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A relatively rapid method for the estimation of the amount of exopolysaccharide produced by lactic acid bacteria during milk fermentation
The available methods for estimation of exopolysaccharide (EPS) produced in fermented milks are very lengthy (several days). A relatively shorter (≤ 1 day) method has been investigated for the estimation of EPS produced during fermentation of non-fat milk at 40°C by two strains of Streptococcus thermophilus (St–143 and St–10255y). Milk samples were analyzed for EPS concentration every 30 min over a fermentation period of 300 min (final pH 4.6). Samples with pH > 5 were adjusted to pH ~5 before protein was removed by heat and acid precipitation. Excess ethanol was added to the neutralized supernatant and precipitated at –20°C for 3 hours. The pellet was dissolved in water at 55°C and centrifuged before residual lactose in the supernatant was removed by repeated precipitation by ethanol. The EPS concentration in the final pellet was estimated by the phenol sulfuric acid method. In milk fermented by S. thermophilus St–143, the EPS content significantly increased (P < 0.05) from 24 to 68 mg/L during the fermentation period from 150 to 300 min. EPS concentration in samples fermented with S. thermophilus St–10255y also significantly varied (P < 0.05) from 16 to 52 mg/L during a similar fermentation period. Interestingly, both of the strains appeared to start producing significant amounts of EPS after 150 min of fermentation time, which corresponded to pH ~5.2 and was close to when milk gelation occured. Thereafter, the EPS concentration continued to increase up to pH ~4.6 (end of fermentation). The total amounts of EPS obtained were comparable to the previously reported results in milks fermented by similar bacterial strains. To explore the recovery of EPS by this method, we added different concentrations of dextran (mol wt: 2x106 Da) to milk and found that up to ~70 % of the added dextran could be recovered, suggesting that this method was reasonably effective in extracting most of the EPS produced in fermented milk.
Keywords: Exopolysaccharides, yogurt, fermented milk