Ó Springer 2005
World Journal of Microbiology & Biotechnology (2005) 21:673–677 DOI 10.1007/s11274-004-3613-2
Exopolysaccharide production by Lactobacillus delbruckii subsp. bulgaricus and Streptococcus thermophilus strains under different growth conditions Belma Aslim1, Zehra Nur Yu¨ksekdag˘1,*, Yavuz Beyatli1 and Nazime Mercan2 1 Department of Biology, Faculty of Science and Arts, Gazi University, Teknikokullar, Ankara, Turkey 2 Department of Biology, Faculty of Science and Arts, Pamukkale University, Kınıklı, Denizli, Turkey *Author for correspondence: Tel.: +0903122126030/2705, Fax: +903122122279, E- mail:
[email protected]
Keywords: Different growth conditions, EPS, exopolysaccharide, Lactobacillus delbruckii subsp. bulgaricus, Streptococcus thermophilus
Summary The optimal temperature, pH and incubation time for production of exopolysaccharide (EPS) by Lactobacillus delbruckii subsp. bulgaricus and Streptococcus thermophilus strains in MRS and M17 media, respectively, were determined. In all strains, the temperature and incubation time for EPS production were 45 °C and 18 h, respectively. At 45 °C, L. delbruckii subsp. bulgaricus B3 and G12 and S. thermophilus W22 strains produced 263, 238 and 127 mg/l, respectively. At 18 h, B3, G12 and W22 strains produced 220, 152 and 120 mg/l, respectively. While the pH for highest EPS production by L. delbruckii subsp. bulgaricus strains was 6.2 (in B3 strain: 211 mg/l, in G12 strain: 175 mg/l), for highest EPS production by S. thermophilus strain it was 6.8 (114 mg/l).
Introduction Several lactic acid bacteria (LAB) are able to produce exopolysaccharides (EPSs) (Looijesteijn et al. 1999). This EPS is economically important because it can impart functional effects to foods and may confer beneficial health effects (Welman & Maddox 2003). LAB producing EPS play an important role in the food industry by improving the viscosity and the texture of fermented products (Ma˚rtensson et al. 2003). In recent years several studies have focused on the rheological and textural properties of yogurt made with EPSproducing strains (Ruas-Madiedo et al. 2002; Lorenzen et al. 2003). The main advantage of LAB EPSs is that they are produced by food-grade microorganisms and can contribute to the proper consistency and texture of fermented foods. However, low yields and high degradation rates of in situ -produced EPS are important bottlenecks for the industrial processor (Degeest et al. 2002). A number of studies on physical and chemical cultivation conditions of LAB to obtain higher EPS yields have been performed (Degeest et al. 2001). The physical factors of greatest importance are incubation temperature, pH, oxygen tension, agitation speed and incubation time (De Vuyst et al. 1998; De Vuyst & Degeest 1999; Degeest et al. 2002). Chemical factors determining the EPS yield of LAB are the carbohydrate source, the nitrogen source, the carbon/nitrogen ratio, and the presence or absence of other medium compo-
nents, e.g. salts and vitamins (Gancel & Novel 1994; De Vuyst et al. 1998; Degeest et al. 2002). Nowadays, a lot of effort is put into the selection of new microbial strains and the optimization of culture conditions to achieve higher yields of those EPSs already commercially successful. Furthermore, there is a considerable interest in finding new EPSs that are suitable for special applications, or that have potential industrial relevance, either by applying different culture conditions or by using novel bacterial strains (Looijesteijn et al. 2000). The objective of the present work was to determine the effects of different culture conditions (temperature, pH and incubation time) on growth and EPS production of our new isolates (L. delbruckii subsp. bulgaricus and S. thermophilus), and the culture conditions suitable for EPS production.
