**3.1 Phenol-sulphuric method (Dubois et al., 1956)**

This method is based on the fact that simple or complex sugar and their derivatives, including methyl esters with free reducing groups or potentially free, when treated with phenol and concentrated sulphuric acid will generate a yellow-orange colour, the reaction is sensible and this colour is stable. The method is simple, quick, sensible, and the results are reproductible.

The changing in colour of solution is measured in visible range and proportional to the quantity of sugar inside the sample.

The carbohydrates are hydrolyzed under heating in strongly acid pH, this reaction produces furan derivative compounds, that when condensed with phenolic compounds produce coloured substances (Figure 1).

The total sugar concentration is determined by spectrophotometry in 490 nm wavelength. Sensitivity of this method range from 10 to 100µg of total sugar and the quantification is made from calibration curve using glucose or pentose as standard and calculation are performed by equation of the linear regression obtained from calibration curve.
