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Streptococcus thermophilus ST 111 produces a stable high-molecular-mass exopolysaccharide in milk-based medium
ARTICLE IN PRESS
International Dairy Journal 14 (2004) 857–864
Streptococcus thermophilus ST 111 produces a stable high-molecular-mass exopolysaccharide in milk-based medium Frederik Vaningelgema, Roel Van der Meulena, Medana Zamfira, Tom Adrianya, Andrew P. Lawsb, Luc De Vuysta,* a Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium b Department of Chemical and Biological Sciences, University of Huddersfield, Queensgate, HDI 3DH Huddersfield, UK
Received 8 December 2003; accepted 12 March 2004
Abstract Streptococcus thermophilus ST 111, grown in milk medium supplemented with whey protein hydrolysate, produced an exopolysaccharide (EPS) composed of galactose and rhamnose in a molar ratio of 2.5:1, and with a molecular mass of more than 5000 kDa. Fermentations in milk medium supplemented with whey protein hydrolysate further demonstrated the stability of this high-molecular-mass EPS, as its molecular mass was affected neither by the pH of the medium nor the fermentation time, indicating no enzymatic degradation. On the other hand, drying of the isolated EPS resulted in a decrease of the molecular mass. The stability of an EPS produced in milk-based media is an important asset for the production of fermented milk and yoghurt products. r 2004 Elsevier Ltd. All rights reserved. Keywords: Exopolysaccharide; Streptococcus thermophilus; Molecular mass; Structure; Stability
1. Introduction Exopolysaccharides (EPS) from lactic acid bacteria (LAB) can be subdivided into two major groups: homopolysaccharides and heteropolysaccharides (De Vuyst & Degeest, 1999; Monsan et al., 2001; De Vuyst & Vaningelgem, 2003). Based on their structure, four groups of homopolysaccharides can be distinguished: a-d-glucans (dextrans, mutans, alternan), b-d-glucans, b-d-fructans (levans, inulin-type fructans), and others like polygalactan. Heteropolysaccharides are produced by LAB in a greater variety concerning chemical composition, monomer ratio, and molecular structure of the repeating unit, as well as the molecular mass of the polymer (De Vuyst & Vaningelgem, 2003). The repeating units of heteropolysaccharides most often contain a combination of d-glucose, d-galactose, and l-rhamnose in different ratios and, in a few cases, fucose, acetylated amino sugars, glucuronic acid, and nononic acid; as well, non-carbohydrate constituents such as glycerol, phosphate, acetyl, and pyruvyl groups *Corresponding author. Tel.: +32-2-629-32-45; fax: 32-2-629-27-20. E-mail address: [email protected] (L.De Vuyst). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.03.007
may be present (De Vuyst, de Vin, Vaningelgem, & Degeest, 2001; De Vuyst & Vaningelgem, 2003). Furthermore, within one repeating unit, different types of linkages can occur. In the backbone of heteropolysaccharides from LAB, a(1-3) linkages are more abundant than b(1-4) ones, which in turn occur more frequently than a(1-2), b(1-3), and b(1-6) linkages (De Vuyst & Vaningelgem, 2003). In general, the types of linkages within an EPS polymer determine the rigidity (Laws & Marshall, 2001) and hence the intrinsic viscosity of the polymer (Ruas-Madiedo, Tuinier, Kanning, & Zoon, 2002b). The apparent viscosity of an EPS solution is primarily determined by the molecular mass of the polymer. The molecular mass of heteropolysaccharides varies from 1.0 104 to 9.0 106 Da. Some strains produce high-molecularmass EPS (Navarini et al., 2001), others produce ! low-molecular-mass EPS (Almiron-Roig, Mulholland, Gasson, & Griffin, 2000), while still others produce both types of EPS (Marshall, Cowie, & Moreton, 1995; Grobben et al., 1997; Degeest & De Vuyst, 1999; Petry et al., 2003). The biosynthesis of the repeating unit of the heteropolysaccharides produced by LAB has been studied on an enzyme level for Streptococcus
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thermophilus (Degeest & De Vuyst, 2000; Degeest, Vaningelgem, Laws, & De Vuyst, 2001b), Lactobacillus casei (Mozzi, Rollan, Savoy de Giori, & Font de Valdez, 2001; Mozzi, Savoy de Giori, & Font de Valdez, 2003), Lb. sakei (Degeest, Janssens, & De Vuyst, 2001a), Lb. delbrueckii subsp. bulgaricus (Grobben, Smith, Sikkema, & de Bont, 1996), and Lactococcus lactis (Looijesteijn, Boels, Kleerebezem, & Hugenholtz, 1999). The genes involved in EPS biosynthesis, regulation, polymerisation, and export appear to be highly conserved among LAB (Jolly & Stingele, 2001; Jolly, Vincent, Duboc, & Neeser, 2002). However, detailed genetic and molecular knowledge about the polymerisation (i.e., chain length determination) of the repeating units and secretion of the final EPS is still lacking (Jolly & Stingele, 2001; Lamothe, Jolly, Mollet, & Stingele, 2002), making it difficult to genetically modify LAB or to design fermentation strategies for the production of EPS with a specific structure or polymer length. Generally, the amounts of EPS produced by LAB in the food matrix are low and their production is often unstable (De Vuyst et al., 2001). This unstable EPS production can be due to the loss of plasmids, especially in the case of mesophilic LAB, the presence of mobile genetic elements or genomic rearrangements (De Vuyst et al., 2001). Also, EPS degradation can occur when intracellular enzymes (e.g., a-d-glucosidase and b-d-glucuronidase) are released into the medium, resulting in a decrease of the molecular mass of the EPS polymers (Pham, Dupont, Roy, Lapointe, & Cerning, 2000; Degeest, Mozzi, & De Vuyst, 2002), which in turn may lead to a decreased intrinsic viscosity (Ruas-Madiedo et al., 2002b). Although the enzymatic degradation can be influenced and hence modulated by the physico-chemical conditions applied (Degeest et al., 2002), strains producing stable high-molecular-mass EPS in situ are of interest for the improvement of texture in food systems. Furthermore, not much is known about the stability of heteropolysaccharides from LAB during food processing operations such as drying, heating, freezing, pumping, etc. S. thermophilus is one of the most important LAB in the dairy industry, applied as a starter culture in yoghurt fermentation and cheese making. The aim of this study was to characterise the EPS of S. thermophilus ST 111, a strain isolated from an artisan Romanian yoghurt, with respect to its monomer composition and molecular mass, when grown in a milk-based medium. The structure of the high-molecular-mass EPS of S. thermophilus ST 111 was elucidated by nuclear magnetic resonance (NMR) spectroscopy. Also, pH-controlled fermentations were performed in a milk-based medium to study the effect of constant pH on the monomer composition and molecular mass of EPS from S. thermophilus ST 111. Finally, the effect of a drying step on the monomer composition and molecular mass of the EPS was studied.