Materials and methods Isolation, identification and growth condition Two strains of L. delbruckii subsp. bulgaricus and one strain of S. thermophilus were used in this study. Isolates were isolated from village type yogurt and were tested by Gram stain, the catalase reaction and cell shape. Also, the carbohydrate fermentation characteristics of all strains were determined by using the API 50 CHL identification system. However, the isolates were
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identified by 16S rDNA sequence (Tilsala-Timisjarvi & Alatossava 1993; Ventura et al. 2000; Roy et al. 2001). The sequences obtained were searched against the GenBank DNA database using the BLAST function.
method (Dubois et al. 1956) using glucose as standard (Torino et al. 2001).
Results and discussion Culture conditions for EPS production Influence of temperature on growth and polymer synthesis To evaluate the influence of temperature, pH and incubation time on the growth and EPS production of L. delbruckii subsp. bulgaricus and S. thermophilus, the strains were grown in MRS and M17 media, respectively (Atlas & Parks 1997). To study the influence of various temperatures on EPS production by the various strains, 30, 37, 42 and 45 °C were incubated at pH 6.2 for L. delbruckii subsp. bulgaricus, pH 6.8 for S. thermophilus for 17 h. Also, for L. delbruckii subsp. bulgaricus strains, the Mann–Rugosa–Sharp (MRS) medium was adjusted to pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.2 and 7.0 with 4 M NaOH and 4 M HCl, separately. For S. thermophilus strain, the M17 medium was adjusted to pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.8 and 7.0 with 4 M NaOH and 4 M HCl, separately. These strains were incubated at 42 °C for 17 h. EPS production of the B3, G12 and W22 strains in MRS and M17 media at different incubation times (5, 8, 10, 14, 16, 18, 24, 32, 48 h). These strains were incubated at pH 6.2, pH 6.8 and 42 °C. Isolation and quantification of EPS After inoculation, broth cultures were incubated at 40 °C for 18 h. The cultures were boiled at 100 °C for 10 min. After cooling, they were treated with 17% (v/v) of 85% trichloracetic acid solution and centrifuged (Frengova et al. 2000). Removal of cells and protein was done by centrifugation. EPS was precipitated with ethanol. It was recovered by centrifugation at 4 °C at 14,000 rev/min for 20 min. Total EPS (expressed as mg/l) was estimated in each sample by phenol–sulphuric
In this study, we determined the effects of different growth conditions (temperature, pH and incubation time) on growth and EPS production. The influence of temperature on bacterial growth and EPS production is presented in Table 1. Four temperatures were tested: 30, 37, 42 and 45 °C. In all strains, there was a direct relationship between EPS production and growth temperature. Maximum polymer amount (263, 238, 127 mg/l) was attained at 45 °C (highest incubation temperature tested). In B3 strain, maximum EPS amount at 30, 37 and 42 °C was 61%, 50% and 20%, respectively, lower than that produced at 45 °C. In G12 strain, maximum EPS amount at 30, 37 and 42 °C was 49%, 41% and 26%, respectively, lower than that produced at 45 °C. In W22 strain, maximum EPS amount at 30, 37 and 42 °C was 14%, 13% and 10%, respectively, lower than that produced at 45 °C. Published efforts to optimize EPS production in LAB have included studies evaluating the effects of such environmental conditions as temperature, pH and incubation time (Cerning et al. 1994; Mozzi et al. 1996; Kimmel et al. 1998). A number of these studies examined the effect of temperature by using various EPSproducing strains of Lactobacillus delbruckii subsp. bulgaricus. Garcia-Garibay & Marshall (1991) found that EPS production by strain NCFB 2772 grown in skim milk was greater at a temperature (48 °C) higher than the optimum for growth (37–42 °C). Mozzi et al. (1996) found a correlation between the optimum growth temperature (37–42 °C) and maximum EPS production by strain CRL 870. In contrast, Schellhaass (1983)
Table 1. Influence of temperature on extracellular polysaccharide (EPS) production. Strains
EPS (mg/l)a
Temperature (°C)
OD600a
30 37 42 z45
1.92 1.94 1.98 2.00
± ± ± ±
0.03 0.02 0.00 0.00
102 131 211 263
± ± ± ±
0.0 0.0 1.0 0.0
30 37 42 45
1.50 1.48 1.76 2.57
± ± ± ±
0.04 0.00 0.03 0.00
123 140 175 238
± ± ± ±
1.0 0.5 0.0 5.0
30 37 42 45
2.26 2.56 2.05 2.26
± ± ± ±
0.04 0.01 0.04 0.01
109 110 114 127
± ± ± ±
0.5 1.0 0.0 0.5
L. delbruckii subsp. bulgaricus B3
L. delbruckii subsp. bulgaricus G12
S. thermophilus W22
a
Values are the means ± SD of triplicate measurements.
