Exopolysaccharides production
EPS was extracted from bacterial cultures grown at 30°C for 48 hours in
TY medium supplemented with 50% v/v fungal spent medium, starting from
OD600 0.1. Cultures were centrifuged at 10,000 rpm for
20 minutes at 4°C and EPS was precipitated by adding 1 volume of cold
absolute ethanol to the supernatant, followed by incubation at 4°C for
24 h. Crude EPS was harvested by centrifugation at 10,000 rpm for 20 min
at 4°C. The pellet was washed and resuspended with dH2O.
Total sugars estimation was evaluated using Dubois method : 200μl of
crude EPS was mixed with 200μl of 5% phenol and 1 ml
H2SO4. The mixture was vortexed and
incubated for 30 minutes at room temperature. Absorbance was measured at
490 nm with a spectrophotometer. Reducing sugars estimation was
evaluated using the dinitrosalicylic acid (DNSA) method . EPS crude
sample and DNSA reagent were mixed in 1:3 ratio. Samples were heated in
a thermostatic bath for 15 minutes at 96°C. After cooling, the
absorbance was measured at 540 nm. The reducing and total sugars
concentration in the cultures was estimated based on a glucose standard
curve.
Data are from 3 biological replicates. Production inhibition index was
calculated as: 1- OD490 treated/OD490control for total sugars and 1- OD540treated/OD540 control for reducing sugars, where OD
represents the value of optical density at the indicated wavelength.