Exopolysaccharide production by Streptococcus thermophilus SY ...

Journal of Applied Microbiology 2002, 92, 297–306

Exopolysaccharide production by Streptococcus thermophilus SY: production and preliminary characterization of the polymer A. Ricciardi, E. Parente, M.A. Crudele, F. Zanetti1, G. Scolari2 and I. Mannazzu3 Dipartimento di Biologia, Difesa e Biotecnologie Agro-forestali, Universita` della Basilicata, Potenza, 1POLYtech Scarl, Trieste, 2Istituto di Microbiologia, Universita` Cattolica del Sacro Cuore, Piacenza and 3Dipartimento di Biotecnologie Agrarie ed Ambientali, Universita` di Ancona, Ancona, Italy 831/07/01: received 24 January 2001, revised 6 August 2001 and accepted 23 August 2001

A . R I C C I A R D I , E . P A R E N T E , M . A . C R U D E L E , F . Z A N E T T I , G . S C O L A R I A N D I . M A N N A Z Z U . 2002.

Aims: To evaluate the effect of yeast extract (YE) concentration, temperature and pH on growth and exopolysaccharide (EPS) production in a whey-based medium by Streptococcus thermophilus SY and to characterize the partially purified EPS. Methods and Results: Factorial experiments and empirical model building were used to optimize fermentation conditions and the chemical composition, average molecular weight (MW) and rheological properties of aqueous dispersions of the EPS were determined. Exopolysaccharide production was growth associated and was higher (152 mg l–1) at pH 6Æ4 and 36°C with 4 g l–1 YE. High performance size exclusion chromatography of the partially purified EPS showed two peaks, with a weight average MW of 2 · 106 and 5 · 104, respectively. The EPS was a heteropolysaccharide, with a glucose : galactose : rhamnose ratio of 2 : 4Æ5 : 1. Its water dispersions had a pseudoplastic behaviour and showed a higher viscosity of xanthan solutions. Significance and Impact of the Study: The fermentation conditions and some properties of an EPS produced by Strep. thermophilus, a dairy starter organism, were described.

INTRODUCTION Lactic acid bacteria (LAB) are used in food fermentations because of their ability to improve the flavour, aroma, texture and safety of perishable raw materials (milk, meat and vegetables). Among the products of their metabolism, extracellular polysaccharides (EPS) have received increasing attention in recent years (Cerning 1995; De Vuyst and Degeest 1999; Ricciardi and Clementi 2000). Lactic acid bacteria usually produce only small amounts of EPS (100–200 mg l–1; Cerning 1995) but yields up to 4 g l–1 have been obtained with Lactobacillus sakei O-1 (van den Berg et al. 1995). Small amounts of EPS produced in fermented milk (Rawson and Marshall 1997) and during cheese production (Low et al. 1998; Perry et al. 1998) have a strong impact on the texture and properties of these products. Although the fermentative yield of EPS produced Correspondence to: A. Ricciardi, Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Universita` della Basilicata, Campus di Macchia Romana, 85100 Potenza, Italy (e-mail: [email protected]). ª 2002 The Society for Applied Microbiology

by LAB is still very low compared with that of commercial EPS, such as xanthan, EPS produced by LAB may provide an interesting alternative to EPS produced from from nonfood-grade organisms (xanthan, gellan, etc.) because of their high thickening power and this has prompted the search for new strains and processes with higher yields (De Vuyst and Degeest 1999; Sutherland 1999). The production of EPS by mesophilic and thermophilic LAB has been reviewed recently (De Vuyst and Degeest 1999; Ricciardi and Clementi 2000). Several strains of Streptococcus thermophilus have been shown to produce EPS in milk or whey-based media (Cerning et al. 1988; Doco et al. 1990; Ariga et al. 1992; Mozzi et al. 1995; Bubb et al. 1997; Lemoine et al. 1997; Ricciardi et al. 1997), complex laboratory media (Petit et al. 1991) or defined media (Gancel and Novel 1994a). The yields of EPS do not usually exceed 400 mg l–1, but yields as high as 1100–3000 mg l–1 have been reported (Petit et al. 1991; De Vuyst et al. 1998). The EPS produced by Strep. thermophilus are heteropolysaccharides made up of branched chains containing glucose and galactose, rhamnose (Lemoine et al. 1997; Bubb et al. 1997)