2. Materials and methods 2.1. Strain and strain propagation S. thermophilus ST 111, a strain isolated from an artisan Romanian yoghurt, was used as the EPSproducing strain throughout this study. Isolation of the strain was accomplished after plating on solid, lactosecontaining (0.5%, w/v) M17 medium (Oxoid, Basingstoke, UK). The strain was identified by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole-cell proteins (Vaningelgem et al., 2004). The strain was stored at 80 C in de Man–Rogosa–Sharpe (MRS) medium (Oxoid) containing 25% (v/v) glycerol as cryoprotectant. To obtain fresh cultures, S. thermophilus ST 111 was propagated twice at 42 C for 12 h in the medium identical to the one used for the fermentations later on. 2.2. Media Milk medium (10.0% skimmed milk powder, w/v; Dairy Industry Inc., Kallo, Belgium), and milk medium supplemented with a whey protein hydrolysate (1.6%, w/v; a commercial product referred to as lactalbumin hydrolysate, Oxoid) were used during this study. 2.3. Isolation and purification of EPS EPS were isolated as described previously (De Vuyst, Vanderveken, Van de Ven, & Degeest, 1998). Briefly, trichloroacetic acid (TCA) and acetone precipitation were carried out. EPS were harvested by centrifugation (25,000 g, 4 C, 20 min), followed by another TCA and acetone precipitation. The isolated EPS material was then dialysed against distilled water for 7 days with water replacement twice a day, using Spectra/Por membranes (VWR International, Darmstadt, Germany) with a molecular-mass-cut-off (MMCO) of 3500 Da, and subsequently concentrated by freeze-drying (Heto Drywinner, Heto-Holten, Allerød, Denmark). To have an idea of the purity of the EPS material, both residual lactose and protein contaminants were determined in solutions of purified EPS. In pure EPS, no residual lactose could be determined by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD, Dionex, Sunnyvale, California, USA), using a CarboPacTM PA 10 column (Dionex). The protein concentration of all EPS samples, determined with a BioRad Protein Assay Kit (BioRad Laboratories, Hercules, California, USA), was always lower than 3% (w/w). 2.4. Molecular mass determination For molecular mass analysis, EPS samples were obtained from fermentations carried out in milk
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medium supplemented with whey protein hydrolysate, isolated, purified, and concentrated as described above. The molecular mass of the purified EPS was determined by gel permeation chromatography (GPC) using two types of columns. First, a 100 cm 1.6 cm Sephacryl S-400 column (Amersham BioSciences AB, Uppsala, Sweden) was used with 200 mL samples containing about 50 mg of lyophilised EPS mL1. The EPS were eluted with 50 mm potassium phosphate buffer (pH 6.8) containing 0.15 m NaCl at a flow rate of 1 mL min1. A dextran standard series (molecular masses of 80, 150, 270, 670, and 1800 kDa; Sigma, St. Louis, Missouri, USA) was used to estimate the molecular mass of the EPS. The linear range of the column was limited to 2000 kDa according to the manufacturer. The standard deviation (expressed in retention time) was 71.5 min based on three injections of one sample. To further characterise the EPS from S. thermophilus ST 111, a 30 cm 7.8 mm UltrahydrogelTMLinear column (Waters Corp., Milford, Massachusetts, USA) was used, applying two types of standards, namely dextrans (molecular masses of 80, 150, 270, 410, 670, 1400, and 4900 kDa; Sigma) and pullulans (molecular masses of 5.9, 11.8, 22.8, 47.3, 112.0, 212.0, 404.0, and 788.0 kDa; Shodex P82, Showa Denko K.K., Tokyo, Japan). Samples of 200 mL containing about 100 mg of lyophilised EPS L1 were applied on the column. The EPS were eluted with 0.1 m NaNO3 at a flow rate of 0.6 mL min1. The linear range of this column was limited to 8000 kDa according to the manufacturer. With both types of columns, the polysaccharide content was determined on line with a Waters 2410 refractive index detector (Waters Corp.) at an internal temperature of 40 C. Molecular mass analysis was always performed in duplicate and values were averaged. 2.5. Monomer analysis For monomer analysis, EPS were obtained from fermentations carried out in milk medium supplemented with whey protein hydrolysate, isolated, purified, and concentrated as described above. The monomer composition of the purified EPS was determined after acid hydrolysis with 6 n trifluoroacetic acid (TFA) at 100 C for 3 h, using HPAEC-PAD (Dionex), as described previously (Degeest et al., 2001b). Monomer analysis was performed in duplicate and values were averaged. 2.6. EPS elucidation by one-dimensional NMR spectroscopy For structure determination, EPS material was obtained from fermentations carried out in milk
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medium (1-L flasks, 42 C, free pH), isolated, purified, and concentrated as described above. The lyophilised polysaccharide was dissolved directly in D2O (99.9% D; Goss Scientific Instruments Ltd., Essex, UK); NMR spectra were recorded at a probe temperature of 70 C. The elevated temperature shifted the HOD signal to a higher field, into a clear region of the spectrum. The higher temperature also increased spectral resolution by reducing the sample viscosity. The NMR spectra were recorded on a Bruker Avance DPX400 MHz spectrometer (Bruker Analytical GmBH, Rheinstetten, Germany) operating with Z-field gradients and using Bruker’s pulse programmes. Chemical shifts ðdÞ were expressed in parts per million relative to internal acetone, d2.225. The 1D 1H spectra were processed with 32,768 data points. The two-dimensional gradient selected Double Quantum Filter COrrelated SpectroscopY (2D gs-DQF-COSY) spectrum was recorded in magnitude mode at 70 C, the time-domain data was multiplied by a squared-sine-bell function (SSB 0). After applying linear prediction and after Fourier transformation, data sets of 1024 1024 points were obtained. The 1 H spectra were recorded for several EPS samples, isolated during the stationary phase. 2.7. Fermentation conditions, on line analysis and sampling To study the influence of the fermentation pH on the monomer composition and molecular mass of EPS produced by S. thermophilus ST 111, large-scale fermentations were carried out in a 15-L computercontrolled laboratory fermentor (Biostat Cs; B. Braun Biotech International, Melsungen, Germany). Milk medium (10 L) was sterilised in an autoclave at 121 C for 20 min, and aseptically pumped into a sterilised fermentor. Previously, the fermentor was filled with water and sterilised in situ at 121 C for 20 min. Afterwards the sterile water was drained. The whey protein hydrolysate was sterilised separately as well and aseptically added to the fermentor containing the sterile milk medium. To keep the medium in the fermentor homogeneous, agitation was performed at 100 rpm with a stirrer composed of three standard impellers. The fermentor was inoculated with 1.0% (v/v) of a fresh culture of the strain. The fermentation temperature was kept constant at 42 C. To study the influence of pH, the initial pH of the milk medium supplemented with whey protein hydrolysate was adjusted to pH 5.5, 5.8, 6.2, and 6.6 using 2 n HCl or 10 n NaOH (a comparison was made with milk medium adjusted to pH 5.8). The pH was kept constant during fermentation through automatic addition of 10 n NaOH. The temperature, pH, and agitation were computer-controlled and monitored on line (Micro MFCS for WindowstNT software; B. Braun Biotech
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International). At different stages during the fermentation, samples were aseptically withdrawn from the fermentor to isolate EPS. The molecular mass of EPS material from several fermentation samples was determined (Sephacryl S-400 gel column, see above). To eliminate differences in growth rate and acidification between these fermentation samples and to have the cells in a similar physiological state, sampling times were linked with the amount of NaOH added to the medium for pH control, namely after addition of 50, 100, and 175 mL of NaOH, i.e., 5.3, 10.6, and 19.5 mL of base per liter of medium. Two other samples were collected after 12 and 24 h of fermentation, respectively. To determine the effect of drying on the EPS characteristics (monomer composition and molecular mass analysis), EPS isolated via TCA and acetone precipitation were dried at 42 C during 48 h. Afterwards, the same purification and concentration steps as described above, i.e., dialysis of the dried EPS dissolved in ultra pure water for 7 days (water replacement twice a day), followed by overnight freeze drying, were carried out. This EPS was compared with purified EPS that had not been subjected to the drying step at 42 C. This experiment has been performed in duplicate.
