Quantification of Exopolysaccharides Produced by Lactic Acid Bacteria in Skim Milk by Periodate - Schiff Reagent Method

LOBO, RENÉ EMANUEL; GEREZ, CARLA LUCIANA; FONT DE VALDEZ, GRACIELA; TORINO, MARÍA INÉS

San Miguel de Tucumán

Simposio; V International Symposium on Lactic Acid Bacteria; 2016

CERELA-CONICET

Certain lactic acid bacteria (LAB) produce sugar extracellular polymers or exopolysaccharides (EPS), which have large applications in the industry of dairy fermented foods. Different methods have been developed to quantify these EPS. However, all of them involve the isolation of polymers, purification and gravimetric determination, resulting in prolonged and expensive procedures with low reproducibility. By contrast, the colorimetric method that uses periodic acid - Schiff reagent (PAS) constitutes a standardized and simple procedure that presents high sensitivity, specificity and reproducibility against different types of carbohydrates using small volumes of sample and reagents. The aim of this study was to evaluate the applicability of the PAS method to directly quantify EPS produced in milk by six strains of Streptococcus thermophilus (CRL419, CRL638, CRL804, CRL810, CRL815 and CRL1190), which synthetize polymers of different size and chemical composition. LAB were cultured in 10% reconstituted skim milk (LDR) for 16 h at 37°C and EPS production was determined in supernatants recovered at the end of incubation. Supernatant from non-inoculated LDR medium was used as control of non-EPS reacting components. The sensitivity of the PAS method (A550nm Biopur diagnostics kit) was evaluated in non-concentrated and in 2, 3, 4, 5 and 6 fold concentrated supernatants. Each supernatant was dialyzed to remove interfering substances (mainly lactose, glucose and galactose) that showed reactivity to PAS method at high levels. EPS concentrations were estimated from standard curves (R2 > 0.99) done with solutions of the corresponding purified EPS (0.0 to 1.0 mg/mL), whose sensitivity to concentration?s changes is given by the slope of curves. As expected, the reactivity of the non-concentrated supernatants (A550nm 0.15 - 0.23), showed no significant differences with the control medium, resulting positive probably due to remaining whey proteins (0.30 mg/mL by Bradford). Supernatants of CRL804, CRL810, CRL815 and CRL1190 were distinguished to the control from the 4-fold concentrates, with values that could be extrapolated in their respective curves (slopes 0.68 to 1.45), given net EPS concentrations ranged from 58.32 to 144.49 mg/L. These results do not keep correlation with the recovered EPS quantified as dry mater (31.67 to 68.47 mg/L). The samples from CRL419 and CRL638 showed low reactivity with PAS (slopes 0.20 and 0.36) and A550nm values were similar to the control even in 6-fold concentrated samples. These results mainly suggest that PAS method could be applied to quantify directly certain EPS from LAB?s cultures, being the molar absorptivity and/or conformational structure of EPS in solution crucial factors affecting the colorimetric reaction.