*3.1. Chemical Characterization of BSG Extract*

In this study, the extract used was produced from brewer's spent grain.

First, the fiber present in the extract was characterized. To determine total soluble and insoluble fiber, the BSG extract was analysed by a gravimetric method involving the elimination of starch and proteins following treatment with α-amylase, amyloglucosidase, and protease, respectively. After this treatment, the BSG extract did not show any precipitate, indicating the absence of insoluble fiber. To determine the soluble fiber, 78% ethanol was added to the sample. The high molecular weight soluble fiber, precipitated and determined by a gravimetric method, resulted to be 7.45 g/100 g BSG extract (Table 2). Since low molecular weight soluble fiber does not precipitate into 78% ethanol, this fiber fraction was not determined with this assay. Therefore, total AXs and β-glucans were determined by hydrolyzing glycosidic bonds with specific enzymes and determining their concentrations on the bases of the concentrations of arabinose and xylose and glucose deriving from their hydrolysis, respectively. The results showed that total AXs and total β-glucans were 7.50 and 1.92 g/100 g of BSG extract, respectively. The same method was also applied to the total fiber dry residue isolated with precipitation under 78% ethanol to determine the content of high molecular weight AXs and β-glucans isolated by the above method. The concentration of AXs was 0.45 g/100 g, but β-glucans were not found to be detectable in the total fiber dry residue. The concentration of low molecular weight AXs soluble in 78% ethanol, and therefore not calculated with the gravimetric method, was calculated by the difference, and resulted to be 7.05 g/100 g of BSG extract.

**Table 2.** Concentrations of free glucose, arabinose, and xylose, total AXs, WEAX and AEAX, total β-glucans, glucose from rapidly and slowly digestible starch, and glucose from resistant starch, in BSG extract, and AXs, β-glucans, and resistant starch determined in the total fiber dry residue.


<sup>1</sup> Data expressed as means ± SD (*<sup>n</sup>* = 3). <sup>2</sup> calculated by the difference between total AXs present in the BSG extract and AXs present in the total fiber dry residue. <sup>3</sup> N.D. not detectable. <sup>4</sup> corresponding to glucose from rapidly and slowly digestible starch, including free glucose. <sup>5</sup> corresponding to glucose from resistant starch, including glucose from rapidly and slowly digestible starch and free glucose.

Gelling properties of AXs are mainly attributed to WEAX, with AEAX showing less gelling and therefore being less active in the modulation of glucose absorption. WEAX and AEAX were determined to have concentrations of 1.23 g/100 g (representing about 16% of total AXs) and 6.36 g/100 g (representing about 84% of total AXs), respectively.

Finally, the concentrations of (1) free glucose, (2) glucose derived from rapidly digestible starch and slowly digestible starch (including free glucose), and (3) total glucose deriving from resistant starch (including glucose derived from rapidly digestible starch, slowly digestible starch, and free glucose) were determined in the BSG extract. The concentration of glucose deriving from resistant starch, calculated by the difference between total glucose concentration determined after 4 h of enzymatic treatment, and after 30 min of enzymatic hydrolysis (corresponding to glucose concentration from rapidly digestible, slowly digestible starch, and free glucose), resulted to be 14.64 g/100 g of BSG extract. In addition, total glucose derived from resistant starch was isolated from the total fiber dry residue and resulted to be 0.62 g/100 g of BSG extract. Thus, the concentration of resistant starch soluble in 78% ethanol, and therefore not calculated with the gravimetric method, was calculated by the difference, and resulted to be 14.02 g/100 g of BSG extract.

In total, the whole BSG extract contained 30.40 g of dietary fiber/100 g of BSG extract, mainly represented by resistant starch (14.64 g/100 g), AXs (7.50 g/100 g), β-glucans (1.92 g/100 g), and other soluble fibers (6.38 g/100 g) isolated with 78% ethanol precipitation and calculated by the difference between total soluble fiber dry residue (7.45 g/100 g) and high molecular weight AXs (0.45 g/100 g) and resistant starch (0.62 g/100 g) weights.

TPC before and after oral-gastric-duodenal digestion were found to be 0.499 ± 0.01 and 1.16 ± 0.03 g GAE/100 g of BSG extract, respectively, suggesting that during digestion polyphenols are released by the food matrix and become bioaccessible. As spectrophotometric methods generally have issues with overestimating the phenolic contents, since Folin–Ciocalteu reagent interacts with non-polyphenolic molecules (i.e., reducing sugars), the ferulic acid content, which is the polyphenol most represented in BSG, was evaluated by means of a validated UHPLC-MS/MS method after oral-gastric-duodenal digestion [31]. Its identification was based on the mass spectrum and fragmentation pattern of the parent ion with m/z 193 (Figure 1). The content of ferulic acid was 64.8 ± 0.06 mg/100 g of digested BSG extract, corresponding to 91.3 ± 0.07 mg/100 g of BSG extract.

**Figure 1.** The chromatogram of an ion product with m/z 193 obtained from the HUPLC-MS/MS analysis of digested BSG extract (**A**). Chromatogram recorded at 320 nm; (**B**). Mass spectrum of parent ion with *m/z* 193; (**C**). Mass spectrum of fragmentation of parent ion with *m/z* 193. LOQ and LOD values determined for ferulic acid were 0.062 and 0.016 μg/mL, respectively.