298 A . R I C C I A R D I ET AL.

and sometimes N-acetyl-glucosammine (Doco et al. 1990) and fucose (Low et al. 1998). As for other EPS produced by LAB, the molecular weight (MW) of the EPS produced by Strep. thermophilus strains is 1–9 · 106 (De Vuyst and Degeest 1999; Ricciardi and Clementi 2000), but two polysaccharides with different MWs have been observed in some strains (Ariga et al. 1992). Exopolysaccharide production by Strep. thermophilus is apparently stimulated at lower temperatures (Cerning et al. 1988; Gancel and Novel 1994a) and, although it is usually growth associated, non-growth-associated EPS production has been observed at low temperatures (Gancel and Novel 1994a) or at low carbohydrate fluxes (1Æ5 mmol h–1) in fedbatch fermentation (Petit et al. 1991). The effect of pH has been evaluated in only a few studies and very rarely in fermentations at controlled pH. Thus, the optimal initial pH for EPS production with Strep. thermophilus S22 has been found to be 7Æ0 in lactose-containing media and 5Æ5 in sucrose-containing media. Few data are also available on the effect of medium composition; lactose availability has been found to influence both the kinetics of EPS production and EPS composition (Petit et al. 1991). Exopolysaccharide production in a defined medium by Strep. thermophilus S22 has been found to be higher with glucose and fructose and lower with lactose and sucrose, although the latter supported better growth (Gancel and Novel 1994a). In previous studies (Ricciardi et al. 1997) we found Strep. thermophilus SY, a strain isolated from yoghurt, to be able to produce EPS (160 mg l–1) in whey-based media both when growing alone and in association with Lact. delbrueckii subsp. bulgaricus. The aim of this work was the optimization of temperature, pH and yeast extract (YE) concentration for EPS production by Strep. thermophilus SY in batch fermentation at controlled pH and to obtain a preliminary characterization of the purified polysaccharide. M A T E R I A LS A N D M E T H O D S Strain Streptococcus thermophilus SY, isolated from yoghurt, was identified as an EPS producer using a method based on the measurement of efflux time in Microhaematocrit capillaries (Ricciardi et al. 1997). The strain was maintained as frozen ()75°C) stock in 10% non-fat dry milk and routinely propagated in whey broth (WB; Ricciardi et al. 1997) at 37°C for 16 h. Media and culture conditions Whey broth, which was used as a basal medium for growth and EPS production, had the following composition: deproteinized whey, 840 ml; YE permeate (YEP), 0–160 ml; tryptone

(Oxoid, Basingstoke, UK), 5 g l–1; distilled water to 1000 ml. Deproteinized whey was obtained by dissolving 40 g spraydried whey powder (Lactoserum Doux; Unilait International, Paris, France) in 1000 ml distilled water, adjusting the pH to 5Æ5 with 6 mol l–1 HCl, heating at 100°C for 30 min, removing the precipitate by centrifugation (6000 g for 10 min at 4°C), adjusting the pH to 7Æ0 with 6 mol l–1 NaOH, heating again at 100°C for 30 min, followed by filtration on GF/D filters (Whatman International, Maidstone, UK). The resulting medium contained 30 g l–1 lactose and remained clear even after sterilization. Yeast extract permeate was obtained by dissolving YE powder (Oxoid) in distilled water to a final concentration of 50 g l–1, followed by ultrafiltration at 4°C with a Sartocon Micro unit (Sartorius GmbH, Goettingen, Germany) with an NMWCO (Nominal Molecular Weight Cut-Off) of 10 000 Da. A single batch of YEP was made and stored frozen (–20°C) until needed. Ultrafiltration was used in order to remove the glucans and mannans which are found in YE and which would have interfered with the purification and determination of EPS composition. For inoculum growth, WB was prepared with a final concentration of 2Æ5 g l–1 YE and the pH buffered by the addition of 0Æ1 mol l–1 3-(N-morpholino) propanesulphonic acid (Fluka, Sigma-Aldrich Srl, Milan, Italy). The pH was adjusted to 6Æ9 and the medium sterilized in screw cap bottles at 121°C for 15 min. Inoculum growth was carried out at 37°C for 16 h. To evaluate the effect of YE, temperature and pH on growth and EPS production, WB was prepared with a YE concentration varying between 0 and 8 g l–1; pH (5Æ6–7Æ2) and temperature (24–48°C) were also varied according to the central composite design described in Table 1. A total of 17 fermentations, including three replicates for treatment 15, were carried out. Experimental runs were carried out in random order. Two stirred tank reactors (Applikon, Schiedam, The Netherlands) with a working volume of 1 L and equipped with instrumentation for the measurement and control of temperature and pH (by addition of 6 mol l–1 NaOH) and agitation (magnetic stirrer, 250 rev min–1) were used for batch fermentations and were inoculated with 10% (v/v) of a 16-h culture of Strep. thermophilus SY in WB. Three further fermentations were carried out using a YE concentration of 4 g l–1, at pH 6Æ4 and 36°C with low agitation speed (100 rev min–1), in a 3-l in-situ sterilizable stirred tank reactor (Chemap AG; Chemap, Volketswil, Switzerland) equipped with a controller (DCU3000; Bbraun Biotech GmbH, Melsungen, Germany) to confirm the results of the factorial experiment. During fermentation the broth was aseptically sampled, treated at 100°C for 15 min to dissolve cell-bound EPS and centrifuged (5000 g for 10 min) in preweighed tubes. The pellet was washed with distilled water and dried at 105°C for 24 h to measure cell dry weight. The supernatant fluid was

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 92, 297–306

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EXOPOLYSACCHARIDE PRODUCTION BY STREPTOCOCCUS THERMOPHILUS

Table 1 Central composite design for the evaluation of the effect of pH, temperature (T, °C) and yeast extract concentration (YE, g l)1) on growth and exopolysaccharide (EPS) production by Streptococcus thermophilus SY in batch fermentation in a whey-based medium

Run

pH (x1)

T (x2)

YE (x3)

l (h)1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

6Æ0 6Æ8 6Æ0 6Æ8 6Æ0 6Æ8 6Æ0 6Æ8 5Æ6 7Æ2 6Æ4 6Æ4 6Æ4 6Æ4 6Æ4

30 30 42 42 30 30 42 42 36 36 24 48 36 36 36



()1) (+1) ()1) (+1) ()1) (+1) ()1) (+1) ()2) (+2) (0) (0) (0) (0) (0)

()1) ()1) (+1) (+1) ()1) ()1) (+1) (+1) (0) (0) ()2) (+2) (0) (0) (0)

()1) ()1) ()1) ()1) (+1) (+1) (+1) (+1) (0) (0) (0) (0) ()2) (+2) (0)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±


Xmax (g l)1)

EPSmax (mg l)1)