3. Results 3.1. Isolation and purification of the EPS produced by S. thermophilus ST 111 When grown in milk medium supplemented with whey protein hydrolysate, the EPS produced by S. thermophilus ST 111 was easily precipitated with acetone. GPC of purified EPS material resulted in a peak with a molecular mass of more than 2000 kDa (Fig. 1a), thus exceeding the linear range of the Sephacryl S-400 gel GPC column. EPS isolated from milk medium gave the same results, while in the case the strain was grown in MRS medium the EPS material was contaminated with polysaccharides derived from yeast extract and peptone (De Vuyst et al., 2003). With an UltrahydrogelTMLinear GPC column with a higher linear range (up to 8000 kDa), the molecular mass of the EPS from S. thermophilus ST 111 was higher compared with the largest molecular mass of each type of standards used, i.e., 4900 kDa for dextran and 788 kDa for pullulan. These data indicate that S. thermophilus ST 111 is producing a high-molecular-mass EPS, the size of which is difficult to determine. Both conformation and branching determine the linear range of a GPC column depending on the standards used.
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Fig. 1. Comparison of the gel permeation chromatograms (Sephacryl S-400 gel) of EPS produced by S. thermophilus ST 111 in milk medium supplemented with whey protein hydrolysate (1.6%, w/v). The EPS were obtained without a drying step (a) and with a drying step at 42 C for 48 h before dialysis and concentration (b). Vertical lines represent the molecular mass markers: (1) 1800 kDa, (2) 670 kDa, (3) 410 kDa, (4) 270 kDa, (5) 150 kDa, and (6) 80 kDa.
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Monomer analysis with HPAEC-PAD gave a galactose/rhamnose ratio of 2.5:1.0 for the EPS material derived from milk medium supplemented with whey protein hydrolysate. This was also found previously for EPS isolated from milk medium fermented with this strain (De Vuyst et al., 2003). 3.2. Structure elucidation of the EPS produced by S. thermophilus ST 111 The 1H spectra recorded for several EPS samples, isolated during the stationary phase from several milk fermentations of S. thermophilus ST 111, were identical (Fig. 2a). There were seven low-field H-1 signals, designated A–G in Fig. 2a (d5.26 A H-1, 5.25 B H-1, 5.16 C H-1, 5.16 D H-1, 5.01 E H-1, 4.65 F H-1 and 4.45 G H-1). The locations of the related H-2 resonances were available from the COSY spectrum (d 4.02 A H-2, 4.05 B H-2, 3.96 C H-2, 4.03 D H-2, 4.35 E H-2, 3.50 F H-2 and 3.53 G H-2). From this information it follows that the polysaccharide repeating unit was a heptasaccharide comprising two rhamnosyl and three a-galactosyl (A–E) linkages with two b-galactosyl linkages (F,G) displaying characteristic trans couplings. The spectra and the H-1 and H-2 chemical shifts resulted in the structure represented in Fig. 2b.
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affected the monomer composition of the EPS produced by S. thermophilus ST 111. The same monomer ratio of 2.5 Gal for 1.0 Rha was determined for all samples. The base consumption during fermentations in milk medium supplemented with whey protein hydrolysate at constant pH 6.2 was the fastest, followed by fermentations at constant pH 6.6, pH 5.8, and pH 5.5 (Fig. 3). When no whey protein hydrolysate was added, acidification and thus base consumption was much slower (Fig. 3). All GPC chromatograms of EPS material isolated from samples obtained during these fermentations showed the presence of high-molecular-mass EPS (>2000 kDa). No other peaks were observed, indicating that no degradation of the EPS took place under the different physico-chemical conditions tested (Table 1). However, when isolated EPS were dried at 42 C during
3.3. Influence of the fermentation pH and drying on the monomer composition and molecular mass of EPS from S. thermophilus ST 111 Neither the addition of whey protein hydrolysate to milk medium nor the pH of the fermentation medium
Fig. 3. Base consumption (mL of 10 n NaOH per liter of fermentation medium) during fermentations with S. thermophilus ST 111 in milk medium at a constant pH of 5.8 (&) and in milk medium supplemented with whey protein hydrolysate (1.6%, w/v) at a constant pH of 5.5 (~), 5.8 (’), 6.2 (m), and 6.6 (). All fermentations were carried out at 42 C. Horizontal lines indicate at which amount of base consumed samples were taken for EPS analysis. In addition, two samples were taken after 12 and 24 h of fermentation.
Table 1 Influence of the fermentation pH on the retention time during gel permeation chromatography of the EPS produced by S. thermophilus ST 111 in milk medium supplemented with whey protein hydrolysate (1.6%, w/v) at a constant temperature of 42 C Fermentation pH Retention timea 5.5 5.8 6.2 6.6 Sampling timeb
Fig. 2. 400-MHz 1H-NMR spectrum of the EPS from S. thermophilus ST 111 (a) and repeating unit of the EPS from S. thermophilus ST 111 (b). The seven low-field H-1 signals (A–G) are specified in the insert of (a).
78.7 75.6 75.0 78.5 5.3 mL L1
77.8 75.0 75.0 78.0 10.6 mL L1
78.8 75.0 76.5 78.1 19.5 mL L1
77.9 77.5 75.0 78.2 12 h
78.3 79.6 75.9 78.1 24 h
a The exact molecular mass could not be determined as all the measurements exceeded the linear range of the column (>2000 kDa). Molecular mass analysis was always performed in duplicate and retention times were averaged. The standard deviation was 71.5 min. b Sample times were linked to the base consumption (10 n NaOH) per liter of fermentation medium (in mL base L1). Two samples were collected after 12 and 24 h of fermentation as well.
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48 h an increased retention time and a broader peak (Fig. 1b) were observed compared with EPS which were not subjected to this drying step (Fig. 1a). This decrease in retention time indicated a decrease of molecular mass and hence some degradation of the EPS, when a drying step was carried out before EPS purification. No differences in monomer composition were observed between dried and non-dried samples. Similar GPC chromatograms were determined in the duplicate experiment.
