Research in Microbiology 154 (2003) 705712 www.elsevier.

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Isolation and characterization of two exopolysaccharides produced by Lactobacillus plantarum EP56

Richard Tallon a , Philippe Bressollier a,b , Maria C. Urdaci a,
a Laboratoire de Microbiologie et Biochimie Applique, ENITA de Bordeaux, 1, cours du Gnral de Gaulle, BP 201, 33175 Gradignan, France b IUT, Dpartement Gnie Biologique, alle Andr Maurois, 87065 Limoges, France

Received 10 April 2003; accepted 5 September 2003 First published online 8 September 2003

Abstract A Lactobacillus plantarum strain producing exopolysaccharides (EPSs) was isolated from corn silage. When this strain, named L. plantarum EP56, was grown on a chemically dened medium, two EPS fractions were isolated. The cell-bound EPS fraction (EPS-b) was composed of a single high-molecular-mass polymer of 8.5 105 Da containing glucose, galactose and N -acetylgalactosamine in a molar ratio of approximately 3:1:1 and traces of glycerol and phosphoglycerol. The released EPS fraction (EPS-r) was composed of the high-molecular-mass bound polysaccharide and a second polymer of 4 104 Da containing glucose, galactose and rhamnose in a molar ratio of 3:1:1 and traces of glycerol and phosphoglycerol. EPS-b and EPS-r contained phosphate which contributes to their negative net charge. Studies on polysaccharide production and location showed that both polymers were synthesized during the exponential growth phase and that the EPS-b polymer was progressively released into the culture medium during the stationary growth phase. Carbon source and temperature inuenced EPS synthesis when L. plantarum EP56 was grown in a chemically dened medium. Lactose was the most efcient carbon source among the ve tested (glucose, galactose, fructose, lactose and sucrose). EPS production was also increased when the incubation temperature is lowered. 2003 Elsevier SAS. All rights reserved.
Keywords: Lactic acid bacteria; Lactobacillus plantarum; Exopolysaccharides

1. Introduction Several bacteria are known to synthesize exopolysaccharides (EPSs). EPSs occur in two forms depending on their location: as a capsule (capsular polysaccharides) where the polymer is closely associated with the cell surface, and as slime polysaccharides that are loosely associated with the cell surface. Such a distinction may be difcult since some strains release capsular polysaccharidic material at the periphery [32]. For the cells, EPSs are thought to play a role in protection against desiccation, toxic compounds, bacteriophages, osmotic stress, and to permit adhesion to solid surfaces and biolm formation [9]. In the food industry, these polymers are used as biothickeners because of their stabilizing, emulsifying or gelling properties. Bacterial polysaccharides can also be differentiated by their chem* Corresponding author.

E-mail address: m-urdaci@enitab.fr (M.C. Urdaci). 0923-2508/$ see front matter 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2003.09.006

ical composition. Homopolysaccharides are composed of only one monosaccharide (glucose or fructose, mainly), and heteropolysaccharides are composed of at least two different monosaccharides. Other residues such as sn-glycerol3-phosphate, N -acetyl-aminosugars, phosphate, and acetyl groups can also be found [21]. Both location and composition, in addition to other criteria such as molecular mass and conformation, inuence the properties of polysaccharides. EPS characteristics and amounts can be inuenced by several factors such as composition of the medium (carbon and nitrogen sources), as well as incubation conditions (temperature, pH, time, etc.) [3]. EPSs produced by lactic acid bacteria (LAB) are the subject of an increasing number of studies. LAB are foodgrade organisms, possessing the generally-recognized-assafe (GRAS) status, and can produce EPSs that are potentially useful as safe additives to improve texture and viscosity of natural fermented milk products and to prevent syneresis. Moreover, it has been suggested that some