YE/X (mg g)1)

1Æ80 2Æ30 2Æ00 2Æ24 2Æ25 2Æ38 2Æ17 2Æ45 1Æ60 1Æ70 2Æ11 0Æ25 1Æ79 2Æ55 2Æ4 ± 0Æ1

80 109 106 117 119 147 102 101 40 49 95 58 21 64 152

46 59 51 53 46 68 42 43 27 30 47 314 16 35 52

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4 3 3 4 4 5 3 4 2 2 1 41 1 3 3

Coded values (xi) for the factors (pH, T and YE) are shown in parenthesis. For run 15 the mean and S.E. of three replicate fermentations are shown while for average specific growth rate (l) and EPS yield for unit biomass, the S.E. of the estimate is shown.

stored at – 20°C. The EPS concentration was measured by the Dubois method, after removing simple carbohydrates with a desalting gel (Ricciardi et al. 1998). Lactose/galactose and L(+)-lactic acid concentrations were measured using enzymatic test combination kits (Boehringer Mannheim, Mannheim, Germany). Preliminary characterization of exopolysaccharide produced by Streptococcus thermophilus SY Culture conditions and isolation of the polysaccharide. The EPS was recovered and purified from the culture broth of a fermentation carried out in the Chemap fermenter, using WB with 4 g l–1 YE (as YEP) and carrying out the fermentation at 36°C and pH 6Æ4. After 8 h, cell-bound EPS was dissolved by raising the temperature to 80°C and the speed to 600 rev min–1 for 15 min. After cooling, the biomass was removed by centrifugation (5000 g for 10 min) and the supernatant fluid concentrated threefold by ultrafiltration at 4°C with a 30-kDa Sartocon Micro (Sartorius) module. The EPS was precipitated by the addition of three volumes of ethanol at – 20°C, followed by centrifugation at 10 000 g for 20 min at 4°C. The pellet was redissolved in 100 ml distilled water and reprecipitated twice as described above. The partially purified polysaccharide was dissolved in distilled water and freeze dried (Minifast 6000; Edwards High Vacuum International, Cranley, UK). Molecular mass determination. A volume of Millipore MQ ultrapure water (Millipore, Bedford, MA, USA),

corresponding to about half of the total volume (10 ml) was added to 50 mg of the freeze-dried sample; the solution was shaken for 12 h and 5Æ0 ml of a 0Æ3-mol l–1 NaCl solution then added to obtain a final molarity of 0Æ15 mol l–1; the sample volume was adjusted with water. The solution was filtered (0Æ45 lm; Sartorius) before high performance liquid chromatography (HPLC) determination. The average molecular mass of the polysaccharide was determined by injecting 100 ll of the solution in an HPLC apparatus (PU880; Jasco Europe srl, Milano, Italy) with a Rheodyne 9125 injector using a TSK PWxl G6000 + G5000 + G3000 column set (TosoHaas GmbH, Stuttgart, Germany), 300 · 7Æ8 mm ID, 13, 10, 6 lm particle size at 40°C using 0Æ15 mol l–1 NaCl as an eluent, with a flow of 0Æ8 ml min–1. A LALLS CMX-100 (P0 ¼ 200 mV; ThermoQuest, Egelsbach, Germany) and a Differential Refractive Index (410; Waters, Milford, MA, USA) with a 128· sensitivity at 32°C were used as detectors. Chemical composition of the exopolysaccharide. The freeze-dried sample was hydrolysed by the following procedure: 200 mg were dissolved in 1 ml 2 mol l–1 trifluoroacetic acid and heated at 100°C for 15 h in sealed ampoules. Before the gas chromatographic analysis, trifluoroacetic acid was removed from samples by freeze drying the hydrolysates in a speed-vac apparatus (Heto V-R1, Allerød, Denmark). Sugar monomers were converted to alditol acetates according to the method of Sawardeker et al. (1965) and injected (25 ll) into a stainless steel column, 1Æ80 m, 32 mm I.D., packed with 2% diethylene glycol adipate on 100/120 mesh Chromosorb W AW-DMCS (Supelco, Milano, Italy) fitted



on an HR PTV 2CH gas chromatograph (model 6800; DANI, Monza, Italy). Raw data were collected by a chromatographic workstation (Maxima 820; Waters, Milford, MA, USA). The column was conditioned at 215°C and the elution carried out in isothermal conditions with a carrier gas (N2) flow rate of 30 ml min–1. The injector and the flame ionization detector (FID) were maintained at 350 and 325°C, respectively. Rheological properties of the exopolysaccharide. Partially purified EPS was dissolved in a volume of hyperpure water to obtain a final concentration of 5 g l–1. Solutions (5 g l–1) of xanthan (Sigma) and high viscosity alginate from Macrocistis pyrifera (Sigma) were used as a control. A digital viscometer (Brookfield DV-I+; Brookfield Engineering Laboratories, Stoughton, MA, USA) with a small sample adapter and a S21C coaxial cylinder spindle was used for viscosity determinations at 0Æ5, 1Æ0, 2Æ0, 5Æ0, 10, 20, 50 and 100 rev min–1 at 20°C. Speed and viscosity values were converted in shear rate and shear stress. Statistics Statistics and graphics were carried out using Systat 7 for Windows (SPSS, Chicago, IL, USA). RESULTS Optimization of exopolysaccharide production Streptococcus thermophilus SY produces EPS in whey-based media (Ricciardi et al. 1997). In order to evaluate the effect of pH (5Æ6–7Æ2), temperature (T; 24–48°C) and YE concentration (0–8 g l–1) on growth and EPS production by Strep. thermophilus SY a composite factorial experiment was carried out. The coded and uncoded values of the factors (pH, T and YE) are shown in Table 1 while the kinetics of growth and EPS production for runs 9, 10, 13, 14 and 15 are shown as an example in Fig. 1. In all fermentations growth started immediately, without a lag phase, and continued exponentially for 4–8 h. The average specific growth rate (l; Table 1) was estimated from the slope of the linear portion of the semilogarithmic growth curves. The maximum biomass concentration (Xmax; Table 1) was obtained within 6–8 h for runs 1–10 and 14–15 and within 12–14 h at 24 and 48°C (data not shown). Exopolysaccharide production paralleled growth and the maximum EPS concentration (EPSmax; Table 1) was obtained at the beginning of the stationary phase. A more or less pronounced decrease in EPS concentration was observed with prolonged incubation (Fig. 1b). Since EPS production was apparently growth associated, the EPS yield for unit biomass was estimated from the slope