4. Discussion Ropy, thermophilic LAB starter cultures for yoghurt production are often used in some countries of the EU, albeit unconsciously or under non-optimal conditions. S. thermophilus ST 111 is a Romanian yoghurt strain that grows in milk medium and other complex media containing glucose, sucrose, or lactose as the sole energy source (Vaningelgem, Zamfir, Adriany, & De Vuyst, unpublished results). It displays a low proteolytic activity and probably this is the main reason for its slow growth in milk. As protein hydrolysates contain peptides or free amino acids that may be more readily available for the cells, milk medium supplemented with whey protein hydrolysate was ideal for good growth and high EPS production as well as for EPS isolation (no interfering polysaccharides) with S. thermophilus ST 111. In milk medium, S. thermophilus ST 111 produces a high-molecular-mass heteropolysaccharide composed of galactose and rhamnose in a 2.5:1 molar ratio (De Vuyst et al., 2003). When the strain was cultivated in milk medium supplemented with whey protein hydrolysate, no changes in molecular mass and monomer composition of the EPS were found. The presence of galactose and rhamnose as the sole sugar residues has been found in EPS of only four S. thermophilus strains and not in EPS of other LAB strains examined so far (Bubb, Urashima, Fujiwara, Shinnai, & Ariga, 1997; Faber, Zoon, Kamerling, & Vliegenthart, 1998; Faber, van den Haak, Kamerling, & Vliegenthart, 2001). This is interesting from a health point of view, because it has been postulated that rhamnose-containing EPS may stimulate the immune system through mannose receptors situated on macrophages (Chabot et al., 2001). The absence of glucose in the repeating unit of the EPS of S. thermophilus ST 111 may indicate an increased activity of UDP-galactose 4-epimerase and no or decreased activity of UDP-glucose pyrophosphorylase, both enzyme activities assuming EPS biosynthesis from . the galactose moiety of lactose (Levander & Ra( dstrom, 2001). The molecular structure of the EPS from S. thermophilus ST 111, as revealed by NMR analysis, consisted of a heptasaccharide repeating unit, identical
to the EPS repeating units from S. thermophilus OR901 (Bubb et al., 1997) and S. thermophilus Rs and Sts (Faber et al., 1998). It is structurally similar to the EPS subunits of S. thermophilus SFi12 (Lemoine et al., 1997) and S. thermophilus MR-1C (Low et al., 1998). All these EPS structures are composed of sugars, which are only linked with each other via a-linkages in the backbone of the repeating unit. These a-linkages may be disadvantageous for the stiffness of the polymer and hence the viscosity of the resulting EPS solutions (Laws & Marshall, 2001, Ruas-Madiedo, Hugenholtz, & Zoon, 2002a). Indeed, the a-linkages in the backbone of the EPS from S. thermophilus ST 111 cause a weak consistency of the EPS solution compared with EPS from S. thermophilus LY03, which contains both a- and b-linkages (Vaningelgem et al., 2004). Of all EPS structures published to date, the repeating unit of the EPS produced by S. thermophilus ST 111 is the second largest one reported, indicating that LAB may not be capable to produce larger building blocks than octasaccharides (De Vuyst et al., 2001). While the molecular mass of the EPS from S. thermophilus ST 111 already had been estimated in previous studies (De Vuyst et al., 2003), the linear range of the GPC column used in this study was greater (up to 8000 kDa); the molecular mass of the EPS from S. thermophilus ST 111 was confirmed to be higher than 5000 kDa. No change in molecular mass and hence no degradation of the polymer nor a change in the monomer composition of the EPS was observed for the high-molecular-mass EPS produced by S. thermophilus ST 111 at varying constant fermentation pH values, independent of the fermentation time, indicating no enzymatic degradation. This shows the production of a stable polymer by S. thermophilus ST 111, which is not the case, e.g., with the high-molecular-mass EPS from S. thermophilus LY03 (Degeest & De Vuyst, 1999). In the latter case both temperature and pH influenced EPS degradation, indicating enzymatic effects (Pham et al., 2000; Degeest et al., 2002). In Lb. rhamnosus R two enzymes, a-d-glucosidase and b-d-glucuronidase, seem to be active in a wide pH range with an optimum around pH 5.0 for a-d-glucosidase and a more acidic pH for b-d-glucuronidase (Pham et al., 2000). It can be assumed that such enzymes are not produced or secreted by S. thermophilus ST 111 or that the environmental conditions are not optimal for their activity. Also, during pH-uncontrolled milk fermentations with Lb. delbrueckii subsp. bulgaricus strains no EPS degradation has been observed; this has been ascribed to the pH decrease and the presence of milk caseins, protecting EPS against degradation (Petry et al., 2003). Alternatively, EPS-degrading enzymes may be specific only towards b-linkages, which are not present in the backbone of EPS from S. thermophilus ST 111. However, drying did influence EPS stability. The use
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of TCA during EPS isolation results in an acid environment, which might have caused physico-chemical degradation instead of enzymatic degradation of the EPS polymer during drying.
5. Conclusions In this study, S. thermophilus ST 111 produced a stable, high-molecular-mass EPS during fermentations in milk-based medium supplemented with whey protein hydrolystate at different constant pH values. This strain can be applicable in yoghurt manufacture, where genetically and physically stable EPS producers—even at low EPS concentrations—are required for in situ improvement of the rheology and texture of fermented food products. A decrease of the EPS molecular mass due to EPS degradation can cause serious problems with respect to the textural and sensorial quality of the end product (Duboc & Mollet, 2001). For industrial exploitation of isolated EPS or EPS-producing microbial strains, more information is required about the stability of EPS during food processing operations.
Acknowledgements Part of this research was financially supported by the Copernicus Program of the Commission of the European Community (Grant IC15-CT-98-0905) and the International Scientific and Technological Cooperation between Flanders and Romania from the Administration of Science and Innovation in Flanders (BWS 02/04). The authors further acknowledge financial support from the Flemish Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), the Fund for Scientific Research (FWO-Flanders), the LINK 2000 and 2002 Actions of the Brussels Capital Region, and the Research Council of the Vrije Universiteit Brussel. Frederik Vaningelgem is a recipient of an IWT fellowship.
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Degeest, B., & De Vuyst, L. (1999). Indication that the nitrogen source influences both amount and size of exopolysaccharides produced by Streptococcus thermophilus LY03 and modeling of bacterial growth and exopolysaccharide production. Applied and Environmental Microbiology, 65, 2863–2870. Degeest, B., & De Vuyst, L. (2000). Correlation of activities of the enzymes a-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase with exopolysaccharide biosynthesis by Streptococcus thermophilus LY03. Applied and Environmental Microbiology, 66, 3519–3527. Degeest, B., Janssens, B., & De Vuyst, L. (2001a). Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0–1: Production kinetics, enzyme activities, and EPS yields. Journal of Applied Microbiology, 91, 470–477. Degeest, B., Mozzi, F., & De Vuyst, L. (2002). Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations. International Journal of Food Microbiology, 79, 161–174. Degeest, B., Vaningelgem, F., Laws, A. P., & De Vuyst, L. (2001b). UDP-N-acetylglucosamine 4-epimerase activity indicates the presence of N-acetylgalactosamine in exopolysaccharides of Streptococcus thermophilus strains. Applied and Environmental Microbiology, 67, 3976–3984. De Vuyst, L., & Degeest, B. (1999). Heteropolysaccharides from lactic acid bacteria. FEMS Microbiology Reviews, 23, 153–177. De Vuyst, L., de Vin, F., Vaningelgem, F., & Degeest, B. (2001). Recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria. International Dairy Journal, 11, 687–707. De Vuyst, L., Vanderveken, F., Van de Ven, S., & Degeest, B. (1998). Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis. Journal of Applied Microbiology, 84, 1059–1069. De Vuyst, L., & Vaningelgem, F. (2003). Developing new polysaccharides. In B. M. Mc Kenna (Ed.), Texture in food, Vol. 2: Semi-solid foods (pp. 275–320). Cambridge: Woodhead Publishing Ltd. De Vuyst, L., Zamfir, M., Mozzi, F., Adriany, T., Marshall, V., Degeest, B., & Vaningelgem, F. (2003). Exopolysaccharide-producing Streptococcus thermophilus strains as functional starter cultures in the production of fermented milks. International Dairy Journal, 13, 707–717. Duboc, P., & Mollet, B. (2001). Applications of exopolysaccharides in the dairy industry. International Dairy Journal, 11, 759–768. Faber, E. J., van den Haak, M. J., Kamerling, J. P., & Vliegenthart, J. F. G. (2001). Structure of the exopolysaccharide produced by Streptococcus thermophilus S3. Carbohydrate Research, 331, 173–182. Faber, E. J., Zoon, P., Kamerling, J. P., & Vliegenthart, J. F. G. (1998). The exopolysaccharides produced by Streptococcus thermophilus Rs and Sts have the same repeating unit but differ in viscosity of their milk cultures. Carbohydrate Research, 310, 269–276. Grobben, G. J., Smith, M. R., Sikkema, J., & de Bont, J. A. M. (1996). Influence of fructose and glucose on the production of exopolysaccharides and the enzyme activities involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772. 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., & de Bont, J. A. M. (1997). Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous
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culture on glucose and fructose. Applied Microbiology and Biotechnology, 48, 516–521. Jolly, L., & Stingele, F. (2001). Molecular organisation and functionality of exopolysaccharide gene clusters in lactic acid bacteria. International Dairy Journal, 11, 733–745. Jolly, L., Vincent, S. J. F., Duboc, P., & Neeser, J.-R. (2002). Exploiting exopolysaccharides from lactic acid bacteria. Antonie van Leeuwenhoek, 82, 367–374. Lamothe, G. T., Jolly, L., Mollet, B., & Stingele, F. (2002). Genetic and biochemical characterization of exopolysaccharide biosynthesis by Lactobacillus delbrueckii subsp. bulgaricus. Archives of Microbiology, 178, 218–228. Laws, A. P., & Marshall, V. M. (2001). The relevance of exopolysaccharides to the rheological properties in milk fermented with ropy strains of lactic acid bacteria. International Dairy Journal, 11, 709–721. Lemoine, J., Chirat, F., Wieruszeksi, J. M., Strecker, G., Favre, N., & Neeser, J.-R. (1997). Structural characterisation of the exocellular polysaccharides produced by S. thermophilus Sfi39 and Sfi12. Applied and Environmental Microbiology, 63, 3512–3518. Levander, F., & R(adstr.om, P. (2001). Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus. Applied and Environmental Microbiology, 67, 2734–2738. Looijesteijn, P. J., Boels, I. C., Kleerebezem, M., & Hugenholtz, J. (1999). Regulation of exopolysaccharide production by Lactococcus lactis subsp. cremoris by the sugar source. Applied and Environmental Microbiology, 65, 5003–5008. Low, D., Ahlgren, J. A., Horne, D., McMahon, D. J., Oberg, C. J., & Broadbent, J. R. (1998). Role of Streptococcus thermophilus MR1C capsular exopolysaccharide in cheese moisture retention. Applied and Environmental Microbiology, 64, 2147–2151. Marshall, V. M., Cowie, E. N., & Moreton, R. S. (1995). Analysis and production of two exopolysaccharides from Lactococcus lactis subsp. cremoris LC330. Journal of Dairy Research, 62, 621–628.
