2.4. Enzymatic Extract

After appropriate culturing periods, the enzymes produced via SsF were extracted. The crude enzymatic extract was prepared by adding 5 g of the homogenized fermented sample (the whole flask homogenization was carried out with a sterile spatula for 5 min) and 40 mL of deionized water in a 250 mL flask. The flask was then placed on a rotary shaker (150 rpm, 30 min at room temperature). To collect the enzymatic extract, the samples were centrifuged at 4800× g for 15 min at 4 °C. The clarified supernatant was filtered using Whatman No. 1 filter paper to quantify total reducing sugars, cellulase activity, glucoamylase activity and pH [30].

The total reducing sugar concentration was measured using the DNS method proposed by Miller in 1959 [34]. This method provides a simple and fast way to handle several samples and was used for the optimization process. The determination is centered on the color reaction between reducing sugars and 3,5-dinitrosalicylic acid. The reaction yield was measured as absorbance of the sample at 540 nm using an LLG–uniSPEC 2 spectrophotometer. The total reducing sugar throughout the studies was expressed as grams of sugar per kilogram of fermented substrate (g/kg_fs).

Cellulase activity was measured using the protocol described by Ghose (1994) and recommended by IUPAC, using filter paper Whatman No. 1 as a substrate [35]. For this determination, a Whatman No. 1 filter paper strip (1 × 6 cm) with a weight of ≈50 mg was used as a substrate. The paper strip was then rolled, placed in a glass tube, and 1 mL of 0.05 M sodium citrate buffer, pH 4.8, was added, immersing and covering the paper, following, 0.5 mL of enzyme solution was added to the same tube. After this step, the tube was vortexed and incubated in a water bath at 50 °C for 60 min. At the end of the incubation, 3 mL of DNS reagent were added, the tubes were also vortexed and incubated in a water bath for 10 min at 95 °C. All the tubes were then placed in a cold water bath for 5 min, and 9 mL of deionized water was added to each tube. Finally, the tubes were vortexed, and the absorbance was measured at 540 nm against reagent blank using an LLG–uniSPEC 2 spectrophotometer. The cellulase activity was estimated, which would have released exactly 2.0 mg of glucose utilizing a plot of glucose liberated against cellulase concentration, was carried out. One FPU/g_ds represents the enzyme unit per gram of initial dry solid substrate [24].

Glucoamylase activity was measured using the protocol described by Melikoglu and et al. in 2013 [30]. The glucoamylase activity of the enzyme solutions was estimated by measuring the amount of glucose released per minute using 1 mL potato starch (6% w/v) as substrate. Glucoamylase activity gave the difference between time zero and 10 min. Throughout the studies, the glucoamylase activity was expressed as U/g_db dry basis. One unit (U) was estimated as the enzyme amount required to produce 1 mg of glucose/minute under the assay conditions.