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EPSs produced by lactic acid bacteria may confer health benets to the consumer. Some studies have indicated that these EPSs may have immunostimulatory [17] and antitumoral [12] activity, and that phosphate groups in EPSs play an important role in the activation of macrophages and lymphocytes [19,20,24]. A large number of LAB have been investigated for EPS production. These studies were concerned with thermophilic species of technological interest: Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Streptococcus thermophilus. However, there is a growing interest in EPS production by mesophilic bacteria, but this mainly concerns bacteria of dairy industry interest such as Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus rhamnosus, and Lactobacillus casei. Among mesophilic LAB, Lactobacillus plantarum is frequently isolated from food products and is also widely used in industry for fermented food (vegetables, sausages, etc.). L. plantarum is one of the most studied LAB species, particularly because some strains are considered to be probiotics due to several of their properties [2]. Some strains have shown an ability to reduce and eliminate potentially pathogenic microorganisms by synthesis of antimicrobial agents and by competition with pathogens for receptor sites at the intestinal mucosa [1]. Thus, L. plantarum EPSproducing strains may be interesting in two respects: for industrial application, such strains may be used in the food industry because of their GRAS status as a textural agent and as a new potentially probiotic strain. Figueroa et al. [13] have isolated ropy strains of L. plantarum from cassava fermentation, but this study principally focused on phenotypic aspects and did not use a molecular approach. In our laboratory, we have isolated a L. plantarum EPS-producing strain from corn silage. The inuence of growth parameters on EPS production, and the isolation and biochemical characterization of two different polymers are described.

where sugars were sterilized separately. CDM with glucose as a carbon source was also used to study the effect of temperature on EPS production and location. CDM was preferred to MRS because this complex medium contains components such as beef extract, peptone, and yeast extract that can interfere with sugar quantication and composition determination [4]. 2.3. Extraction and purication of exopolysaccharides Two methods were used to differentiate bound EPS (EPS-b) from EPS released into the culture medium (EPS-r). The EPS-b extraction method was adapted from Toba et al. [30]. Ten milliters of culture samples were centrifuged at 15 000 g for 15 min at 4 C. The pellet containing cells was washed in 5 ml of sterile physiological solution and then centrifuged at 15 000 g for 15 min at 4 C. The viscous pellet was suspended in 5 ml of 1 M NaCl. EPS-b was dissociated from cells by sonication at 40 W for 3 min at 4 C with a Vibra cell 75021 sonicator (Bioblock Scientic, Illkirch, France) equipped with a SM 1298 probe. Samples were then centrifuged at 6000 g for 30 min at 4 C to eliminate insoluble material. The EPS was precipitated from the supernatant by addition of two volumes of cold ethanol followed by an overnight incubation at 4 C. After centrifugation, 30 min at 6000 g , 4 C, the pellet containing EPS-b was resuspended in 2 ml of distilled water and dialyzed (molecular weight cut-off: 60008000 Da) against 5 l of distilled water for 2 days with three water changes per day. An alternative method using ethylenediamine tetraacetic acid (EDTA) has also been tested to isolate EPS-b fraction. The viscous pellets were resuspended in 5 ml of 0.05 M EDTA. The mixture was incubated under gentle agitation for 4 h at 4 C and then centrifuged at 6000 g for 30 min at 4 C. EPS were precipitated with ethanol as described above. To isolate the EPS released into the culture broth, supernatants obtained from 10-ml samples, after centrifugation, were treated with trichloroacetic acid at a nal concentration of 20% and incubated for 2 h at 4 C under gentle agitation. Precipitated proteins were removed by centrifugation at 25 000 g for 20 min at 4 C. The EPS was precipitated from the supernatant with two volumes of cold ethanol followed by an overnight incubation at 4 C. After centrifugation, 6000 g for 30 min at 4 C, the pellet containing EPS-r was dialyzed as described for the EPS-b fraction. 2.4. Analytical methods The total amount of carbohydrate in the EPS was determined using the phenol/sulfuric acid method [11] with glucose as a standard. Results were expressed in mg equivalent of glucose per liter of growth medium. To compare EPS production under different culture conditions, EPS amounts were expressed as specic production dened as the amount of polysaccharides produced per optical density (OD)600