Monsan, P., Bozonnet, S., Albenne, C., Joucla, G., Willemot, R.-M., & Remaud-Sim!eon, M. (2001). Homopolysaccharides from lactic acid bacteria. International Dairy Journal, 11, 675–685. Mozzi, F., Rollan, G., Savoy de Giori, G., & Font de Valdez, G. (2001). Effect of galactose and glucose on the exopolysaccharide production and the activities of biosynthetic enzymes in Lactobacillus casei CRL 87. Journal of Applied Microbiology, 91, 160–167. Mozzi, F., Savoy de Giori, G., & Font de Valdez, G. (2003). UDPgalactose 4-epimerase: A key enzyme in exopolysaccharide formation by Lactobacillus casei CRL 87 in controlled pH batch cultures. Journal of Applied Microbiology, 94, 1–9. Navarini, L., Abatangelo, A., Bertocchi, C., Conti, E., Bosco, M., & Picotti, F. (2001). Isolation and characterization of the exopolysaccharide produced by Streptococcus thermophilus SFi20. International Journal of Biological Macromolecules, 28, 219–226. Petry, S., Furlan, S., Waghorne, E., Saulnier, L., Cerning, J., & Maguin, E. (2003). Comparison of the thickening properties of four Lactobacillus delbrueckii subsp. bulgaricus strains and physicochemical characterization of their exopolysaccharides. FEMS Microbiology Letters, 221, 285–291. Pham, P. L., Dupont, I., Roy, D., Lapointe, G., & Cerning, J. (2000). Production of exopolysaccharide by Lactobacillus rhamnosus R and its enzymatic degradation during prolonged fermentation. Applied and Environmental Microbiology, 66, 2302–2310. Ruas-Madiedo, P., Hugenholtz, J., & Zoon, P. (2002a). An overview of the functionality of exopolysaccharides produced by lactic acid bacteria. International Dairy Journal, 12, 163–171. Ruas-Madiedo, P., Tuinier, R., Kanning, M., & Zoon, P. (2002b). Role of exopolysaccharides produced by Lactococcus lactis subsp. cremoris on the viscosity of fermented milks. International Dairy Journal, 12, 689–695. Vaningelgem, F., Zamfir, M., Mozzi, F., Adriany, T., Vancanneyt, M., Swings, J., & De Vuyst, L. (2004). Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics. Applied and Environmental Microbiology, 70, 900–912.
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Streptococcus thermophilus ST 111 produces a stable high-molecular-mass exopolysaccharide in milk-based medium Frederik Vaningelgema, Roel Van der Meulena, Medana Zamfira, Tom Adrianya, Andrew P. Lawsb, Luc De Vuysta,* a Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium b Department of Chemical and Biological Sciences, University of Huddersfield, Queensgate, HDI 3DH Huddersfield, UK
Received 8 December 2003; accepted 12 March 2004
Abstract Streptococcus thermophilus ST 111, grown in milk medium supplemented with whey protein hydrolysate, produced an exopolysaccharide (EPS) composed of galactose and rhamnose in a molar ratio of 2.5:1, and with a molecular mass of more than 5000 kDa. Fermentations in milk medium supplemented with whey protein hydrolysate further demonstrated the stability of this high-molecular-mass EPS, as its molecular mass was affected neither by the pH of the medium nor the fermentation time, indicating no enzymatic degradation. On the other hand, drying of the isolated EPS resulted in a decrease of the molecular mass. The stability of an EPS produced in milk-based media is an important asset for the production of fermented milk and yoghurt products. r 2004 Elsevier Ltd. All rights reserved. Keywords: Exopolysaccharide; Streptococcus thermophilus; Molecular mass; Structure; Stability
1. Introduction Exopolysaccharides (EPS) from lactic acid bacteria (LAB) can be subdivided into two major groups: homopolysaccharides and heteropolysaccharides (De Vuyst & Degeest, 1999; Monsan et al., 2001; De Vuyst & Vaningelgem, 2003). Based on their structure, four groups of homopolysaccharides can be distinguished: a-d-glucans (dextrans, mutans, alternan), b-d-glucans, b-d-fructans (levans, inulin-type fructans), and others like polygalactan. Heteropolysaccharides are produced by LAB in a greater variety concerning chemical composition, monomer ratio, and molecular structure of the repeating unit, as well as the molecular mass of the polymer (De Vuyst & Vaningelgem, 2003). The repeating units of heteropolysaccharides most often contain a combination of d-glucose, d-galactose, and l-rhamnose in different ratios and, in a few cases, fucose, acetylated amino sugars, glucuronic acid, and nononic acid; as well, non-carbohydrate constituents such as glycerol, phosphate, acetyl, and pyruvyl groups *Corresponding author. Tel.: +32-2-629-32-45; fax: 32-2-629-27-20. E-mail address: [email protected] (L.De Vuyst). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.03.007
may be present (De Vuyst, de Vin, Vaningelgem, & Degeest, 2001; De Vuyst & Vaningelgem, 2003). Furthermore, within one repeating unit, different types of linkages can occur. In the backbone of heteropolysaccharides from LAB, a(1-3) linkages are more abundant than b(1-4) ones, which in turn occur more frequently than a(1-2), b(1-3), and b(1-6) linkages (De Vuyst & Vaningelgem, 2003). In general, the types of linkages within an EPS polymer determine the rigidity (Laws & Marshall, 2001) and hence the intrinsic viscosity of the polymer (Ruas-Madiedo, Tuinier, Kanning, & Zoon, 2002b). The apparent viscosity of an EPS solution is primarily determined by the molecular mass of the polymer. The molecular mass of heteropolysaccharides varies from 1.0 104 to 9.0 106 Da. Some strains produce high-molecularmass EPS (Navarini et al., 2001), others produce ! low-molecular-mass EPS (Almiron-Roig, Mulholland, Gasson, & Griffin, 2000), while still others produce both types of EPS (Marshall, Cowie, & Moreton, 1995; Grobben et al., 1997; Degeest & De Vuyst, 1999; Petry et al., 2003). The biosynthesis of the repeating unit of the heteropolysaccharides produced by LAB has been studied on an enzyme level for Streptococcus
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thermophilus (Degeest & De Vuyst, 2000; Degeest, Vaningelgem, Laws, & De Vuyst, 2001b), Lactobacillus casei (Mozzi, Rollan, Savoy de Giori, & Font de Valdez, 2001; Mozzi, Savoy de Giori, & Font de Valdez, 2003), Lb. sakei (Degeest, Janssens, & De Vuyst, 2001a), Lb. delbrueckii subsp. bulgaricus (Grobben, Smith, Sikkema, & de Bont, 1996), and Lactococcus lactis (Looijesteijn, Boels, Kleerebezem, & Hugenholtz, 1999). The genes involved in EPS biosynthesis, regulation, polymerisation, and export appear to be highly conserved among LAB (Jolly & Stingele, 2001; Jolly, Vincent, Duboc, & Neeser, 2002). However, detailed genetic and molecular knowledge about the polymerisation (i.e., chain length determination) of the repeating units and secretion of the final EPS is still lacking (Jolly & Stingele, 2001; Lamothe, Jolly, Mollet, & Stingele, 2002), making it difficult to genetically modify LAB or to design fermentation strategies for the production of EPS with a specific structure or polymer length. Generally, the amounts of EPS produced by LAB in the food matrix are low and their production is often unstable (De Vuyst et al., 2001). This unstable EPS production can be due to the loss of plasmids, especially in the case of mesophilic LAB, the presence of mobile genetic elements or genomic rearrangements (De Vuyst et al., 2001). Also, EPS degradation can occur when intracellular enzymes (e.g., a-d-glucosidase and b-d-glucuronidase) are released into the medium, resulting in a