Analysis of reducing sugar by 3,5-dinitrosalicylic acid (DNS)

The analysis of residual reducing sugars from the bacterial metabolic activity on different carbon sources was carried out through the analysis of reducing sugars by 3,5-dinitro-salicylic acid (DNS) as described by Miller [42]. After cultivation described in the item Cultivation conditions for growth analysis of CB10 bacteria on different substrates, the cultures were centrifuged (Sorvall centrifuge at 16,266 xg for 30 minutes at 4°C) and a 100 μL aliquot of the supernatant, plus the same proportion of the DNS reagent, was incubated in the temperature cycling apparatus PCR type Thermo scientific Bio-Rad PTC-100® termal cycler (95°C, 5 min; 4°C, 1 min.; and 20°C, 10 min.). After the reaction, the absorbance was read at 540 nm in a Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer. The measured absorbance was compared to the values of a standard mannose curve (180.2 mw) at 0 to 20 mM to detect the presence of reducing sugars released in each culture.