2. Materials and methods 2.1. Bacterial strains In a preliminary screening for EPS-producing Lactobacillus strains based on mucoid phenotype criteria observed in MRS agar medium [8] at 25 C, a strain identied by the polymerase chain reaction (PCR)-specic reaction [25] as L. plantarum was isolated. This strain, isolated from corn silage, was named L. plantarum EP56. 2.2. Culture conditions for EPS production Carbon source assays were carried out in 400 ml of chemically dened medium (CDM) [18] containing 20 g/l of either glucose (CDM-glu), galactose (CDM-gal), lactose (CDM-lac), fructose (CDM-fru) or sucrose (CDM-suc),

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unit. The value was calculated when maximum OD was attained, i.e., at the beginning of the stationary phase. Protein content was determined by the Bradford method, using the Bio-Rad assay reagent (Bio-Rad, Munich, Germany) and bovine serum albumin as a standard. Phosphorus content was determined using ammonium molybdate according to the procedure of Rouser et al. [28] on 400 g of EPS, previously hydrolyzed with 2 M triuoroacetic acid for 2 h at 120 C. Na2 HPO4 was used as a standard. 2.5. Analysis of cells and puried EPS net charge Alcian blue is a cationic dye used to stain acidic polysaccharides. Ten microliters of a 0.1% alcian blue solution in acetic acid were directly mixed on a slide with 10 l of an 18-h-old culture of L. plantarum EP56 in CDM. The dye induces cell aggregation with strains that can produce negatively charged polymers and therefore allows a direct observation of the polymer producers. The negative net charge of the puried EPS was analyzed by anion exchange chromatography (AEC) on a quaternary methyl ammonium (QMA) Accel plus column (1.5 m 20 cm, Waters). The column was eluted at a 2 ml/min ow rate, with 0.05 M NH4 HCO3 , pH 8.0, and followed by a linear gradient of 0.05 to 2 M NaCl in the same buffer. Polysaccharides were monitored by UV detection at 210 nm. Five-milliters fractions were collected, and sugar composition and molecular mass were determined. 2.6. Monosaccharide analysis Sugar composition of isolated EPSs was determined by two procedures using myo-inositol as an internal standard. For neutral and amino sugars, 100 g equivalent of glucose of puried polysaccharide was submitted to methanolysis with methanolic HCl, 0.9 M, for 16 h at 80 C. The resulting mixture of methylglycosides dried under nitrogen at room temperature was re-N -acetylated by addition of 50 l of dry methanol, 5 l of pyridine, and 5 l of acetic anhydride [5]. After drying and derivation with 15 l of trimethylsilylimidazole (TMSIM, Alltech France) at room temperature for 30 min, the re-N -acetylated trimethylsilylated (TMS) glycosides were analyzed (i) on a BP1 fused-silica capillary column (12 m 0.32 mm, SGE) with a Peri 2000 gas-liquid chromatography, ame ionization detector (GLC-FID) chromatograph (Perichrom, France), the temperature program was 140 to 240 C at 6.1 C/min followed by an isothermal elution, a split injector (split ow 1/25) was used, and (ii) on a PTE 5 fused-silica capillary column (30 m 0.32 mm, Supelco) with an 8060/MD 800 GLC-mass spectometry (GLC-MS) system (Fisons instruments, Rodano, Italy). Before analysis on the GLC-MS system, TMS samples were dried under nitrogen, dissolved in 500 l of hexane, and 1 l was injected using a splitless injector. The oven temperature program included an initial temperature of 80 C for 2 min,