decrease of the molecular mass of the EPS polymers (Pham, Dupont, Roy, Lapointe, & Cerning, 2000; Degeest, Mozzi, & De Vuyst, 2002), which in turn may lead to a decreased intrinsic viscosity (Ruas-Madiedo et al., 2002b). Although the enzymatic degradation can be influenced and hence modulated by the physico-chemical conditions applied (Degeest et al., 2002), strains producing stable high-molecular-mass EPS in situ are of interest for the improvement of texture in food systems. Furthermore, not much is known about the stability of heteropolysaccharides from LAB during food processing operations such as drying, heating, freezing, pumping, etc. S. thermophilus is one of the most important LAB in the dairy industry, applied as a starter culture in yoghurt fermentation and cheese making. The aim of this study was to characterise the EPS of S. thermophilus ST 111, a strain isolated from an artisan Romanian yoghurt, with respect to its monomer composition and molecular mass, when grown in a milk-based medium. The structure of the high-molecular-mass EPS of S. thermophilus ST 111 was elucidated by nuclear magnetic resonance (NMR) spectroscopy. Also, pH-controlled fermentations were performed in a milk-based medium to study the effect of constant pH on the monomer composition and molecular mass of EPS from S. thermophilus ST 111. Finally, the effect of a drying step on the monomer composition and molecular mass of the EPS was studied.
2. Materials and methods 2.1. Strain and strain propagation S. thermophilus ST 111, a strain isolated from an artisan Romanian yoghurt, was used as the EPSproducing strain throughout this study. Isolation of the strain was accomplished after plating on solid, lactosecontaining (0.5%, w/v) M17 medium (Oxoid, Basingstoke, UK). The strain was identified by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole-cell proteins (Vaningelgem et al., 2004). The strain was stored at 80 C in de Man–Rogosa–Sharpe (MRS) medium (Oxoid) containing 25% (v/v) glycerol as cryoprotectant. To obtain fresh cultures, S. thermophilus ST 111 was propagated twice at 42 C for 12 h in the medium identical to the one used for the fermentations later on. 2.2. Media Milk medium (10.0% skimmed milk powder, w/v; Dairy Industry Inc., Kallo, Belgium), and milk medium supplemented with a whey protein hydrolysate (1.6%, w/v; a commercial product referred to as lactalbumin hydrolysate, Oxoid) were used during this study. 2.3. Isolation and purification of EPS EPS were isolated as described previously (De Vuyst, Vanderveken, Van de Ven, & Degeest, 1998). Briefly, trichloroacetic acid (TCA) and acetone precipitation were carried out. EPS were harvested by centrifugation (25,000 g, 4 C, 20 min), followed by another TCA and acetone precipitation. The isolated EPS material was then dialysed against distilled water for 7 days with water replacement twice a day, using Spectra/Por membranes (VWR International, Darmstadt, Germany) with a molecular-mass-cut-off (MMCO) of 3500 Da, and subsequently concentrated by freeze-drying (Heto Drywinner, Heto-Holten, Allerød, Denmark). To have an idea of the purity of the EPS material, both residual lactose and protein contaminants were determined in solutions of purified EPS. In pure EPS, no residual lactose could be determined by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD, Dionex, Sunnyvale, California, USA), using a CarboPacTM PA 10 column (Dionex). The protein concentration of all EPS samples, determined with a BioRad Protein Assay Kit (BioRad Laboratories, Hercules, California, USA), was always lower than 3% (w/w). 2.4. Molecular mass determination For molecular mass analysis, EPS samples were obtained from fermentations carried out in milk
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medium supplemented with whey protein hydrolysate, isolated, purified, and concentrated as described above. The molecular mass of the purified EPS was determined by gel permeation chromatography (GPC) using two types of columns. First, a 100 cm 1.6 cm Sephacryl S-400 column (Amersham BioSciences AB, Uppsala, Sweden) was used with 200 mL samples containing about 50 mg of lyophilised EPS mL1. The EPS were eluted with 50 mm potassium phosphate buffer (pH 6.8) containing 0.15 m NaCl at a flow rate of 1 mL min1. A dextran standard series (molecular masses of 80, 150, 270, 670, and 1800 kDa; Sigma, St. Louis, Missouri, USA) was used to estimate the molecular mass of the EPS. The linear range of the column was limited to 2000 kDa according to the manufacturer. The standard deviation (expressed in retention time) was 71.5 min based on three injections of one sample. To further characterise the EPS from S. thermophilus ST 111, a 30 cm 7.8 mm UltrahydrogelTMLinear column (Waters Corp., Milford, Massachusetts, USA) was used, applying two types of standards, namely dextrans (molecular masses of 80, 150, 270, 410, 670, 1400, and 4900 kDa; Sigma) and pullulans (molecular masses of 5.9, 11.8, 22.8, 47.3, 112.0, 212.0, 404.0, and 788.0 kDa; Shodex P82, Showa Denko K.K., Tokyo, Japan). Samples of 200 mL containing about 100 mg of lyophilised EPS L1 were applied on the column. The EPS were eluted with 0.1 m NaNO3 at a flow rate of 0.6 mL min1. The linear range of this column was limited to 8000 kDa according to the manufacturer. With both types of columns, the polysaccharide content was determined on line with a Waters 2410 refractive index detector (Waters Corp.) at an internal temperature of 40 C. Molecular mass analysis was always performed in duplicate and values were averaged. 2.5. Monomer analysis For monomer analysis, EPS were obtained from fermentations carried out in milk medium supplemented with whey protein hydrolysate, isolated, purified, and concentrated as described above. The monomer composition of the purified EPS was determined after acid hydrolysis with 6 n trifluoroacetic acid (TFA) at 100 C for 3 h, using HPAEC-PAD (Dionex), as described previously (Degeest et al., 2001b). Monomer analysis was performed in duplicate and values were averaged. 2.6. EPS elucidation by one-dimensional NMR spectroscopy For structure determination, EPS material was obtained from fermentations carried out in milk
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medium (1-L flasks, 42 C, free pH), isolated, purified, and concentrated as described above. The lyophilised polysaccharide was dissolved directly in D2O (99.9% D; Goss Scientific Instruments Ltd., Essex, UK); NMR spectra were recorded at a probe temperature of 70 C. The elevated temperature shifted the HOD signal to a higher field, into a clear region of the spectrum. The higher temperature also increased spectral resolution by reducing the sample viscosity. The NMR spectra were recorded on a Bruker Avance DPX400 MHz spectrometer (Bruker Analytical GmBH, Rheinstetten, Germany) operating with Z-field gradients and using Bruker’s pulse programmes. Chemical shifts ðdÞ were expressed in parts per million relative to internal acetone, d2.225. The 1D 1H spectra were processed with 32,768 data points. The two-dimensional gradient selected Double Quantum Filter COrrelated SpectroscopY (2D gs-DQF-COSY) spectrum was recorded in magnitude mode at 70 C, the time-domain data was multiplied by a squared-sine-bell function (SSB 0). After applying linear prediction and after Fourier transformation, data sets of 1024 1024 points were obtained. The 1 H spectra were recorded for several EPS samples, isolated during the stationary phase. 