Effects of incubation condition on the growth characteristics and exopolysaccharide production by ropy Lactobacillus delbrueckii subsp. bulgaricus

  (Pengaruh kondisi inkubasi terhadap sifat pertumbuhan dan produksi eksopolisakarida oleh Lactobacillus delbruesckii subsp. bulgaricus ropi)

Ratmawati Malaka· and Effendi Abustam·

            A study was conducted to exam the effects incubation condition on the growth characteristics and exopolysaccharide production by ropy Lactobacillus delbrueckii subsp. bulgaricus. Temperature and time of incubation defined as incubation condition, which are independent variables and EPS production, lactic acid and pH as dependent variables.

Ropy strains of Lactobacillus delbrueckii subsp. bulgaricus, were grown in 10% of Reconstitution Skim Milk (RSM) medium, produces exopolysaccharides (EPS) during the early stationary phase of growth.  Temperature and incubation time affected the EPS production and growth characteristics.  The optimal exopolysaccharide production was at 30^oC for 16 h of incubation time.

(Key words :  Exopolysaccharide, Lactobacillus delbrueckii subsp. bulgaricus)

            Suatu penelitian telah dilakukan untuk melihat pengaruh kondisi inkubasi terhadap karakteristik pertumbuhan dan produksi EPS oleh Lactobacillus delbrueckii subsp. bulgaricus. Temperatur dan waktu inkubasi didefinisikan sebagai kondisi inkubasi yang merupakan faktor bebas dan produksi EPS, asam laktat dan pH sebagai faktor dependen.

            Lactobacillus delbrueckii subsp. bulgaricus strain ropi yang ditumbuhkan dalam medium susu skim rekonstitusi (SSR) 10% menghasilkan EPS pada fase awal stasioner dalam pertumbuhan.  Suhu dan waktu inkubasi mempengaruhi produksi EPS dan karakteristik pertumbuhan.  Produksi polisakarida optimal pada suhu 30^oC selama waktu inkubasi 16 jam.

(Kata kunci : Eksopolisakarida, Lactobacillus delbrueckii subsp. bulgaricus)

            The effect of environmental condition on the exopolysaccharide (EPS) production by lactic acid bacteria has mainly been studied in thermophilic species of technological interest.  This type of polymers prevents the “wheying-off” and improves the texture of the final product of fermented milk (Mozzi et al., 1994).   The most mucoid microorganisms produce EPS under all growth conditions, but production is maximal under particular growth conditions on a defined medium (Ganzel and Novel, 1994).  All parameters increasing and decreasing growth rates influence the extracellular concentration of EPS precursors and therefore EPS synthesis. 

Growth temperature can also affect the synthesis of EPS.  Several reports informed that low temperatures markedly induced slime production of yoghurt culture and other microorganisms (Schellhaass, 1983; Mozzi et al., 1995; Van den Berg et al., 1995).  However, most investigators reported that EPS production was effective in high temperature (Garcia-Garibay and Marshall, 1991; Grobben et al., 1995).

Little information, however, exists in relation to Lactobacillus delbrueckii subsp. bulgaricus culture incubation condition that affecting the ability of the organism to produce EPS and relation to its growth characteristics.  The present work was to study the influence of the temperature and incubation time on the EPS production and growth characteristics of ropy Lb. delbrueckii subsp. bulgaricus.

Ropy Lactobacillus delbrueckii subsp. bulgaricus used in this study was obtained from the collection of the Microbiology of Biotechnology Agriculture Laboratory, Research Center of Hasanuddin University, cultured from March to November 2003.  Stock culture was kept in 10% sterile skim milk reconstitution (SMR) at –20^oC.  Bromochresolpurple Agar and Skim Milk Agar was chosen to estimate the total number of bacteria.  SMR 10% was used as growth medium for EPS production.

            Lb. delbrueckii subsp bulgaricus with ropy strain was grown in 10% SMR sterilized at 115^oC for 15 min, using 1% (v/v) inoculums and 16 h of incubation at 37^oC.  Each culture was sub cultured at least 3 times prior the experiment.  Fermentations were performed in erlenmeyer containing 200 ml of 10% SMR, incubated at 20°, 25°, 30°, 35° and 40°C for 8, 10, 12, 14 and 16 h.  Samples were taken after the incubation period and cooled in cold water before assayed.

            The cell viability was determined by the plate dilution method using Bromochresolpurple Agar (BCPA).  Serial dilutions of each sample were plated in duplicate and the plates were incubated at 37°C for 48 h.  Results were expressed as colony forming units (cfu/ml).