followed by an increase to 235 C at a rate of 20 C/min, then held at 235 C for 2 min. Detection of phosphorylated components was adapted from Robijn et al. [26]. EPS (500 g) was hydrolyzed with 2 M TFA, reduced with NaBH4 , neutralized and lyophilized. After a rst trimethylsilylation of the dry residue with trimethylchorosilane (TMCS, Alltech, France), free phosphate groups were methylated in the presence of a diazomethane saturated ether solution. A second trimethylsilylation was performed on the dried residue, and the resulting mixture of TMS (phosphate-methylated) alditols was qualitatively analyzed by GLC-MS on a PTE 5 column. Five microliter samples were dried under N2 , dissolved in 20 l of hexane. One microliter was injected using the splitless injector, and a temperature program including an initial temperature of 80 C for 2 min followed by an increase to 220 C at a rate of 4 C/min was applied. 2.7. Molecular mass determination The apparent molecular mass of puried EPSs was determined by high-performance size-exclusion chromatography (HPSEC). Chromatography was performed on a 600E system (Waters) equipped with a PL aquagel-OH 60, 8 m column (30 cm 7.5 mm, Polymer Laboratories). The column was eluted at a ow rate of 0.8 ml/min with a 0.2 M sodium acetate buffer (pH 5.1). The sample volume was 20 l, containing 25 g of puried EPS. Compounds were detected using refractive index monitoring (Model 475, Kontron Instruments, Schlieren, Switzerland), and standard dextrans (7 104 to 4.9 106 Da, Sigma) were used to determine molecular weights. 2.8. Glycohydrolase activity assays L. plantarum EP56 was grown at 37 C in CDM. Two milliliters of culture broth were centrifuged at 10 000 g for 10 min at 4 C at different time intervals of growth (exponential, beginning of stationary phase, and end of stationary phase). The extracellular glycohydrolase activity was measured on supernatants. Pellets were washed with 2 ml of physiological saline buffer and resuspended in 2 ml of the same solution. Cells were disrupted by six 15-s treatments in a bead beater at 4 C using 0.1-mm diameter microbeads interrupted by 30-s periods to cool samples. Cell debris were pelleted by centrifugation. Intracellular glycohydrolase activities were measured in supernatants. Pellets containing cell debris were resuspended in 400 l of 50 mM phosphate-buffered saline (PBS) buffer and were used to determine cell-linked glycohydrolase activities. Glycohydrolase activities, in regard to -D-galactosidase, -D-galactosidase, -D-glucosidase, -D-glucosidase, N -acetyl--D-galactosaminidase, N -acetyl--D-glucosaminidase and -L-rhamnosidase, were determined at 37 C at pH 4.0 and 7.0 by measuring the rate of p -nitrophenol (PNP)

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released from the appropriate p -nitrophenyl sugars. The reaction was stopped by addition of 0.5 ml of 1 M Na2 CO3 , and the amount of PNP released was determined by spectrophotometry at 405 nm. One unit of enzyme activity was dened as the amount of cells required to liberate 1 mol of p -nitrophenol per min. The ability of produced glycohydrolases to degrade L. plantarum EP56 EPSs was determined by incubating EPS-b and EPS-r with the active fraction of glycohydrolase in 50 mM PBS buffer at pH 7.0. Changes in apparent molecular mass were determined by HPSEC as described above as a function of incubation time.

