APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2005, p. 6398–6400 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.10.6398–6400.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 10
Metabolically Improved Exopolysaccharide Production by Streptococcus thermophilus and Its Influence on the Rheological Properties of Fermented Milk Malin Svensson,1 Elisabet Waak,2 Ulla Svensson,2 and Peter Rådstro ¨m1* Applied Microbiology, Lund Institute of Technology, Lund University, SE-221 00 Lund,1 and Arla Foods Innovation Center, SE-105 46 Stockholm,2 Sweden Received 31 January 2005/Accepted 13 May 2005
Altered levels of enzymes in the central carbon metabolism in Streptococcus thermophilus increased the exopolysaccharide (EPS) production 3.3 times over that of the parent strain. The influence of enhanced EPS production on the rheological properties of fermented milk is described for engineered strains of S. thermophilus which produce different levels of EPSs. Dairy strains of Streptococcus thermophilus that produce exopolysaccharides (EPSs) have attracted interest recently, since the EPSs act as in situ-produced natural biothickeners that improve the texture of fermented foods (5, 6, 10). Thus, a high EPS production in situ during the fermentation of milk to yogurt could be an advantage for the food industry. However, EPSs from S. thermophilus strains are produced at relatively low levels, i.e., 50 to 400 mg per liter (3). We have previously described the selection of a galactosefermenting (Gal⫹) mutant (TMB 6010) from the galactosenegative (Gal⫺) strain S. thermophilus LY03 with enhanced activities of the Leloir enzymes (Fig. 1), which generates an EPS yield 1.4 times higher than that of the parent strain (7). However, a drawback was the accumulation of glucose-1-phosphate (G1P), which was observed in this mutant. We have also reported on a successful metabolic engineering strategy to enhance EPS production in a Gal⫺ strain of S. thermophilus by a factor of 2 by increasing the activities of ␣-phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (GalU) (7), which are part of the central carbon metabolism (Fig. 1). The aims of this study were (i) to improve EPS production by S. thermophilus LY03 even further than in our previously constructed mutants (7) and (ii) to investigate the influence of EPSs on the rheological properties of fermented milk. To investigate the combined effect of increased activities of PGM, GalU, and the Leloir enzymes, S. thermophilus TMB 6010 was transformed with pFL46 (7) by electroporation as described previously (9), yielding TMB 6013. The strains S. thermophilus LY03 (commercial yogurt strain from Vrije University, Brussels, Belgium), TMB 6010, and TMB 6013 were cultivated in MST medium (8) at 42°C and a pH of 6.2 (Table 1), according to Levander et al. (7). In the late exponential phase, the EPS production by TMB 6013 was 3.3 times higher than that by LY03 and at least 1.5 times higher than that by our previously metabolically engineered strains (7). All these strains produced EPSs with identical structures (Andrew
Laws, personal communication). To gain a better understanding of how the metabolism was affected by the altered activities of PGM, GalU, and the Leloir enzymes, the intracellular concentrations of G1P, glucose-6-phosphate (G6P), and UDPglucose (UDPglc) were measured in the late exponential phase (Table 2) as previously described (7). Higher levels of G1P and G6P were found in TMB 6010 than in LY03, in agreement with our previous observations (7). This result suggests an increased flux through the Leloir pathway in TMB 6010 compared with LY03. Interestingly, a higher flux through the Leloir pathway in combination with increased activities of PGM and GalU in TMB 6013 resulted in levels of both G6P and G1P similar to those of LY03. When the metabolites could be used more efficiently in TMB 6013, a higher EPS yield was also obtained than with TMB 6010. Similar concentrations of UDPglc were found in the three strains, which indicates that the increased flux from glycolysis and the Leloir pathway in TMB 6013 does not lead to an accumulation of UDPglc.
FIG. 1. Schematic representation of lactose catabolism in S. thermophilus. Galactose is degraded by the Leloir enzymes (GalK, GalT, and GalE). Abbreviations: LacS, lactose transporter; LacZ, ␤-galactosidase; GalM, mutarotase; GalK, galactokinase; GalT, galactose-1phosphate uridylyltransferase; GalE, UDP-glucose 4-epimerase; Gal1P, galactose-1-phosphate.