Fig. 1 Kinetics of (a) growth (X) and (b) exopolysaccharide (EPS) production during batch fermentation of a whey-based medium by Streptococcus thermophilus SY at 36°C. s, pH 5Æ6, yeast extract (YE) 4 g l–1; n, pH 6Æ4, YE 4 g l–1; h, pH 7Æ2, YE 4 g l–1; d, pH 6Æ4, YE 0 g l–1; j, pH 6Æ4, YE 8 g l–1

of the linear regression equations of EPS–EPS0 vs X–X0 (where 0 indicates the initial values for EPS and X, respectively). The estimates are shown in Table 1. The effect of the factors on the response variables was estimated using a quadratic polynomial equation (Box and Draper 1987): y ¼ a0 þ a1 x1 þ a2 x2 þ a3 x3 þ a11 x21 þ a12 x1 x2 þ a13 x1 x3 þ a22 x22 þ a23 x2 x3 þ a33 x23



where x1 ¼ (pH–6Æ4)/0Æ4, x2 ¼ (T–36)/6, x3 ¼ (YE–4)/2 and y is the response variables (Xmax, EPSmax, YE/X and the square root of lmax; the use of the untransformed value of lmax produced a poorer fit). Moreover, the inhibition at temperatures higher than the maximum for growth could not be fitted by the polynomial equation and, therefore, run 12 was discarded. If only those coefficients significantly different from 0 (P < 0Æ05) are taken into account, the following equations are obtained: Xmax ¼ 229 þ 008x1 þ 015x3 ÿ 015x21

R2 ¼ 081 ð1Þ

sqrðlÞ ¼ 088 þ 010x1 þ 0:07x2 þ 011x3

R2 ¼ 084 ð2Þ

EPSmax ¼ 173 ÿ 30x21 ÿ 14x22 ÿ 30x23

R2 ¼ 070

ð3Þ

No significant relationship was obtained for YE/X. The average percentage error for predictions was 6, 13 and 25% for Xmax, l and EPS, respectively. The predicted values for Xmax, lmax and EPSmax as a function of pH and YE concentration at 36°C are shown as a contour plot in Fig. 2. According to eqn 1, the maximum biomass concentration was significantly affected only by pH and YE. This was only in partial agreement with the experimental results since, at 48°C, which is probably higher

Fig. 2 Contour plot of predicted values (on the basis of eqns 1–3; see text for details) of maximum biomass concentration (……, scale ranging from 1Æ5 to 2Æ5 g l–1), maximum exopolysaccharide concentration (––, scale ranging from 0 to 150 mg l–1) and average specific growth rate (- - -, scale ranging from 0Æ2 to 1Æ4 h–1) for Streptococcus thermophilus SY during batch fermentation at 36°C of a whey-based medium as a function of pH and yeast extract concentration

301

than the optimum temperature for growth (see below), a lower value of Xmax was observed. Both high and low pH decreased Xmax, which increased with YE. The square root of the specific growth rate during the exponential phase was found to be linearly related to pH, temperature and YE concentration. This simple model did not provide reliable predictions at temperatures higher than 42°C and it was impossible to establish the optimum temperature for growth. However, the optimum temperature for Strep. thermophilus SY is probably close to 42°C (see l values in Table 1) and the decrease in growth at temperatures higher than 45°C is in good agreement with data presented by Martley (1983) for other strains. pH, T and YE all had significant effects (P < 0Æ05) on the predicted EPS production, which was maximum at pH 6Æ4, 36°C and 4 g l–1. Although the model for EPS concentration had the lowest predictive values, it is in good agreement with the trend shown by data in Table 1, with the exception of the experimental result obtained at 30°C, pH 6Æ8 and 6 g l–1, which is significantly higher than the predicted value (127 mg l–1) and close to the maximum, which was obtained at pH 6Æ4, 36°C and 4 g l–1 as predicted by the model. The effect of the three factors (pH, T and YE) on YE/X was not significant; at 48°C, a stressful condition, a high YE/X was observed. However, growth at a low temperature (24°C), pH (5Æ6) or YE did not result in an increase in YE/X. To study in more detail the kinetics of growth and EPS production and to provide fermentation broth for the recovery and purification of the EPS, three replicate fermentations were carried out in optimal conditions in a 3-l stirred tank reactor. The kinetics of the three replicate fermentations were very similar and growth, EPS, lactic acid and galactose production and lactose consumption are shown in Fig. 3. As shown previously, EPS production was growth associated, with a yield of EPS for unit biomass of 59 ± 5 mg g–1. The maximum EPS concentration was 140 ± 5 mg l–1 (mean and S.E. for three replicate fermentations), which is lower than the value predicted by eqn 3 (173 mg l–1) but in good agreement with that found for the fermentations in 1-l reactors. As in previous fermentations, a slight decrease in EPS concentration was observed at the end of growth. Since Strep. thermophilus SY is galactose negative, both galactose and lactic acid were produced from lactose. Both growth and EPS production ended when the lactose was exhausted. Preliminary characterization of the exopolysaccharide produced by Streptococcus thermophilus SY The EPS produced by Strep. thermophilus SY was partially purified by precipitation with ethanol followed by



