioreactor. CRV values obtained for immobilized cells were slightly lower than for the free cells. Lactate concentrations for both treatments were similar.
The production of biomass by growing lactic cultures in alginate beads had previously been suggested (Champagne et al. 1992; Champagne et al. 1993), particularly for cultures which were sensitive to oxygen or centrifugation stresses. For EPS producers, the immobilization techniques may facilitate the recovery of the cells even in a viscous environment as they are entrapped in beads (Table 2) and this presents the main advantage. Thus, recovery of the beads could easily be carried out with a simple filtration unit. However, an important disadvantage with this technology was the lower biomass yields. A method to improve populations in beads, and reduced cell release into the medium, must be developed.
During fed-batch cultures, the medium viscosity increased in parallel with the cell counts (Table 2). However, the final CRV of 3.25 was almost 40% lower than the viscosity obtained during control batch fermentation (Table 2) and for equal biomass levels. At the end of the culture, glucose concentrations were low (0.4 g/l) and the lactate concentration was at 38.7 g/l for both the immobilized and free cells.
quantity of carbohydrates in the medium affects EPS yields (Prasher
et al. 1997) and high initial carbohydrate levels tend to enhance
final EPS levels (De Vuyst et al. 1998; Cheirsilp
et al. 2003; Korakli et al. 2003). It was therefore
our strategy to add glucose at a level allowing maximum growth, and
to prevent an excess of carbohydrate which would result in enhanced
EPS production, particularly in the fed-batch assays. As mentioned
previously, a 6% glucose level was chosen using the AS data. As expected,
lower CRV were obtained for the fed batch productions, during which
glucose concentration was equal or lower than 12 g/l. However similar
final lactate concentrations and populations as for the batch production
were obtained. The excess of glucose at the end of the fed batch process
indicates that the feeding rate of glucose should be lowered towards
the end of the fermentation. Because a lower viscosity was obtained
in the fed-batch MMRS cultures, compared to control batch fermentations,
it can be expected that cell recovery by centrifugation at
The current formulation of MMRS is expensive and other formulations could be used commercially. If defined media are used, these data show that it would be useful to add all the nitrogen-based compounds (peptones, yeast extracts) at the beginning of the fermentation and add the carbohydrate under fed-batch.
study showed the effects of different culture conditions on growth
of L. rhamnosus RW-9595M and on viscosity of fermented media.
Lower viscosities and good growth were obtained by maintaining the
growth temperature at
This study shows that many approaches are possible for the production of concentrated suspensions of valuable probiotic or starter EPS-producing cultures, which can be adapted to the equipment and media available or the strains involved.
Gratitude is expressed to Ms. Cathy St-Laurent for technical assistance.
BERGMAIER, D.; CHAMPAGNE, C.P. and LACROIX, C. Exopolysaccharide production during batch cultures with free and immobilized Lactobacillus rhamnosus RW-9595M. Journal of Applied Microbiology, November 2003, vol. 95, no. 5, p. 1049-1057. [CrossRef]
BROADBENT, Jeffery R.; MCMAHON, Donald J.; OBERG, Craig J. and WELKER, Dennis L. Use of exopolysaccharide-producing cultures to improve the functionality of low fat cheese. International Dairy Journal, July 2001, vol. 11, no. 4-7, p. 433-439. [CrossRef]
CACHON, R. and DIVIÈS, C. Modeling of growth and lactate fermentation by Lactococcus lactis ssp. lactis biovar. diacetylactis in batch culture. Applied Microbiology and Biotechnology, October 1993, vol. 40, no. 1, p. 28-33. [CrossRef]
CHAMPAGNE, C.P.; GAUDRAULT, H. and CONWAY, J. Effect of ultrafiltration of bakers and brewers yeast extracts on their nitrogen content and turbidity. Acta Alimentaria, November 1999, vol. 28, no. 4, p. 321-328. [CrossRef]
CHEIRSILP, Benjamas; SHIMIZU, Hiroshi and SHIOYA, Suteaka. Enhanced kefiran production by mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae. Journal of Biotechnology, January 2003, vol. 100, no. 1, p. 43-53. [CrossRef]
DE VUYST, L.; VANDERVEKEN, F.; VAN DE VEN, S. and DEGEEST, B. Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their grown-associated biosynthesis. Journal of Applied Microbiology, June 1998, vol. 84, no. 6, p. 1059-1068. [CrossRef]
DE VUYST, Luc and DEGEEST, Bart. Heteropolysaccharide from lactic acid bacteria. FEMS Microbiology Reviews, April 1999, vol. 23, no. 2, p. 153-177. [CrossRef]
DE VUYST, Luc; DE VIN, Filip; VANINGELGEM, Frederik and DEGEEST, Bart. Recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria. International Dairy Journal, 2001, vol. 11, no. 9, p. 687-707. [CrossRef]
DOLEYRES, Y. and LACROIX, C. Technologies with free and immobilised cells for probiotic bifidobacteria production and protection. International Dairy Journal, October 2005, vol. 15, no. 10, p. 973-988. [CrossRef]
GAMAR-NOURANI, L.; BLONDEAU, K. and SIMONET, J.-M. Influence of culture conditions on exopolysaccharide production by Lactobacillus rhamnosus strain C83. Journal of Applied Microbiology, October 1998, vol. 85, no. 4, p. 664-672. [CrossRef]
GROBBEN, G.J.; SIKKEMA, J.; SMITH, M.R. and DE BONT, J.A.M. Production of extracellular polysaccharides by Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 grown in a chemically defined medium. Journal of Applied Bacteriology, 1995, vol. 79, no. 1, p. 103-107.
