3.2.1. Color
Table 3 presents the differences in color (ΔE) between the BSG powders obtained through Processes A and B (
Figure 1).
As shown in
Table 3, the largest total color difference (
p < 0.05) was observed between powders A2 and B1. This difference may be influenced by the use of ultrasound (U1 and U2) in Process B, combined with fraction separation (1 and 2), as illustrated in
Figure 1 for both processes.
According to the classification by Adekunte et al. [
21], very different colors (ΔE > 3) were observed between powders A1 and A2 (
Table 3), suggesting that the color difference could be attributed to the structural composition of the two fractions of dried BSG obtained after sieving, which separates particles larger than 2.36 mm (Fraction 1) from those smaller than 2.36 mm (Fraction 2). The powders B1 and B2 showed smaller differences in color (1.5 < ΔE < 3) between each other, following the aforementioned classification. It is proposed that the structural modifications in particle size distribution (
Figure 3) produced by ultrasound (U1 and U2) in Process B resulted in powders with smaller color differences compared to Process A.
When comparing powders obtained from the same fractions (1 and 2, above and below 2.36 mm, respectively) but through different processes, the comparison between A1 and B1 (
Table 3) showed smaller color differences (ΔE < 1.5) than between A2 and B2 (1.5 < ΔE < 3), according to the Adekunte et al. [
21] classification. Our findings suggest that the color changes in the powders due to the ultrasound treatments (U1 and U2) applied in Process B, but not in Process A, affected Fraction 2 (particles smaller than 2.36 mm) more significantly. It is likely that the chemical composition of Fraction 2 particles was more susceptible to the effects of ultrasound than Fraction 1 particles. Ultrasound has been reported to induce various chemical changes in biomass or waste material, as described by Bundhoo and Mohee [
28], including alterations in lignin, hemicellulose, cellulose content, cellulose crystallinity, polymerization extent, and organic matter solubilization. It is possible that the particles in Fraction 2, subjected to ultrasound, were more affected by the subsequent operations in Process B than those in Fraction 1. As a result, the impact on color was greater in Fraction 2, likely due to the Maillard reaction or caramelization occurring during the drying step [
12,
38,
39] and the effect of decreased particle size caused by milling [
39,
40,
41] in Process B.
3.2.2. Total Phenolic Content and Antioxidant Activity against ABTS•+ Radical
Table 4 presents the total phenolic content (TPC) and antioxidant activity against the ABTS•+ radical in BSG powders (A1, A2, B1, B2) obtained through Processes A and B.
The application of ultrasound and variations in particle size did not significantly influence the extraction of phenolic compounds from BSG, as indicated by the lack of significant differences in total phenolic content between the treatments (
Table 4).
Regarding the total phenolic content (TPC) of BSG, our findings align with previous results reported by Carciochi et al. [
43], who found TPC values ranging from 1.59 to 3.57 mg GAE g
−1 BSG d.m., as well as with the results reported by Alonso-Riaño et al. [
29], Petrón et al. [
44], and Patrignani et al. [
45]. Other studies have reported higher TPC values for BSG extracts, such as those found by Meneses et al. [
23] and Bonifácio-Lopes et al. [
46], which were 7.13 and 13 mg GAE g
−1 BSG d.m., respectively. It is important to note that in this study, it was analyzed only the free phenolic compounds, as no hydrolysis step was employed during the extraction process. Since a portion of the phenolic acids present in BSG are bound to the cell wall of lignins, they can be more readily released during alkaline extraction, where the solubility of lignin is enhanced under such conditions [
29].
The ABTS assay is a widely used method for evaluating antioxidant activity in vitro, measuring the capacity of antioxidants to neutralize ABTS•+ radicals through electron donation, relative to a standard such as Trolox [
24]. The results obtained using the ABTS method showed that antioxidant activity slightly increased in treatments without ultrasound application. This may be explained by the release of antioxidant substances, such as phenolic compounds, capable of scavenging the ABTS•+ radical from the solid to the liquid fraction during Process B, where ultrasound was employed. The initial application of ultrasound likely facilitated the extraction of these ABTS•+ radical scavengers retained in the BSG, which were possibly lost to the liquid fraction during subsequent pressing in Process B (
Figure 1). Indeed, the application of ultrasound has been shown to increase the extraction of phenolic compounds from BSG when using water and ethanol as solvents [
30]. Additionally, no significant differences (
p > 0.05) were observed between the different particle size fractions (A1 and A2; B1 and B2) within the processes for antioxidant activity, as measured by the capacity to scavenge ABTS•+ radicals.