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Exopolysaccharides from lactic acid bacteria reported higher polymer production by strain RR at temperatures below the optimum for growth. In our study, EPS production by B3, G12 and W22 strains was evaluated at 30, 37, 42 and 45 °C. In all strains, the rate of EPS production was lower at 30, 37 and 42 °C than at 45 °C. Influence of pH In order to improve EPS production of strains, the influence of pH was studied. Results for growth and EPS production are shown in Table 2. pH affects both growth and EPS production. There was a general increase in EPS production and growth of all strains with increasing pH. All strains showed maximum growth at pH 7. L. delbruckii subsp. bulgaricus B3 and G12 strains produced maximum EPS amounts of 211 and 175 mg/l at pH 6.2, respectively. S. thermophilus W22 strain produced maximum EPS amounts of 114 mg/l at pH 6.8. When the pH was adjusted to 4, EPS production dramatically decreased to 65 (G12 strain), 62 (B3 strain) and 58 (W22 strain) mg/l. For these strains best growth was demonstrated at pH 7. However, best EPS production for these strains was at pH 6.2 for L. delbruckii subsp. bulgaricus and pH 6.8 for S. thermophilus. Little work has been done examining the effects of pH on EPS production by LAB. Mozzi et al. (1995) measured maximum EPS synthesis (488 mg/l) and highest cell numbers at a constant pH of 6.0 for L. casei CRL 87. In addition, the amount of EPS (mg/l) was 3.6 times as high in fermentations with pH control as in Table 2. Influence of pH on extracellular polysaccharide (EPS) production. Strains
pH
OD600a
L. delbruckii subsp. bulgaricus B3 4.0 0.98 ± 0.00 4.5 1.05 ± 0.01 5.0 1.24 ± 0.02 5.5 1.64 ± 0.04 6.0 1.70 ± 0.01 6.2 1.98 ± 0.00 7.0 2.04 ± 0.02 L. delbruckii subsp. bulgaricus G12 4.0 0.50 ± 0.00 4.5 1.00 ± 0.02 5.0 1.05 ± 0.04 5.5 1.61 ± 0.01 6.0 1.72 ± 0.00 6.2 1.76 ± 0.03 7.0 2.26 ± 0.02 S. thermophilus W22 4.0 0.81 ± 0.02 4.5 1.13 ± 0.04 5.0 1.36 ± 0.02 5.5 1.74 ± 0.00 6.0 1.95 ± 0.03 6.8 2.05 ± 0.04 7.0 2.10 ± 0.00 a
EPS (mg/l)a
62 75 111 123 154 211 198
± ± ± ± ± ± ±
0.0 2.0 0.0 3.0 0.0 1.0 0.0
65 100 121 126 150 175 131
± ± ± ± ± ± ±
0.0 2.0 0.0 3.0 0.0 0.0 2.0
58 69 72 84 98 114 101
± ± ± ± ± ± ±
0.0 1.0 2.0 0.0 3.0 0.0 2.0
Values are the means ± SD of triplicate measurements.
