Effect of culture pH on the growth characteristics ...

Effect of culture pH on the growth characteristics and exopolysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus Ropy strain

(Pengaruh pH kultur terhadap sifat pertumbuhan dan produksi eksopolisakarida oleh Lactobacillus bulgaricus subsp. bulgaricus strain ropy)

Ratmawati Malaka 

ABSTRACT

            The effect of environmental condition on EPS production by Lactobacillus bulgaricus  was studied using initial pH from 4.5 to 8.5 in SMR 10% at 30^oC for 16 h of incubation time.  The initial pH influence the EPS production, that the optimal pH condition was 6.5 – 8.5.

Key Words : Exopolysaccharides,  Lactobacillus bulgaricus, initial pH.

ABSTRAK

            Pengaruh kondisi lingkungan terhadap produksi EPS oleh L. bulgaricus dipelajari dengan menggunakan pH media awal mulai dari pH 4,5 sampai 8,5 pada SMR 10% dengan menggunakan suhu inkubasi 30^oC selama 16 jam.  PH awal media mempengaruhi produksi EPS dimana pH optimal untuk produksi EPS adalah pH 6,5 sampai 8.5.

Kata Kunci :  Eksopolisakarida, L. bulgaricus, pH awal

INTRODUCTION

            Lacrobacillus delbrueckii subsp. bulgaricus are used in the dairy industry to transform milk into yoghurt (Chervaux et al., 2000).  Several strains which are capable of forming exopolysaccharide (EPS) have been isolated and analyzed (Grobben et al., 1998). 

            The EPS production by lactic acid bacteria is an important characteristic to be considered when selecting starter culture for fermented milks.  This type of polymers prevents the wheing-off and improves the texture of the final product.  However, there is no single set of culture conditions that guarantees high EPS yields in all producing microorganism since cells differ in their carbon source utilization, mineral requirements and even temperature for an optimal EPS synthesis.  Mozzi et al.,  (1994) have been reported that pH of medium is a critical factor for the EPS production by Lactobacillus casei.

            The effect of environmental conditions on the EPS formation by lactic acid bacteria was mainly studied in thermophilic species of technological interest (Malaka, 1997).  In resent years, mesophilic lactic acid bacteria like Lactobacillus delbrueckii became important microorganisms as producer of EPS.  However little attention has been paid to the ability of these lactic acid bacteria to produce EPS under different pH culture.

            According the above considerations, the aim of this study was to determine the influence of the initial pH on the EPS production by Lactobacillus delbrueckii subsp bulgaricus ropy strain when growing in milk.

 MATERIALS AND METHODS

Microorganism

L. delbrueckii subsp. bulgaricus ropy strain was obtained from the culture collection of Research Center of Hasanuddin University.  Stock cultures were kept in 10% sterile reconstitution skim milk (RSM) at –20^oC.

            10% RSM (sterilized at 121^oC for 15 min) were used as growth media.  The pH was adjusted after sterilization with lactic acid to initial values of 4.5, 5.5, 6.5, 7.5 and 8.5.

            Fermentations were performed at 30^oC (according to our experiment before for temperature optimal to EPS production) in Erlenmeyer containing 100 ml of growth media inoculated with a 16 h active culture at the rate of 1% (v/v) (about 10⁸ cfu/ml) inoculums.  Samples were taken at 16 h, and cooled before the assays.  Cell viability was determined by the agar plate method using BCP agar.  Serial dilutions of each sample were plated in duplicate and plates were incubated at 37^oC for 48 h.  Results were expressed as colony forming units (cfu/ml). 

            The EPS obtained from the cell free supernatants and isolated according Mozzi et al. methods (1994).  The EPS production was expressed as mg/l.  EPS was freeze-dried and stored at 4^oC.  The EPS production was expresses as mg/l (Mozzi et al., 1994).

            Titratable acidity, expressed as lactic acid percentage, was measured according to Mozzi et al. (1994).  PH was measured using pH-meter (Hanna-pH meter). 

            The effect of pH of media on activity and EPS production of L. bulgaricus was studied using RSM 10%.  Initial pH values were 4.5, 5.5, 6.5, 7.5, and 8.5.  pH value of 4.5 and 5.5 represented as acid conditions, 6.5 represented as pH of milk and 7.5 and 8.5 represented as base condition.  After incubation for 16 h, the amount of nutrient consumed as well as the colony counts, lactic acid produced and the EPS formed increased when increasing the initial pH from 4.5 to 6.5 and constant at pH 7.5 (Fig. 1 – 3).

Fig. 1.  Exopolysaccharide production by L. bulgaricus in different initial pH   medium.

            The EPS production in skim milk reconstitution 10% after 16 of incubation time was increased from 128.6 mg/l to 326.2 mg/l (with pH 4.5 to 6.5).  When comparing the behaviour of L. bulgaricus in SMR 10% on pH change (0 h and 16 h of incubation time), was valuable change.  In 4.5 of initial pH cause of increased the pH medium to 4.8.  This effect maybe cause of L. bulgaricus would exist in it subcritis environment.  However, with increase the initial pH, L. bulgaricus more activated to produce of lactic acid so the pH of medium decrease (Fig 2).

Fig. 2.  The pH change in intial pH (0 h of incubation time)  and final pH (16 of incubation time) by L. bulgaricus in SMR 10%.

                        The growth of L. bulgaricus in SMR 10% medium was related to the metabolism of glucose to lactate in the pH range assayed, while in the latter one a partial uncoupling between growth and lactate formation was observed.  This could be attributed to the effect of the initial pH and certain nutrient limitation on the cell growth, which would reduce the viability of the cells with low effect on the rate of carbohydrate metabolism. 

Fig.  3.  Production of lactic acid in SMR 10% by L. bulgaricus in different of initial pH.

CONCLUSION

            A pH range of 6.5 – 7.5 was the optimal one for EPS production by L. bulgaricus ropy strain when growing in SMR 10% at 30^oC at 16 h of incubation.

ACKNOWLEDGEMENTS

            This study was partially supported by Hibah Bersaing, DIKTI.  Jakarta.

Malaka, R.  1997.  The effect of Curdlan, a bacteria polysaccharide on Physical Properties and Microstructure of Acid Milk Curd by Lactic Acid Fermentation.  A Master Thesis.  Miyazaki University.  Japan.

Mozzi, F., G. De Giori, G. Oliver, G. F. Fe Valdez.  1994.  Effect of pH on the growth

characteristics and polysaccharide production by Lactobacillus casei.  Milchwissenschaft 49 (12) : 667 – 670.

Dipublikasi di Buletin Penelitian (Seri Hayati) 10 (1): 36 - 40.

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