2.7. Fermentation conditions, on line analysis and sampling To study the influence of the fermentation pH on the monomer composition and molecular mass of EPS produced by S. thermophilus ST 111, large-scale fermentations were carried out in a 15-L computercontrolled laboratory fermentor (Biostat Cs; B. Braun Biotech International, Melsungen, Germany). Milk medium (10 L) was sterilised in an autoclave at 121 C for 20 min, and aseptically pumped into a sterilised fermentor. Previously, the fermentor was filled with water and sterilised in situ at 121 C for 20 min. Afterwards the sterile water was drained. The whey protein hydrolysate was sterilised separately as well and aseptically added to the fermentor containing the sterile milk medium. To keep the medium in the fermentor homogeneous, agitation was performed at 100 rpm with a stirrer composed of three standard impellers. The fermentor was inoculated with 1.0% (v/v) of a fresh culture of the strain. The fermentation temperature was kept constant at 42 C. To study the influence of pH, the initial pH of the milk medium supplemented with whey protein hydrolysate was adjusted to pH 5.5, 5.8, 6.2, and 6.6 using 2 n HCl or 10 n NaOH (a comparison was made with milk medium adjusted to pH 5.8). The pH was kept constant during fermentation through automatic addition of 10 n NaOH. The temperature, pH, and agitation were computer-controlled and monitored on line (Micro MFCS for WindowstNT software; B. Braun Biotech
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International). At different stages during the fermentation, samples were aseptically withdrawn from the fermentor to isolate EPS. The molecular mass of EPS material from several fermentation samples was determined (Sephacryl S-400 gel column, see above). To eliminate differences in growth rate and acidification between these fermentation samples and to have the cells in a similar physiological state, sampling times were linked with the amount of NaOH added to the medium for pH control, namely after addition of 50, 100, and 175 mL of NaOH, i.e., 5.3, 10.6, and 19.5 mL of base per liter of medium. Two other samples were collected after 12 and 24 h of fermentation, respectively. To determine the effect of drying on the EPS characteristics (monomer composition and molecular mass analysis), EPS isolated via TCA and acetone precipitation were dried at 42 C during 48 h. Afterwards, the same purification and concentration steps as described above, i.e., dialysis of the dried EPS dissolved in ultra pure water for 7 days (water replacement twice a day), followed by overnight freeze drying, were carried out. This EPS was compared with purified EPS that had not been subjected to the drying step at 42 C. This experiment has been performed in duplicate.
3. Results 3.1. Isolation and purification of the EPS produced by S. thermophilus ST 111 When grown in milk medium supplemented with whey protein hydrolysate, the EPS produced by S. thermophilus ST 111 was easily precipitated with acetone. GPC of purified EPS material resulted in a peak with a molecular mass of more than 2000 kDa (Fig. 1a), thus exceeding the linear range of the Sephacryl S-400 gel GPC column. EPS isolated from milk medium gave the same results, while in the case the strain was grown in MRS medium the EPS material was contaminated with polysaccharides derived from yeast extract and peptone (De Vuyst et al., 2003). With an UltrahydrogelTMLinear GPC column with a higher linear range (up to 8000 kDa), the molecular mass of the EPS from S. thermophilus ST 111 was higher compared with the largest molecular mass of each type of standards used, i.e., 4900 kDa for dextran and 788 kDa for pullulan. These data indicate that S. thermophilus ST 111 is producing a high-molecular-mass EPS, the size of which is difficult to determine. Both conformation and branching determine the linear range of a GPC column depending on the standards used.
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Fig. 1. Comparison of the gel permeation chromatograms (Sephacryl S-400 gel) of EPS produced by S. thermophilus ST 111 in milk medium supplemented with whey protein hydrolysate (1.6%, w/v). The EPS were obtained without a drying step (a) and with a drying step at 42 C for 48 h before dialysis and concentration (b). Vertical lines represent the molecular mass markers: (1) 1800 kDa, (2) 670 kDa, (3) 410 kDa, (4) 270 kDa, (5) 150 kDa, and (6) 80 kDa.
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Monomer analysis with HPAEC-PAD gave a galactose/rhamnose ratio of 2.5:1.0 for the EPS material derived from milk medium supplemented with whey protein hydrolysate. This was also found previously for EPS isolated from milk medium fermented with this strain (De Vuyst et al., 2003). 3.2. Structure elucidation of the EPS produced by S. thermophilus ST 111 The 1H spectra recorded for several EPS samples, isolated during the stationary phase from several milk fermentations of S. thermophilus ST 111, were identical (Fig. 2a). There were seven low-field H-1 signals, designated A–G in Fig. 2a (d5.26 A H-1, 5.25 B H-1, 5.16 C H-1, 5.16 D H-1, 5.01 E H-1, 4.65 F H-1 and 4.45 G H-1). The locations of the related H-2 resonances were available from the COSY spectrum (d 4.02 A H-2, 4.05 B H-2, 3.96 C H-2, 4.03 D H-2, 4.35 E H-2, 3.50 F H-2 and 3.53 G H-2). From this information it follows that the polysaccharide repeating unit was a heptasaccharide comprising two rhamnosyl and three a-galactosyl (A–E) linkages with two b-galactosyl linkages (F,G) displaying characteristic trans couplings. The spectra and the H-1 and H-2 chemical shifts resulted in the structure represented in Fig. 2b.
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affected the monomer composition of the EPS produced by S. thermophilus ST 111. The same monomer ratio of 2.5 Gal for 1.0 Rha was determined for all samples. The base consumption during fermentations in milk medium supplemented with whey protein hydrolysate at constant pH 6.2 was the fastest, followed by fermentations at constant pH 6.6, pH 5.8, and pH 5.5 (Fig. 3). When no whey protein hydrolysate was added, acidification and thus base consumption was much slower (Fig. 3). All GPC chromatograms of EPS material isolated from samples obtained during these fermentations showed the presence of high-molecular-mass EPS (>2000 kDa). No other peaks were observed, indicating that no degradation of the EPS took place under the different physico-chemical conditions tested (Table 1). However, when isolated EPS were dried at 42 C during
3.3. Influence of the fermentation pH and drying on the monomer composition and molecular mass of EPS from S. thermophilus ST 111 Neither the addition of whey protein hydrolysate to milk medium nor the pH of the fermentation medium
Fig. 3. Base consumption (mL of 10 n NaOH per liter of fermentation medium) during fermentations with S. thermophilus ST 111 in milk medium at a constant pH of 5.8 (&) and in milk medium supplemented with whey protein hydrolysate (1.6%, w/v) at a constant pH of 5.5 (~), 5.8 (’), 6.2 (m), and 6.6 (). All fermentations were carried out at 42 C. Horizontal lines indicate at which amount of base consumed samples were taken for EPS analysis. In addition, two samples were taken after 12 and 24 h of fermentation.