* Corresponding author. Mailing address: Applied Microbiology, Lund Institute of Technology, Lund University, P.O. Box 124, SE221 00 Lund, Sweden. Phone: 46 46 222 3412. Fax: 46 46 222 4203. E-mail: [email protected]
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IMPROVED EXOPOLYSACCHARIDE PRODUCTION
TABLE 1. Combined effect of PGM, GalU, and the Leloir enzymes on growth and EPS yield in MST medium at pH 6.2 Strain
GalT activity (U mg of protein⫺1)
GalK activity (U mg of protein⫺1)
PGM activity (U mg of protein⫺1)
GalU activity (mU mg of protein⫺1)
YEPS/lactoseb (g mol of carbon⫺1)
LY03c TMB 6010c TMB 6013d
1.5 ⫾ 0.1 15.8 ⫾ 1.9 18.4 ⫾ 3.0
0.05 ⫾ 0.03 0.69 ⫾ 0.03 0.81 ⫾ 0.05
0.90 ⫾ 0.04 0.92 ⫾ 0.05 1.93 ⫾ 0.07
5.0 ⫾ 0.6 4.0 ⫾ 0.7e 10.0 ⫾ 0.9
1.4 ⫾ 0.2 1.2 ⫾ 0.0 1.2 ⫾ 0.0
0.16 ⫾ 0.04 0.25 ⫾ 0.03 0.53 ⫾ 0.04
7 7 This study
max, maximum specific growth rate. YEPS/lactose, EPS yield expressed as grams per mole of carbon from consumed lactose. All the strains consumed similar amounts of lactose. c Values are averages ⫾ standard deviations based on the results obtained with more independent cultivations than described in reference 7; in total, at least four independent cultivations. d Values are averages ⫾ standard deviations based on the results obtained with at least four independent cultivations. e This value is not presented in reference 7. a b
Instead, UDPglc can be metabolized, for example, by the enzymes involved in EPS biosynthesis. To investigate whether the enhanced EPS production by TMB 6013 would affect the rheological properties of fermented milk, LY03, TMB 6010, and TMB 6013 were cultivated in enriched milk medium (10% [wt/vol] skim milk powder; Arla Foods, Stockholm) with 0.5% (wt/vol) yeast extract (Merck, Darmstadt, Germany) and 1.0% (wt/vol) peptone (Oxoid, Basingstoke, United Kingdom). The milk medium was enriched with yeast extract and peptone because previous reports have shown that S. thermophilus strains are stimulated by the addition of extra amino acids (1, 2). Batch cultures were grown (at least in duplicate) at 42°C with an initial volume of 2.0 liters. Incubation was performed without agitation and without pH buffering, and growth was followed by monitoring the decline in the pH from 6.5 to 4.5. Under these conditions, the three strains grew in similar ways (data not shown). It has been reported that EPS production by S. thermophilus in milk is low under conditions in which the pH is not controlled (4), and since the EPS synthesis in milk at pH 4.5 was too low to measure, we determined EPS production at pH 4.5 during batch cultures in MST medium without pH buffering. The EPS yield in MST medium was 6.3 times higher from TMB 6013 (0.038 ⫾ 0.005 g mol of carbon⫺1) than from LY03 (0.006 ⫾ 0.002 g mol of carbon⫺1). It was therefore assumed that TMB 6013 also produced more EPS than the other strains at pH 4.5 in enriched milk. In addition, EPS biosynthesis has been shown to be growth associated in S. thermophilus (4), and there was no significant difference in growth between the strains in the two media (data not shown). The amount of expelled whey from the milk cultures was determined after 2 days of storage at 8°C by a pipetting and weighing method, but
TABLE 2. Concentrations of intracellular metabolites in MST medium in the late exponential phase Concn (mol g
no difference in syneresis was observed between the cultures (Table 3). Samples from the enriched milk cultures were gently stirred by hand using a spoon, prior to rheological analysis. The elastic modulus and the viscous modulus were measured using a CarriMed CSL 100 rheometer having a 4-cm-diameter parallel-plate geometry with a gap of 2 mm. The method was based on oscillation stress sweeps at a frequency of 1 Hz. The elastic modulus showed significantly lower values in TMB 6013 cultures than in cultures with LY03 or TMB 6010 (Table 3). The viscosity of the samples was measured using a Brookfield instrument. The spindles used were RV models 2 to 4, and the shear rate was 10 rpm. No significant difference was observed between the three strains during analysis of the viscous modulus and the viscosity. These results indicate that the increased EPS production in TMB 6013 reduces the elasticity of the fermented milk but does not affect the viscosity of the product. The result might have been different if EPSs with other structures had been studied; however, lower values of the elastic modulus have been reported for yogurts made with EPS-producing strains than with yogurts made with non-EPS-producing strains (6). The results of this study show that it is possible to increase EPS production by S. thermophilus LY03 through enhancement of the activities of the Leloir enzymes together with PGM and GalU. Our results suggest that increased EPS production by this genetically modified strain can lower the elastic modulus in milk cultures but does not affect the viscosity. To the best of our knowledge, this is the first time that the influence of improved EPS production on the rheological properties of fermented milk has been investigated through cultivation of an S. thermophilus mutant and its parent strain, producing different levels of EPSs with identical structures.
TABLE 3. Rheological properties of cultures grown to pH 4.5 in enriched milk medium
LY03a TMB 6010 a TMB 6013b
2.0 ⫾ 0.4 10.2 ⫾ 2.4 3.7 ⫾ 1.3
5.0 ⫾ 1.0 12.1 ⫾ 3.5 4.2 ⫾ 1.3
0.9 ⫾ 0.1 0.8 ⫾ 0.5 0.8 ⫾ 0.3
a Values are averages ⫾ standard deviations based on the results obtained with more independent cultivations than described in reference 7; in total, at least four independent cultivations. b Values are averages ⫾ standard deviations based on the results obtained with at least four independent cultivations.
Rheological property b
Syneresis (%) Elastic modulus (Pa) Viscous modulus (Pa) Viscosity (Pa s)
18 ⫾ 0 33 ⫾ 5 10 ⫾ 1 2.7 ⫾ 0.4
18 ⫾ 1 38 ⫾ 3 13 ⫾ 2 2.9 ⫾ 0.1
18 ⫾ 0 23 ⫾ 1 9⫾0 2.7 ⫾ 0.1
a All the values are averages ⫾ standard deviations based on the results obtained with at least two independent cultivations. b Separated whey was expressed as a percentage (wt/wt).
SVENSSON ET AL.
This work was financially supported by Vinnova, the Swedish Agency for Innovation Systems. ¨ hman for technical assistance. We thank Eva O 1. 2. 3. 4.
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