up to 97 h to reach 79.3 mg/l, while the amount of EPS-b decreased. All assays conducted under other conditions of incubation temperature and carbon source showed the same evolution for both EPS-b and EPS-r fractions (data not shown). 3.2. Inuence of temperature on growth and polymer synthesis L. plantarum EP56 was grown in static asks in CDM. Four temperatures were tested: 18, 25, 30 and 37 C. Results are presented in Table 1. Incubation temperature appeared to have no effect on maximum OD. Cultures of L. plantarum EP56 reached a maximum OD of approximately 5.0 (which corresponds to about 2.3 109 cells/ml) whatever the temperature. Nevertheless, growth kinetics were affected by incubation temperature. Maximum growth rate (max ) ranged from 0.30 to 1.02/h when temperature varied from 18 to 37 C. Concerning EPS synthesis, the same total EPS production prole was observed for all conditions, as described above. The EPS yield expressed in mg/l EPS per OD unit showed comparable variations with total EPS production. Values presented in Table 1 result from sampling at the beginning of the stationary growth phase when maximum OD and EPS amounts are attained. There was an inverse relationship between EPS production and growth temperature. The maximum polymer amount (135.8 mg/l) was attained at the lowest incubation temperature tested (18 C). At 25 and 30 C, the maximum EPS amount was 16% and 44% lower than that produced at 18 C. When incubation temperature approached the optimal growth temperature of 37 C, the maximum EPS amount decreased sharply and was 4 times lower than the amount of EPS produced at 18 C. Just as the maximum EPS amount was affected by incubation temperature, specic EPS production was inuenced by growth conditions also (Table 1). The yield of EPS increased from 7.0 to 27.2 mg/l/OD unit when the incubation temperature varied from 37 to 18 C. An incubation temperature of 25 C
Table 1 Effect of temperature and carbon source on growth and EPS production of L. plantarum EP56 grown in CDM Incubation temperature ( C) 18 25 30 37 25 25 25 25 25 Carbon source Glucose Glucose Glucose Glucose Glucose Galactose Fructose Sucrose Lactose Maximum max EPSmax EPS yield (/h)a (mg/l)a (mg/l/OD unit)a,b ODa 5.0 5.0 4.9 5.1 5.0 4.9 4.1 5.1 4.9 0.30 0.58 0.98 1.02 0.58 0.53 0.36 0.56 0.37 135.8 114.5 75.6 35.6 114.5 121.1 46.3 129.2 140.2 27.2 22.9 15.4 7.0 22.9 24.7 11.3 25.3 28.6

3. Results 3.1. Localization of polysaccharides Results of the investigation on EPS localization in L. plantarum EP56 grown at 25 C in CDM are presented in Fig. 1. The presence of polysaccharide was observed both in culture medium supernatants and in pellets which show a mucous aspect. Sonication treatment of pellets resuspended in 1 M NaCl revealed that a polysaccharide fraction was loosely linked to cells. The total amount of EPS (sum of cellbound and released EPS fractions) increased rapidly with the number of cells and reached a maximum of 126.4 mg/l in the stationary phase. Finally, after this maximum, the total EPS amount was stable. Two steps were distinguished for the cell-bound EPS (EPS-b) fraction production. The rst lasted from 0 to 25 h and showed an increase to 73.6 mg/l. As was noted for total EPS, the maximum EPS-b amount was attained at the beginning of the stationary growth phase and was followed by a second step, from 25 h to the end of fermentation, consisting of an important decrease from 73.6 to 44.6 mg/l. The amount of EPS-r fraction showed a different behavior throughout fermentation. At 25 h, the EPS-r amount reached 36.8 mg/l, but continued to increase

Fig. 1. Prole of growth and EPS production by L. plantarum EP56 in glucose-containing CDM at 25 C. (F), log OD; (2), total EPS; (Q), cell-bound EPS (EPS-b); ("), released EPS (EPS-r).

a Each value represents the average of three measurements. The maximal standard deviation between the three measurements was 7%. b One OD unit corresponds to approximately 4.5 108 colony-forming unit (CFU)/ml.