Fig. 3 Kinetics of growth (s, X, g l–1), exopolysaccharide (EPS) (n, EPS, mg l–1), lactic acid (h, g l–1) and galactose production (m, g l–1) and lactose consumption (d, g l–1) by Streptococcus thermophilus SY grown in batch culture in a whey-based medium with 4 g l–1 yeast extract at pH 6Æ4 and 36°C

ultrafiltration and its MW, composition and rheological properties determined. The elution pattern of the EPS during high performance size exclusion chromatography was very complex; the main peak (22 min) had a shoulder from 27 to 30 min; a second, sharp peak was centred at 35 min, while the total volume corresponded to 40 min. Assuming a value of 0Æ15 ml g–1 for the differential refractive index increment (dn/dc), a value found for similar EPS polymers, the weight average MW was estimated to be 2Æ04 · 106, while the number average MW was 1Æ70 · 106, with a polydispersity index of 1Æ2. The second peak, corresponding to an elution time of 35 min, had an MW value of about 5 · 104. The monomer composition of the partially purified polysaccharide, as determined by gas–liquid chromatography, was 55% galactose, 24Æ4% glucose, 11Æ6% rhamnose and 2Æ3% arabinose. Several minor unidentified components accounted for 6Æ7% of the total sugars. The relationship between viscosity and shear stress of 5 g l–1 aqueous dispersions of the partially purified EPS and two commercially available polysaccharides (xanthan and alginate) and shear rate (in the range 0–93 s–1) is shown in Fig. 4. All the solutions showed a pseudoplastic behaviour. The EPS from Strep. thermophilus SY had the highest viscosity at all shear rates (from 7000 to 870 mPa’s with shear rate from 0Æ465 to 46Æ5 s–1).

DISCUSSION Streptococcus thermophilus SY produced EPS in whey-based media throughout the growth phase. At the end of growth a reduction in the concentration of EPS was sometimes observed. Growth-associated production has been observed with most EPS from LAB, including Strep. thermophilus strains (De Vuyst and Degeest 1999; Ricciardi and Clementi 2000). The rare cases of non-growth-associated EPS production (Petit et al. 1991; Gancel and Novel 1994a) have been attributed to the use of optical density rather than cell dry weight for biomass measurement (De Vuyst and Degeest 1999). Exopolysaccharide degradation upon prolonged incubation has been observed with other EPS produced by LAB (Mozzi et al. 1996; Gassem et al. 1997; De Vuyst et al. 1998) and has been attributed to the production of glycohydrolases (Cerning et al. 1988; Cerning et al. 1992; Gancel and Novel 1994b). Both growth and EPS production were affected by pH, YE and temperature. An attempt to model the combined effect of temperature, pH and YE concentration was carried out using a central composite design and empirical model building with quadratic polynomial equations. Statistical methods have frequently been applied in predictive microbiology to evaluate the effect of several factors on pathogenic and spoilage micro-organisms (Whiting 1995) and their use for the optimization of media and fermen-



303

Fig. 4 Flow curves for 5 g l–1 dispersions in distilled water of purified exopolysaccharide from Streptococcus thermophilus SY (s), medium viscosity alginate from Macrocystis pyrifera (Sigma; n) and xanthan (Sigma; h). Closed symbols, shear stress; open symbols, viscosity

tation conditions for LAB is not new (Parente and Hill 1992; Monteagudo et al. 1993; Rincoń et al. 1993; Oh et al. 1995; Wijtzes et al. 1995; Dobmann et al. 1996). Polynomial quadratic models are often insufficient to provide a satisfactory representation of the relationship between experimental factors and response variables (final population, lmax and lag time) and polynomial cubic or quartic models (Rincoń et al. 1993; Monteagudo et al. 1993; Oh et al. 1995; Dobmann et al. 1996) or other non-linear models (Wijtzes et al. 1995) have sometimes been used. However, the use of higher level models was not appropriate for the experimental design used in this study and would have resulted in overfitting; the models for growth and EPS production provide an indication of the effect of pH, temperature and YE concentration on growth and EPS production by Strep. thermophilus SY which is in good agreement with experimental data. The poor predictive ability of the models for EPS production and the high value for EPS concentration (147 mg l–1 compared with the predicted value of 127 mg l–1) and yield per unit biomass observed at 30°C, pH 6Æ8 and 6 g l–1 YE may be a result of an interaction between factors (which, however, was not statistically significant) and/or of the relatively low accuracy of the method used for the measurement of EPS concentration which has a coefficient of variation as high as 10% (Ricciardi et al. 1998). Optimization of the YE supplementation, temperature and pH of the fermentation by factorial experiments and empirical modelling did not