            Titratable acidity, expressed as lactic acid percentage, was measured according to Marshall (1993).  The pH value was measured by using pH-meter.

The EPS obtained from the cell free supernatants of broth cultures (6000 rpm, 10 min) were precipitated at 4^oC for 24 h with 2 volumes of cold 95% ethanol.  The precipitates were dialyzed against distilled water at 4^oC during 24 h in order to eliminate residual sugars from the culture medium, and then freeze-dried and stored at 4^oC.  The EPS production was expressed as mg/l (Mozzi et al., 1994).

The means of the EPS production, pH and lactic acid based on the temperature and time of incubation are shown in Table 1.

            The effect of temperature and incubation time on the EPS production of Lactobacillus bulgaricus in 10% skim milk reconstitution is shown in Table 1 and Fig. 1.  The temperature and time of incubation were significantly affecting EPS production.  Incubation temperature in 30^oC had a higher significantly EPS production than another temperatures.  While EPS production incubated at 16 h was higher significantly than anothers time of incubation.  Fig. 1 is shown that, after incubation for 8 h a good EPS production was observed at 30^oC.   This microorganism

could produce EPS of 359,96 mg/l when incubated at this temperature for 16 h incubation time, while a low production occurred in another incubation temperature.  The EPS synthesis slowed down when increasing the temperature from 37^oC to 42^oC was found by Mozzi et al. (1995) who used L. delbrueckii ssp. bulgaricus.  These results are in disagreement with those of Garcia-Garibay and Marshall (1991), who found that the maximal synthesis of polymers by L. delbrueckii ssp. bulgaricus occurred at 45^oC.

            The kinetics of EPS production by L. bulgaricus was determined at 25^oC and 30^oC.  No EPS was produced during the exponential growth phase (0 – 6 h).  The EPS was produced thereafter to reach an early stationary phase between 12 – 16 h (Fig. 2).  This result was similar with Ganzel and Novel (1994) experiment  that the maximum EPS production was found between 14 – 18 h.  Polysaccharide production was higher at temperatures unfavorable for growth and followed by chilling at 5^oC (unpublished data).  Cerning (1990) reviewed that the EPS production has often been found to be greater at lower growth temperatures.  If the cells are growing more slowly, then wall polymer formation will be slower, thereby making more isoprenoid phosphate available for EPS synthesis.

            Cell viability (CFU/ml) increased at increasing incubation time until 16 h for all incubation temperature, however, at 40^oC the total of CFU was reached the highest (9,224 log CFU), it indicated that the growth phase was an early stationary phase. 

Incubation time is essential for EPS production.  Mozzi et al. (1996) found that lengthening the incubation time up to 72 h at 30^oC decreased EPS production.  This might be due to the activation of certain hydrolyzing agents such as glucohydrolases that capable to degrade the polysaccharide.  Polymer degradation by these enzymes has also been reported by other investigators.  Pham et al.  (2000) also found that maximum EPS production by Lactobacillus rhamnosus R was observed in 24 h of incubation time at 37^oC. 

            As shown in Table 1, the lactec acid and pH were significantly affected by temperature and time of incubation, respectively. Lactic acid production in incubation time at 35 and 40^oC were high significantly than that at 20, 25 and 30^oC. Incubation time at 16 h had a higher significantly lactic acid production with compared the an others incubation time.

            The results of lactic acid production and pH growth are shown in Fig. 3 and 4.  The lactic acid production and pH, however, showed a temperature dependency; lactic acid synthesis increased at increasing temperature, while pH decreased at increasing temperature and incubation time.  Lactic acid production by L. bulgaricus increased from 0,96 (8 h) to 1,22 % (16 h), and indicated that the optimum growth temperature of these bacteria ranged between 35 – 40^oC.  The lower pH value was reached at 35^oC for 16 h of incubation time (3,479).

            Starters for the dairy industry containing ropy strains are available, because they are essential for proper consistence of fermented milks and yoghurt.  Incubation temperature and time of incubation affected the growth characteristics and exopolysaccharide production by ropy Lactobacillus delbrueckii subsp. bulgaricus.  The temperature of 30^oC for 16 h produced higher EPS than another incubation condition, while highest growth characteristics (cell viability, % lactic acid) were obtained in incubation temperature at 35^oC and lowest at 20 to 25^oC.

            We thank the Director of Research Center Hasanuddin University for facilitating us the equipment for this study, Is Suryanti and Fatma Mahruddin for technical assistance.  This work was financially supported by Hibah Bersaing DIKTI DEPDIKNAS Jakarta.

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