2.7 Kinetics of colorant production
In order to describe the kinetics of colorant production by the three selected isolates, submerged bioprocesses were performed (SeeSubmerged bioprocesses for colorant production ). Samples were collected in triplicate at intervals of 5 days for 20 days with the view to determination of dry cell weight (g/L) and extracellular colorants (Abs extra). To determine the biomass, the mycelial masses were collected and filtered with filter paper previously dried in a desiccator and then weighed. After filtration, both were dried in an oven at 100 ºC for 24 h. The biomass concentration was expressed by the dry weight of the mycelial mass using the difference between the final weight and the initial weight. The production of colorants was estimated by analyzing the maximum absorbance (400 nm) in ethyl acetate using a UV/VIS spectrophotometer.
2.8 Analysis of the colorants using liquid chromatography coupled to mass spectrometry (LC/MS-MS)
The extracted colorants were diluted in methanol (HPLC-Honeywell) to a concentration of 1 mg/mL. Then, the samples were analyzed using a liquid chromatograph (Prominence UFLC, Shimadzu), which was equipped with a C18 Shim-pack CLC-ODS(M)® column (5 µm, 250 x 4.6 mm), and coupled to a mass spectrometer (Amazon Speed Ion trap, Brucker Daltonics) with an ESI source operating in positive and negative modes. Samples were eluted in gradient of 20-100% methanol over 24 minutes at a flow rate of 1 mL/min. The data obtained were processed using the DataAnalysis program (Bruker Daltonics).