result in significant increases in yield, with maximum EPS concentrations in the range 140–160 mg l–1. The values obtained in this study for the maximum EPS production and specific growth rate were comparable with data obtained in previous experiments with the same strain (Ricciardi et al. 1997): l and EPS values, respectively, of 0Æ39 h–1 and 106 mg l–1 were obtained at 42°C, pH 6Æ0 and 2 g l–1 YE compared with values of 0Æ34 h–1 and 130 mg l–1 obtained at pH 6Æ0, 2Æ5 g l–1 YE and pH 6Æ0 in previous fermentations. Although EPS production was growth associated, conditions that stimulated growth did not necessarily result in a proportional stimulation of EPS production. In fact, while the optimal pHs for growth (as measured by Xmax) and EPS production were both close to 6Æ4, an increase in YE concentration resulted in increased growth but did not necessarily result in increased EPS production. The optimal pH for EPS production has been found to vary in different strains of LAB (De Vuyst and Degeest 1999; Ricciardi and Clementi 2000). However, the optimal pH for EPS production is often close to 6Æ0 (van den Berg et al. 1995; Gassem et al. 1997; De Vuyst et al. 1998) which is in agreement with our findings. While an increase in EPS yield at low temperatures has been observed in many LAB (De Vuyst and Degeest 1999; Ricciardi and Clementi 2000), including Strep. thermophilus strains (Cerning et al. 1988; Gancel and Novel 1994a), maximum EPS production by Strep. thermophilus SY was obtained at 36–42°C, while EPS yield per unit biomass was increased at high (48°C) rather than low



temperatures (24°C), a pattern which has been observed in other LAB (Grobben et al. 1995; Mozzi et al. 1995; De Vuyst et al. 1998). However, as the growth of Strep. thermophilus SY was very poor at 48°C, the high yield may simply be a calculation artefact. Although casein hydrolysates have been found to be stimulatory for EPS production in milk (Cerning et al. 1990) or whey (Gassem et al. 1997) and YE has been used as a growth supplement for EPS production by Strep. thermophilus LY03 in a milk-based medium (De Vuyst et al. 1998), to our knowledge, no systematic study on the effect of YE on EPS production has been published. Yeast extract is a stimulatory ingredient which is often included in whey media for growth and lactic acid production by LAB (Aeschlimann and von Stockar 1990; Chiarini et al. 1992; Amrane and Prigent 1998). Although growth and lactic acid production by thermophilic LAB have been found to increase with increasing YE concentration (Chiarini et al. 1992; Amrane and Prigent 1998), increasing YE above 4 g l–1 stimulated the growth of Strep. thermophilus SY but did not result in a similar stimulation of EPS production, which decreased at the highest YE concentration tested (8 g l–1). The overall effect of temperature, YE and pH on growth and EPS production is in good agreement with the current paradigm that considers growth and EPS production as two phenomena which are somewhat competitive; in fact, since the same polyisoprenoid carrier is used for both EPS and cell wall polymer synthesis (De Vuyst and Degeest 1999) a decreased availability of the limited pool of carrier for EPS production is expected when growth is stimulated. As for many other EPS-producing LAB (De Vuyst and Degeest 1999; Ricciardi and Clementi 2000), the low yield of EPS from Strep. thermophilus SY may be dependent on several limitations, including the competition of energy-yielding metabolism with EPS synthesis for sugar monomers and the limited availability of polyisoprenoid carrier for EPS synthesis and export. Improvement of media and better control of the fermentation process may increase the yield. In fact, the whey media used in this study contained 30 g l–1 lactose; since EPS production stopped when lactose was exhausted, the addition of extra lactose (as described in De Vuyst et al. 1998) or the use of higher concentrations of whey powder may increase the yield up to 450–500 mg l–1, although non-competitive inhibition of growth by lactate may pose a further limitation. However, genetic and metabolic engineering of the energy-yielding and EPS synthesis pathways are probably needed to obtain yields high enough to justify large-scale production (de Vos 1996; De Vuyst and Degeest 1999). The EPS produced by Strep. thermophilus SY was polydisperse, with two main MW fractions. The occurrence of high (1–6 · 106) and low (0Æ1–1 · 105) MW fractions in EPS produced by LAB is well documented (De Vuyst and Degeest 1999; Ricciardi and Clementi 2000). The EPS

produced by Strep. thermophilus have MW ranging from 1 to 9 · 106 (Doco et al. 1990; Ariga et al. 1992; Lemoine et al. 1997) but the occurrence of two polymers with different MW, which occurs in some Lactococcus lactis subp. cremoris (Marshall et al. 1995) and Lactobacillus delbrueckii subsp. bulgaricus (Grobben et al. 1997) strains, has been reported only for another Strep. thermophilus strain (Ariga et al. 1992). Although ultrafiltration (NMWCO 1 · 104) was used to remove potentially contaminating polysaccharide components of YE, further experiments in chemically defined media are needed to demonstrate that the smaller fraction of Strep. thermophilus SY EPS (SY) (MW 5 · 104) does not derive from media components. The gross composition of the EPS was similar to that determined for the polysaccharides produced by other Strep. thermophilus strains (Cerning et al. 1988; Doco et al. 1990; Ariga et al. 1992; Lemoine et al. 1997; Low et al. 1998), with glucose, galactose and rhamnose as the main components. As the chemical analysis was performed on a partially purified polysaccharide, the minor components could be contaminant compounds originating from the complex ingredients added to the medium. On the basis of the principal sugars, the chemical composition of the EPS produced by Strep. thermophilus SY shows a glucose : galactose : rhamnose ratio of 2 : 4Æ5 : 1. Production in chemically defined media and more thorough purification of the EPS are needed to confirm the composition of the SY. As with many other EPS produced by LAB and other bacteria, the EPS of Strep. thermophilus SY showed shear thinning properties, with higher viscosities at all shear rates compared with xanthan (van den Berg et al. 1995; Bubb et al. 1997); both properties are desirable for use as a viscosifying agent in food products (De Vuyst and Degeest 1999). Even if the purified EPS shows promising rheological properties, as for many other heteropolysaccharides produced by LAB, its large-scale production in a semi-purified form is not interesting because of the low yield compared with other commercial bacterial heteropolysaccharides, such as xanthan (De Vuyst and Degeest 1999). However, it is tempting to speculate that, if the yield can be increased to 0Æ5–1 g l–1 by manipulation of the initial lactose content of the medium or by better process control, this strain could be used, alone or in combination with Lact. delbrueckii subsp. bulgaricus or Lact. helveticus, in a process for the joint production of ammonium lactate (a source of non-protein nitrogen for feeds) or lactic acid and of the partially purified polysaccharide, to be used as a viscosifying agent in dairy products. In fact, continuous processes for the production of ammonium lactate from whey permeate media using cell recycle by microfiltration have already proven to be technically and economically feasible (Timmer and Kromkamp 1994; Tejayadi and Cheryan