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was then chosen for further assays in order to obtain a high maximum growth rate with a signicant EPS production. 3.3. Inuence of carbon source In order to improve EPS production by L. plantarum, the inuence of carbon source was studied. Five carbon sources were tested. Results for growth and polymer production are shown in Table 1. Maximum OD was affected by carbon source. Values of approximately 5.0 were attained except with fructose, whose maximum OD was 4.1. As described for the temperature assays, the carbon source affects both growth kinetics and EPS production. Glucose, galactose and sucrose showed a maximum growth rate of 0.58, 0.53 and 0.56/h, respectively. Fructose and lactose were less favorable carbon sources for growth of L. plantarum EP56: the maximum growth rate decreased to 0.36 and 0.37/h, respectively, when these sugars were used for fermentation. The carbon source also inuenced EPS production. Glucose, galactose and sucrose produced maximum EPS amounts of 114.5, 121.1 and 129.2 mg/l, which means values from 22.9 to 25.3 mg/l/OD unit. When fructose was used as a carbon source, EPS production dramatically decreased to 46.3 mg/l (11.3 mg/l/OD unit). However, lactose, which is a poor carbon source in regard to the max value, resulted in the best EPS production: 140.2 mg/l EPS produced, which corresponds to an EPS yield of 28.6 mg/l/OD unit. 3.4. Biochemical characterization of EPS fractions EPS-b and EPS-r produced by L. plantarum EP56 in CDM at 25 C were isolated at the beginning of the stationary phase. The HPSEC elution pattern and size distribution analysis of the different EPSs synthesized by the strain showed only one peak for the EPS-b fraction and the average molecular mass was 8.5 105 Da. Analysis of the EPS-r fraction revealed two peaks which corresponded to EPSs with apparent molecular mass of 8.5 105 and 4 104 Da, respectively. The apparent molecular mass of both high- and low-molecular mass EPSs was not modied during growth. The protein content of each EPS extract (bound and released) was 2.5 and 1.1% (w/w), respectively. Both bound and released EPS fractions had a negative net charge because they were strongly adsorbed on an anion exchange QMA Sep-Pak cartridge (Waters). EPSs were eluted with NaCl concentrations from 0.5 to 2 M. At the same time, no retention was observed when EPSs were loaded on a cation exchange CM Sep-Pak cartridge. In an attempt to assess the charge distribution of each EPS fraction, EPS-r and EPS-b were loaded on an anion exchange column and eluted using a linear ionic strength gradient. The presence of only one chromatographic peak attested to the fact that EPS-b was homogenous in charge whereas EPS-r contained at least two species for this same criterium (Fig. 2).

(A)

(B) Fig. 2. Anion exchange chromatograms of bound EPS (A) and released EPS (B) produced by L. plantarum EP56.

3.5. Monosaccharide analysis and phosphorus content The sugar composition of puried EPS produced by L. plantarum EP56 grown at 25 C in CDM containing glucose was analyzed by GLC-FID and GLC-MS after methanolysis. Results are shown in Table 2. High-molecularmass bound EPS contained N -acetylgalactosamine, glucose and galactose in an approximate molar ratio of 1:3:1, respectively. The released EPS fraction was composed of a high-molecular-mass polymer whose composition was approximately 1:3:1 for N -acetylgalactosamine, glucose and galactose, respectively. This was similar to the bound polymer. The low-molecular-mass EPS which was found only in the released EPS fraction did not contain N -acetylgalactosamine but rhamnose, resulting in a molar ratio of approximately 1:3:1 for rhamnose, glucose and galactose, respectively. Traces of glycerol were observed in both the high- and low-molecular-mass HPSEC fractions. A qualitative GLC-MS analysis focusing on the detection of phospho-

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Table 2 Composition of puried EPSs from L. plantarum EP56 grown at 25 C in CDM with glucose as carbon source EPS fraction EPS MM (Da) 4 104 8.5 105 8.5 105 Galactose 19.9 17.4 17.8 Composition of EPSa in mol/100 mol Glucose Rhamnose 58.5 64.5 60.9 21.6 N -acetyl galactosamine 18.1 21.3 Phosphate content (% w/w) 1.6 1.6 1.6

Released EPS (EPS-r) Bound EPS (EPS-b)

a Each value represents the average of three measurements.

Fig. 3. Specic activity of glycohydrolases produced by L. plantarum EP56 in cell-bound fraction. (Q), -D-galactosidase; (P), N -acetyl-D-galactosaminidase; (!), -L-rhamnosidase; ("), -D-glucosidase; (1), N -acetyl--D-glucosaminidase; (2), -D-glucosidase.