Table 1 Influence of the fermentation pH on the retention time during gel permeation chromatography of the EPS produced by S. thermophilus ST 111 in milk medium supplemented with whey protein hydrolysate (1.6%, w/v) at a constant temperature of 42 C Fermentation pH Retention timea 5.5 5.8 6.2 6.6 Sampling timeb
Fig. 2. 400-MHz 1H-NMR spectrum of the EPS from S. thermophilus ST 111 (a) and repeating unit of the EPS from S. thermophilus ST 111 (b). The seven low-field H-1 signals (A–G) are specified in the insert of (a).
78.7 75.6 75.0 78.5 5.3 mL L1
77.8 75.0 75.0 78.0 10.6 mL L1
78.8 75.0 76.5 78.1 19.5 mL L1
77.9 77.5 75.0 78.2 12 h
78.3 79.6 75.9 78.1 24 h
a The exact molecular mass could not be determined as all the measurements exceeded the linear range of the column (>2000 kDa). Molecular mass analysis was always performed in duplicate and retention times were averaged. The standard deviation was 71.5 min. b Sample times were linked to the base consumption (10 n NaOH) per liter of fermentation medium (in mL base L1). Two samples were collected after 12 and 24 h of fermentation as well.
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48 h an increased retention time and a broader peak (Fig. 1b) were observed compared with EPS which were not subjected to this drying step (Fig. 1a). This decrease in retention time indicated a decrease of molecular mass and hence some degradation of the EPS, when a drying step was carried out before EPS purification. No differences in monomer composition were observed between dried and non-dried samples. Similar GPC chromatograms were determined in the duplicate experiment.
4. Discussion Ropy, thermophilic LAB starter cultures for yoghurt production are often used in some countries of the EU, albeit unconsciously or under non-optimal conditions. S. thermophilus ST 111 is a Romanian yoghurt strain that grows in milk medium and other complex media containing glucose, sucrose, or lactose as the sole energy source (Vaningelgem, Zamfir, Adriany, & De Vuyst, unpublished results). It displays a low proteolytic activity and probably this is the main reason for its slow growth in milk. As protein hydrolysates contain peptides or free amino acids that may be more readily available for the cells, milk medium supplemented with whey protein hydrolysate was ideal for good growth and high EPS production as well as for EPS isolation (no interfering polysaccharides) with S. thermophilus ST 111. In milk medium, S. thermophilus ST 111 produces a high-molecular-mass heteropolysaccharide composed of galactose and rhamnose in a 2.5:1 molar ratio (De Vuyst et al., 2003). When the strain was cultivated in milk medium supplemented with whey protein hydrolysate, no changes in molecular mass and monomer composition of the EPS were found. The presence of galactose and rhamnose as the sole sugar residues has been found in EPS of only four S. thermophilus strains and not in EPS of other LAB strains examined so far (Bubb, Urashima, Fujiwara, Shinnai, & Ariga, 1997; Faber, Zoon, Kamerling, & Vliegenthart, 1998; Faber, van den Haak, Kamerling, & Vliegenthart, 2001). This is interesting from a health point of view, because it has been postulated that rhamnose-containing EPS may stimulate the immune system through mannose receptors situated on macrophages (Chabot et al., 2001). The absence of glucose in the repeating unit of the EPS of S. thermophilus ST 111 may indicate an increased activity of UDP-galactose 4-epimerase and no or decreased activity of UDP-glucose pyrophosphorylase, both enzyme activities assuming EPS biosynthesis from . the galactose moiety of lactose (Levander & Ra( dstrom, 2001). The molecular structure of the EPS from S. thermophilus ST 111, as revealed by NMR analysis, consisted of a heptasaccharide repeating unit, identical
to the EPS repeating units from S. thermophilus OR901 (Bubb et al., 1997) and S. thermophilus Rs and Sts (Faber et al., 1998). It is structurally similar to the EPS subunits of S. thermophilus SFi12 (Lemoine et al., 1997) and S. thermophilus MR-1C (Low et al., 1998). All these EPS structures are composed of sugars, which are only linked with each other via a-linkages in the backbone of the repeating unit. These a-linkages may be disadvantageous for the stiffness of the polymer and hence the viscosity of the resulting EPS solutions (Laws & Marshall, 2001, Ruas-Madiedo, Hugenholtz, & Zoon, 2002a). Indeed, the a-linkages in the backbone of the EPS from S. thermophilus ST 111 cause a weak consistency of the EPS solution compared with EPS from S. thermophilus LY03, which contains both a- and b-linkages (Vaningelgem et al., 2004). Of all EPS structures published to date, the repeating unit of the EPS produced by S. thermophilus ST 111 is the second largest one reported, indicating that LAB may not be capable to produce larger building blocks than octasaccharides (De Vuyst et al., 2001). While the molecular mass of the EPS from S. thermophilus ST 111 already had been estimated in previous studies (De Vuyst et al., 2003), the linear range of the GPC column used in this study was greater (up to 8000 kDa); the molecular mass of the EPS from S. thermophilus ST 111 was confirmed to be higher than 5000 kDa. No change in molecular mass and hence no degradation of the polymer nor a change in the monomer composition of the EPS was observed for the high-molecular-mass EPS produced by S. thermophilus ST 111 at varying constant fermentation pH values, independent of the fermentation time, indicating no enzymatic degradation. This shows the production of a stable polymer by S. thermophilus ST 111, which is not the case, e.g., with the high-molecular-mass EPS from S. thermophilus LY03 (Degeest & De Vuyst, 1999). In the latter case both temperature and pH influenced EPS degradation, indicating enzymatic effects (Pham et al., 2000; Degeest et al., 2002). In Lb. rhamnosus R two enzymes, a-d-glucosidase and b-d-glucuronidase, seem to be active in a wide pH range with an optimum around pH 5.0 for a-d-glucosidase and a more acidic pH for b-d-glucuronidase (Pham et al., 2000). It can be assumed that such enzymes are not produced or secreted by S. thermophilus ST 111 or that the environmental conditions are not optimal for their activity. Also, during pH-uncontrolled milk fermentations with Lb. delbrueckii subsp. bulgaricus strains no EPS degradation has been observed; this has been ascribed to the pH decrease and the presence of milk caseins, protecting EPS against degradation (Petry et al., 2003). Alternatively, EPS-degrading enzymes may be specific only towards b-linkages, which are not present in the backbone of EPS from S. thermophilus ST 111. However, drying did influence EPS stability. The use
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of TCA during EPS isolation results in an acid environment, which might have caused physico-chemical degradation instead of enzymatic degradation of the EPS polymer during drying.
5. Conclusions In this study, S. thermophilus ST 111 produced a stable, high-molecular-mass EPS during fermentations in milk-based medium supplemented with whey protein hydrolystate at different constant pH values. This strain can be applicable in yoghurt manufacture, where genetically and physically stable EPS producers—even at low EPS concentrations—are required for in situ improvement of the rheology and texture of fermented food products. A decrease of the EPS molecular mass due to EPS degradation can cause serious problems with respect to the textural and sensorial quality of the end product (Duboc & Mollet, 2001). For industrial exploitation of isolated EPS or EPS-producing microbial strains, more information is required about the stability of EPS during food processing operations.
Acknowledgements Part of this research was financially supported by the Copernicus Program of the Commission of the European Community (Grant IC15-CT-98-0905) and the International Scientific and Technological Cooperation between Flanders and Romania from the Administration of Science and Innovation in Flanders (BWS 02/04). The authors further acknowledge financial support from the Flemish Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), the Fund for Scientific Research (FWO-Flanders), the LINK 2000 and 2002 Actions of the Brussels Capital Region, and the Research Council of the Vrije Universiteit Brussel. Frederik Vaningelgem is a recipient of an IWT fellowship.
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