1995). Recovery of the partially purified EPS by ultrafiltration of the permeate from a cell recycle reactor, followed by spray drying, could result in an economically valuable by-product from the lactic acid fermentation of cheese whey. ACKNOWLEDGEMENTS This work was funded by Consiglio Nazionale delle Ricerche (Rome), special grant no. 99Æ03192.CT06. REFERENCES Aeschlimann, A. and von Stockar, U. (1990) The effect of yeast extract supplementation on the production of lactic acid from whey permeate by Lactobacillus helveticus. Applied Microbiology and Biotechnology 32, 398–402. Amrane, A. and Prigent, Y. (1998) Lactic acid production rates during different growth phases of Lactobacillus helveticus cultivated on whey supplemented with yeast extract. Biotechnology Letters 20, 379–383. Ariga, H., Urashima, T., Michichata, E., Ito, M., Morizono, N., Kimura, T. and Takahashi, S. (1992) Extracellular polysaccharide from encapsulated Streptococcus salivarius subsp. thermophilus OR901 isolated from commercial yoghurt. Journal of Food Science 57, 625–628. Box, E.P. and Draper, N.R. (1987) Empirical Model-Building and Response Surfaces. New York: John Wiley. Bubb, W.A., Urashima, T., Fujiwara, R., Shinnai, T. and Ariga, H. (1997) Structural characterization of the exocellular polysaccharide produced by Streptococcus thermophilus OR 901. Carbohydrate Research 301, 41–50. Cerning, J. (1995) Production of expolysaccharides by lactic acid bacteria and dairy propionibacteria. Lait 75, 463–472. Cerning, J., Bouillane, C., Lando, M. and Desmazeaud, M.J. (1992) Isolation and characterization of exopolysaccharides from slime-forming lactic acid bacteria. Journal of Dairy Science 75, 692– 699. Cerning, J., Bouillanne, C., Desmazeaud, M.J. and Landon, M. (1988) Exocellular polysaccharide production by Streptococcus thermophilus. Biotechnology Letters 10, 255–260. Cerning, J., Bouillanne, C., Landon, M. and Desmazeaud, M.J. (1990) Comparison of exopolysaccharide production by thermophilic lactic acid bacteria. Science des Aliments 10, 443–451. Chiarini, L., Mara, L. and Tabacchioni, S. (1992) Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus. Applied Microbiology and Biotechnology 36, 461–464. de Vos, W.M. (1996) Metabolic engineering of sugar catabolism in lactic acid bacteria. Antonie Van Leeuwenhoek 70, 223–242. De Vuyst, L. and Degeest, B. (1999) Heteropolysaccharides from lactic acid bacteria. FEMS Microbiology Reviews 23, 153–157. De Vuyst, L., Vanderveken, F., Van de Ven, S. and Degeest, B. (1998) Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growthassociated biosynthesis. Journal of Applied Microbiology 84, 1059–1068.