rylated compounds in puried EPS revealed the presence of phosphoglycerol both in the high- and low-molecular-mass HPSEC fractions. The phosphate content of all fractions was colorimetrically determined to be 1.6% (w/w). This phosphate content in the high-molecular-mass EPS fraction corresponded to a 0.15 mol phosphate/mol repeating subunit, taking into account three glucose, one galactose and one N -acetylgalactosamine as constitutive monosaccharides for the EPS subunit. Growth of L. plantarum EP56 on different carbon sources and at different temperatures did not change the sugar composition of the EPS produced (data not shown). 3.6. Glycohydrolase production and localization In an attempt to localize glycohydrolases, enzyme activities (for enzymes -D-galactosidase, N -acetyl--D-galactosaminidase, -L-rhamnosidase, -D-glucosidase, N -acetyl--D-glucosaminidase, and -D-glucosidase) in extracellular, intracellular and cell-bound fractions were determined (Fig. 3). No glycohydrolase activity was present in the extracellular and intracellular fractions, whereas the presence of four enzyme (-D-galactosidase, -Dglucosidase, N -acetyl--D-glucosaminidase, and -D-glucosidase) activities were found in the cell-bound fraction. These four enzyme activities increased during growth, reached a maximum around 25 h of culture for -D-ga-

lactosidase, N -acetyl--D-glucosaminidase, and -D-glucosidase activities and around 13 h for -D-glucosidase activity, and then decreased. No activity was detected after 50 h. The cell-bound extracts were tested for their ability to degrade EPSs (high- and low-molecular-mass) by incubating polymers with crude enzymatic extracts. HPSEC chromatograms indicated that the apparent molecular mass of puried EPSs produced by L. plantarum EP56 was not affected after 36 h of incubation with crude enzymatic extracts.

4. Discussion This study reports for the rst time the isolation and biochemical characterization of EPSs produced by a strain of L. plantarum species. Strain EP56 synthesizes and excretes polysaccharides and shows a mucoid phenotype on MRS agar plates. The original aspect of EPS production by this strain consists of the presence of two EPS fractions when the strain is grown in liquid CDM: EPSs were isolated in culture supernatants as in most studies of EPS production by LAB; moreover, a second EPS fraction was isolated from viscous pellets resulting from the centrifugation of L. plantarum EP56 culture broth. This cell-linked EPS can be compared to capsular polysaccharides dened by Whiteld [32], because it is easily detached from cells by mild sonication. Moreover, incubating cell pellets with 0.1 M EDTA permitted

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isolation of this cell-linked EPS fraction. This indicates that the polysaccharide is weakly linked to cells by electrostatic interactions (ionic, hydrogen bonds, or hydrophobic interactions) instead of covalent bonds. This phenomenon has already been observed by Wicken et al. [33] on L. casei subsp. rhamnosus NCTC 10302 which synthesizes a rhamnosecontaining polysaccharide present as cell-bound material. Toba et al. [30] also isolated a cell-linked polysaccharide by sonication from L. lactis subsp. cremoris LAPT 3001. The layer of cell-associated EPS in addition to released EPS may confer interesting properties to L. plantarum EP56. Cell-linked polysaccharides produced by L. lactis MZ4010 protect cells against bacteriophages and lysozyme, whereas released EPS also protects against copper and nisin [22]. Analysis of EPSs revealed that the EPS-b fraction consisted of only one negatively charged polymer of 8.5 105 Da composed of glucose, galactose and N -acetylgalactosamine in an approximate ratio of 3:1:1. Small amounts of glycerol and phosphoglycerol were also detected. The negative net charge observed by AEC may be due to the presence of phosphate in a proportion of 1.6% (w/w). This was reinforced by dying cell suspensions with Alcian blue. Peripheral coloration and aggregation in the presence of the cationic dye was only observed in strain EP56, but not observed with the non-EPS-b-producing strain NCIMB 8826 or with strain EP56 after sonication (data not shown). The EPS-r fraction contained two polymers: a high-molecularmass polymer whose composition was identical to EPS-b, and a negatively charged low-molecular-mass polymer (4 104 Da) consisting of glucose, galactose and rhamnose in an approximate ratio of 3:1:1. Such heterogeneity in polysaccharide synthesis has already been observed [6,16,19]. Marshall et al. [23] reported that L. lactis subsp. cremoris LC330 is able to produce two polysaccharides which composition and molecular mass differ: one polymer closely associated with cell wall material and a second released into the culture medium. These observations on L. plantarum EP56 and the evolution of the ratios of both fractions during prolonged fermentation indicate that the high-molecular-mass polysaccharide is synthesized and remains attached to cells by weak bonds. A decrease in the amount of EPS-b observed in the second step may be due to desorption of the polymer rather than to enzymatic degradation, because glycohydrolases produced by L. plantarum EP56 have no effect on the polymer molecular mass. The progressive release of the EPS-b fraction could explain the continuous increase in EPS-r during the stationary growth phase which is concomitant with a decrease in EPS-b. Fermentation assays, under our dened conditions, suggest that EPS production is growth-associated. The yield of EPS increased during the exponential growth phase and no further production was observed in the stationary growth phase. These results are in agreement with observations on other species [10,14,31]. However, a number of studies have indicated that EPS production by LAB is inuenced by culture conditions. As described in several cases of mesophilic