those without pH control. In a study conducted with L. sake 0-1, the optimum pH for EPS production was 5.8 (Van den Berg et al. 1995). Ricciardi et al. (2002) reported that the best pH for EPS production in S. thermophilus SY strain was 6.4. De Vuyst et al. (1998) also found that optimal pH was 6.2 compared with the results obtained at pH 4.9, 5.5 and 6.9. The effect of pH on EPS production depends on the experimental conditions and the strains used. The optimal pH for EPS production has been found to vary in different strains of LAB (De Vuyst & Degeest 1999; Ricciardi et al. 2002). However, the optimal pH for EPS production is often close to 6.0 (De Vuyst et al. 1998; Van den Berg et al. 1995) which is in agreement with our findings. Influence of incubation time In this study, EPS production of L. delbruckii subsp. bulgaricus B3, G12 and S. thermophilus W22 strains was detected between 5 and 48 h in MRS and M17 media, respectively (Figures 1–3). It was determined that EPS production of these strains increased (220, 152, 120 mg/l, respectively) until 18 h. In all strains, maximum EPS production was obtained at 18 h. After 24 h of incubation, EPS production started to decrease. In all strains, maximum EPS production was attained in the exponential phase of growth. EPS production increased during the exponential growth phase and no further production was observed in the stationary growth phase. These results are in agreement with observations on other researchers (De Vuyst et al. 1998; Grobben et al. 1998; Tallon et al. 2003). Marshall et al. (1995) indicated that the onset of EPS production from a strain of L. lactis subsp. cremoris is observed toward the end of the exponential phase of growth. Other investigators observed continued EPS production beyond or only in the stationary phase of growth (Manca De Narda et al. 1985; Gancel & Novel 1994; Bouzar et al. 1996). Such conclusions were however often based on optical density measurements of microbial growth, which is not always a valid parameter when using complex media (De Vuyst & Degeest 1999).
Conclusion The present study describes the influence of different conditions (temperature, pH and incubation time) on growth and EPS production of three strains isolated from Turkish yogurt. EPS production is an important element of characteristics of yogurt bacteria is forming starter cultures. In 45 °C, pH 6.2 for Lactobacillus strains, pH 6.8 for Streptococcus strain and 18 h, all three strains have high EPS production. Before using our new isolates as yogurt starters, it will be suitable to determine the best conditions for the production of EPS. The results can provide the basis for future investigations towards
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OD (600nm)
2
200
EPS (mg/L)
1,5
150
1
100
0,5
EPS (mg/l)
OD (600nm)
2,5
50
0
0 5
10
8
14
16
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Time (hours)
3
OD (600nm)
2,5
OD (600nm)
160
EPS (mg/L)
140 120
2
100
1,5
80 60
1
EPS (mg/l)
Figure 1. Growth and EPS production (mg/l) of the B3 strain in different incubation times.
40 0,5
20
0
0 5
8
10
14
16
18
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32
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Time (hours) Figure 2. Growth and EPS production (mg/l) of the G12 strain in different incubation times.
2,5
OD (600nm)
140
OD (600nm)
120
EPS (mg/L)
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2
80
1,5
60
1
EPS (mg/l)
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40
0,5
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0
0 5
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48
Time (hours) Figure 3. Growth and EPS production (mg/l) of the W22 strain in different incubation times.
defining the structure and physical and chemical characteristics of polymers. Acknowledgment This research has been supported by TUBITAK through Project No. TBAG-2090 (101T129). References Atlas, R.M. & Parks, L.C. 1997 Handbook of Microbiological Media, 2nd edn. pp. 783–965. New York: CRC Press. ISBN 0849326389. Bouzar, F., Cerning, J. & Desmazeaud, M. 1996 Expoloysaccharide production in milk by Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187 and by two colonial variants. Journal of Dairy Science 79, 205–211. Cerning, J., Renard, C.M.G.C., Thiboult, J.F., Bouillanne, C., London, M. & Desmazeaud, M. 1994 Carbon source requirements
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