305

Dobmann, M.U., Vogel, R.F. and Hammes, W.P. (1996) Mathematical description of the growth of Lactobacillus sake and Lactobacillus pentosus under conditions prevailing in fermented sausages. Applied Microbiology and Biotechnology 46, 334–339. Doco, T., Wieruszeski, J.-M., Fournet, B., Carcano, D., Ramos, P. and Loones, A. (1990) Structure of an exocellular polysaccharide produced by Streptococcus thermophilus. Carbohydrate Research 198, 313–321. Gancel, F. and Novel, G. (1994a) Exopolysaccharide production by Streptococcus salivarius ssp. thermophilus cultures. 1. Conditions of production. Journal of Dairy Science 77, 685–688. Gancel, F. and Novel, G. (1994b) Exopolysaccharide production by Streptococcus salivarius ssp. thermophilus cultures. 2. Distinct modes of polymer production and degradation among clonal variants. Journal of Dairy Science 77, 689–695. Gassem, M.A., Schmidt, K.A. and Frank, J.F. (1997) Exopolysaccharide production from whey lactose by fermentation with Lactobacillus delbrueckii ssp. bulgaricus. Journal of Food Science 62, 171–173. Grobben, G.J., Sikkema, J., Smith, M.R. and de Bont, J.A.M. (1995) Production of extracellular polysaccharides by Lactobacillus delbrueckii ssp. bulgaricus NCFB2772 grown in a chemically defined medium. Journal of Applied Bacteriology 79, 103–107. Grobben, G.J., Smith, M.R., Sikkema, J. and de Bont, J.A.M. (1996) Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB2772. Applied Microbiology and Biotechnology 46, 279–284. Grobben, G.J., van Casteren, W.H.M., Schols, H.A., Oosterveld, A., Sala, G., Smith, M.R., Sikkema, J. and de Bont, J.A.M. (1997) Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB2772 grown in continuous culture on glucose and fructose. Applied Microbiology and Biotechnology 48, 516–521. Lemoine, J., Chirat, F., Wieruszeski, J.-M., Strecker, G., Favre, N. and Neeser, J.R. (1997) Structural characterization of the exocellular polysaccharides produced by Streptococcus thermophilus SFi39 and Sfi12. Applied and Environmental Microbiology 63, 3512–3518. Low, D., Ahlgren, J.A., Horne, D., McMahon, D.J., Oberg, C. and Broadbent, J.R. (1998) Role of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retention. Applied and Environmental Microbiology 64, 2147–2151. Marshall, V.M.E., Cowie, E.N. and Moreton, R.S. (1995) Analysis and production of two exopolysaccharides from Lactococcus lactis subsp. lactis LC330. Journal of Dairy Research 62, 621–628. Martley, F.G. (1983) Temperature sensitivities of thermophilic starter strains. New Zealand Journal of Dairy Science and Technology 18, 191–196. Monteagudo, J.M., Rincoń, J., Rodrı´guez, L., Fuertes, J. and Moya, A. (1993) Determination of the best nutrient medium for the production of L-lactic acid from beet molasses. A statistical approach. Acta Biotecnologica 13, 103–110. Mozzi, F., De Giori, G., Oliver, G. and De Valdez, F.G. (1996) Exopolysaccharide production by Lactobacillus casei in milk under different growth conditions. Milchwissenschaft 51, 670–673. Mozzi, F., Oliver, G., De Giori, G.S. and De Valdez, G.F. (1995) Influence of temperature on the production of exopolysaccharides by thermophilic lactic acid bacteria. Milchwissenschaft 50, 80–82.



Oh, S., Rheem, S., Sim, J., Kim, S. and Baek, K. (1995) Optimizing conditions for the growth of Lactobacillus casei YIT in TryptoneYeast Extract-Glucose medium by using response surface methodology. Applied and Environmental Microbiology 61, 3809–3814. Parente, E. and Hill, C. (1992) A comparison of factors affecting the production of two bacteriocins from lactic acid bacteria. Journal of Applied Bacteriology 73, 290–298. Perry, D.B., McMahon, D.J. and Oberg, C.J. (1998) Manufacture of low fat Mozzarella cheese using exopolysaccharide-producing starter cultures. Journal of Dairy Science 81, 563–566. Petit, C., Grill, J.P., Maazouzo, N. and Marczak, R. (1991) Regulation of polysaccharide formation by Streptococcus thermophilus in batch and in fed-batch cultures. Applied Microbiology and Biotechnology 36, 216–221. Rawson, H.L. and Marshall, V.M. (1997) Effect of ‘ropy’ strains of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus on rheology of stirred yogurt. International Journal of Food Science and Technology 32, 213–220. Ricciardi, A., Parente, E., Aquino, M. and Clementi, F. (1998) Use of desalting gel for the rapid separation of simple sugars from exopolysaccharides produced by lactic acid bacteria. Biotechnology Techniques 12, 649–652. Ricciardi, A., Parente, E. and Clementi, F. (1997) Produzione di esopolisaccaridi da Streptococcus thermophilus e Lactobacillus delbrueckii subsp. bulgaricus in coltura pura e in associazione, in un substrato a base di siero. Annali di Microbiologia 47, 213–222. Ricciardi, A. and Clementi, F. (2000) Exopolysaccharides from lactic acid bacteria: structure, production and technological applications. Italian Journal of Food Science 12(23), 45.

Rincoń, J., Fuertes, J., Moya, A., Monteagudo, J.M. and Rodrı´guez, L. (1993) Optimization of the fermentation of whey by Lactobacillus casei. Acta Biotechnologica 13, 323–331. Sawardeker, J.S.F., Sloneker, J.H.A. and Jeanes, A. (1965) Quantitative determination of monosaccharides as their alditol acetates by gas chromatography. Analytical Chemistry 37, 1602–1604. Sutherland, I.W. (1999) Extracellular polysaccharides. In Biotechnology, Vol. 6. ed. Roehr, M. pp. 613–657. New York: VCH. Tejayadi, S. and Cheryan, M. (1995) Lactic acid from cheese whey permeate. Productivity and economics of a continuous membrane bioreactor. Applied Microbiology and Biotechnology 43, 242–248. Timmer, J.M.K. and Kromkamp, J. (1994) Efficiency of lactic acid production by Lactobacillus helveticus in a membrane cell recycle reactor. FEMS Microbiology Reviews 14, 29–38. van den Berg, D.J.C., Robijn, G.W., Janssen, A.C., Giuseppin, M.L.F., Vreeker, R., Kamerling, J.P., Vliegenthart, J.F.C., Ledeboer, A.M. and Verrips, C.T. (1995) Production of a novel extracellular polysaccharide by Lactobacillus sake 0–1 and characterization of the polysaccharide. Applied and Environmental Microbiology 61, 2840–2844. Whiting, R.C. (1995) Microbial modeling in foods. Critical Reviews in Food Science and Nutrition 35, 467–494. Wijtzes, T., de Wit, J.C., Huis in’t Veld, J.H.J., van’t Riet, K. and Zwietering, M.H. (1995) Modelling bacterial growth as a function of acidity and temperature. Applied and Environmental Microbiology 61, 2533–2539.