HOUTSMA, P.C.; DE WIT, J.C. and ROMBOUTS, F.M. Minimum inhibitory concentration (MIC) of sodium lactate and sodium chloride for spoilage organisms and pathogens at different pH values and temperatures. Journal of Food Protection, December 1996, vol. 59, no. 12, p. 1300-1304.
LAMBOLEY, Laurence; ST-GELAIS, Daniel; CHAMPAGNE, Claude P. and LAMOUREUX, Maryse. Growth and morphology of thermophilic dairy starters in alginate beads. The Journal of General and Applied Microbiology, June 2003, vol. 49, no. 3, p. 205-214. [CrossRef]
MACEDO, M.G.; LACROIX, C.; GARDNER, N.J. and CHAMPAGNE, C.P. Effect of medium supplementation on exopolysaccharide production by Lactobacillus rhamnosus RW-9595M in whey permeate. International Dairy Journal, 2002, vol. 12, no. 5, p. 419-426. [CrossRef]
MORISHITA, Takashi; DEGUCHI, Yoriko; YAJIMA, Masako; SAKURAI, Toshizo and YURA, Takashi. Multiple nutritional requirements of lactobacilli: genetic lesions affecting amino acids biosynthesis pathways. Journal of Bacteriology, October 1981, vol. 148, no. 1, p. 64-71.
Fernanda; OLIVER, Guillermo; SAVOY DE GIORI, Graciela and FONT DE
Fernanda; SAVOY DE GIORI, Graciela; OLIVER, Guillermo and FONT DE
PHAM, P.L.; DUPONT, I.; ROY, D.; LAPOINTE, G. and CERNING, J. Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation. Applied and Environmental Microbiology, June 2000, vol. 66, no. 6, p. 2302-2310. [CrossRef]
PRASHER, R.; MALIK, R.K. and MATHUR, D.K. Utilization of whey for the production of extracellular polysaccharide by a selected strain of Lactococcus lactis. Microbiologie, Aliments, Nutrition, 1997, vol. 15, no. 1, p. 79-88.
RUAS-MADIEDO, Patricia; HUGENHOLTZ, Jeroen and ZOON, Pieternela. An overview of the functionality of exopolysaccharides produced by lactic acid bacteria. International Dairy Journal, 2002, vol. 12, no. 2-3, p. 163-171. [CrossRef]
RUAS-MADIEDO, Patricia; ALTING, Arno C. and ZOON, Pieternela. Effect of exopolysaccharides and proteolytic activity of Lactococcus lactis subsp. cremoris strains on the viscosity and structure of fermented milks. International Dairy Journal, February 2005, vol. 15, no. 2, p. 155-164. [CrossRef]
SHIHATA, A. and SHAH, N.P. Influence of addition of proteolytic strains of Lactobacillus delbrueckii subsp. bulgaricus to commercial ABT starter cultures on texture of yogurt, exopolysaccharide production and survival of bacteria. International Dairy Journal, 2002, vol. 12, no. 9, p. 765-772. [CrossRef]
STIEBER, R.W.; COULMAN, G.A. and GERHARDT, P. Dialysis continuous process for ammonium-lactate fermentation of whey: Experimental tests. Applied and Environmental Microbiology, December 1977, vol. 34, no. 6, p. 733-739.
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