2.2.1. Total Dietary Fiber Determination

Total dietary fiber content (TDF) was determined using the Total Dietary Fiber Assay Kit (Neogen, Lansing, MI, USA) according to the manufacturer's protocol [22], which represents a simplified version of the official AOAC 985.29 method for the determination of total fiber. TDF was determined on quintuplicate samples. Five aliquots of BSG extract (1.000 ± 0.005 g) were incubated at about 100 ◦C with 50 μL heat-stable α-amylase solution to allow starch gelatinization, hydrolysis, and depolymerization. The samples were then incubated at 60 ◦C with 100 μL protease solution (to solubilize and depolymerize the proteins) and 200 μL amyloglucosidase solution (to hydrolyze the starch fragments into glucose). The samples were filtered to separate the insoluble fiber. The supernatants were then treated with approximately four volumes of ethanol to allow precipitation of the soluble fibers and remove the depolymerized proteins and glucose (from starch). The samples were then filtered and the residues, corresponding to the soluble fiber, were washed with 78% ethanol, 95% ethanol, and acetone, and dried overnight in a microwave oven at 103 ◦C and then weighed. The first residue was analyzed for proteins, determined using the Bradford method [23], and the second one was incubated at 525 ◦C to determine the ash content. TDF was calculated as the sum of the weight of insoluble fiber and soluble fiber dry residue minus protein and ash weights.

The other three residues were stored at room temperature pending analysis to determine the content of AXs, β-glucans, and resistant starch.