LAB [4,7,14,23,31], a lowering of incubation temperature positively affects EPS production and negatively affects growth of L. plantarum EP56. This may be due to the limiting effects of availability of lipid carriers essential for both cell wall synthesis and polysaccharide production. The mechanism proposed by Sutherland [29] suggests that at a low growth rate, cells exhibit more isoprenoid glycosyl lipid carriers, whereas at a high growth rate, these precursors are used for cell wall synthesis and are less available for EPS production. Like the incubation temperature, the carbon source affects both growth and EPS synthesis. This was markedly observed with fructose. Grobben et al. [15] noted that regulation of the EPS biosynthetic pathway in L. bulgaricus NCFB 2772 may be dependent on the carbohydrate source. Growth of this strain on fructose inhibits the activity of some key enzymes in EPS synthesis. Degeest et al. [7] have also demonstrated that a linkage exists between enzyme activity in L. sakei 0-1 and EPS yield. Every other carbon source tested contains at least one of the monomeric constituents of EPSs. The pool of enzymes involved in EPS synthesis in these cases may be less complex and probably allow higher EPS yields. In the case of fermentation on CDM supplemented with lactose, the low growth rate could be explained by a decient transport system in this strain and may be related to the low -D-galactosidase activity measured. The nature of the monosaccharides constituting the two polymers is commonly found in LAB polysaccharides. The presence of glycerol has already been reported in polysaccharides produced by L. sakei [26] and L. paracasei [27]. However, glycerol molar ratios were higher (0.8 and 0.9 mol/mol repeating subunit for L. sakei and L. paracasei, respectively) compared to 0.15 in the case of L. plantarum EP56. This means that glycerol is not present in each subunit. Phosphate in polysaccharides, also observed in a few studies [19,20,26,27], may confer important properties to the L. plantarum EP56 polysaccharide, because phosphate groups are required for activation of lymphocytes [20,24] and antitumor activity [12,19]. Phosphate residues that confer a net negative charge to polymers may also have an important role in sequestering positively charged toxic compounds such as nisin and metal ions [22]. As L. plantarum is an important species for probiotic utilization [2], strains that are able to produce exopolysaccharides may also present an important interest. In conclusion, L. plantarum EP56 is able to produce a low-molecular-mass polysaccharide which is released into the culture medium and a high-molecular-mass polysaccharide loosely, partially and temporarily linked to cells. The nature of the bonds between this polymer and cells remains to be established. We must also study the potential properties of adhesion which could result from the presence of this cell-bound polysaccharide. The relationship between the heterogeneity of EPS synthesis and genetic determinants must be determined in order to understand the biosynthetic regulation mechanisms.

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Acknowledgements We thank Bernard Verneuil from IUT Limoges, Dpartement Gnie Biologique for his help in the analysis of mass spectrometry results. We also thank Kathryn Mayo for critical reading of the manuscript. References
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