3.2.3. Proximate Composition
Table 5 shows the proximate composition (in dry matter, %
w/
w) of BSG powders obtained from Processes A and B. The results indicate significant differences (
p < 0.05) in total dietary fiber, soluble and insoluble fiber, and available carbohydrates among the four types of powder (A1, A2, B1, and B2). However, total lipid, protein, and ash contents did not differ statistically (
p > 0.05) among them. Lynch et al. [
1] provide an extensive compilation of various authors’ reports on the chemical composition of BSG, based on dry matter, noting lipid content of 3–13%, protein content of 14.2–31%, ash content of 1.1–4.2%, hemicellulose content of 19.2–41.9%, cellulose content of 12–33%, starch content of 1–12%, and lignin content of 11.5–27.8%. Our findings for total lipids, protein, total dietary fiber, and ash are within the ranges reported in these studies. Similarly, Shih et al. [
12] produced BSG flours using two drying methods to prepare muffins, reporting chemical compositions of 7.1–12.4 g 100 g
−1 d.m. for lipids, 14.5–18 g 100 g
−1 d.m. for protein, 2.7–4.1 g 100 g
−1 d.m. for ash, 8.1–15.9 g 100 g
−1 d.m. for soluble sugar, and 29.2–49.7 g 100 g
−1 d.m. for dietary fiber. More recently, Garrett et al. [
27] reported the proximate composition of BSG used to prepare flour by drying (65 °C for 72 h) and grinding, with dry basis contents of 4.9% for fat, 16.8% for protein, 2.9% for ash, and 75.4% for carbohydrates.
Total lipid, protein, and ash content in the powders were not significantly affected by the differences between Processes A and B, whether due to the application of ultrasound or the fraction separation methods used, as shown in
Figure 1. It is believed that further improvements will be necessary in future studies, particularly concerning the application of ultrasound treatment and sieving, to achieve noticeable differences, especially in protein content. The intensity of cell wall component breakdown and the consequent release of protein during ultrasound treatment of BSG could influence these differences [
13,
31]. Kissell and Prentice [
47] found that in brewers’ spent grain, the finer fractions had a higher protein concentration compared to the coarser fractions, suggesting that the smaller particles may originate from regions of the grain with higher protein concentrations.
The choice of sieve used for fraction separation contributed to differences in the fiber and available carbohydrate content of the powders. Finer fractions (Fraction 2: particles < 2.36 mm) retained more dietary fibers in treatments without ultrasound (Process A). The use of ultrasound, combined with the finer fractions, may be an effective method for increasing the efficiency of dietary fiber extraction from BSG.
According to the study by Hassan et al. [
9], the application of ultrasound can increase the concentration of reducing sugars. In our study, for the same fraction, the powders produced without ultrasound (Process A) showed a higher (
p < 0.05) percentage of available carbohydrates compared to the powders obtained using Process B (
Table 5). This behavior could be explained by the release of carbohydrates from the solid to the liquid fraction, promoted by the ultrasonic treatment and subsequent pressing applied after U1 (
Figure 1). As a result, more sugar was transferred to the liquid fraction, and less sugar was retained in the solid BSG fraction obtained in Process B.
When considering either of the processes (A or B), it can be observed (
Table 5) that fraction separation (
Figure 1) into coarse (Fraction 1: equal to or larger than 2.36 mm) and fine (Fraction 2: smaller than 2.36 mm) particle sizes affects the proximate composition of total dietary, soluble, and insoluble fiber, as well as available carbohydrates in the BSG powders obtained in our study. This behavior can be attributed to the fact that Fraction 1 consists of whole grains and agglomerates, while Fraction 2 consists of broken and/or segmented grains. BSG consists of layers of husk, pericarp, and seeds with residual amounts of barley endosperm and aleurone [
48]. It is a lignocellulosic material, and its main components are dietary fiber (50%) and protein (30%) [
1]. Therefore, sieving and fraction separation into coarse and fine particle sizes of dried BSG (
Figure 1) before milling would allow for the production of powders with different proximate compositions. These powders could be applied as specific ingredients in food products, depending on the requirements for higher levels of dietary fiber (Fraction 2) or carbohydrates (Fraction 1).
Table 5 also shows the soluble and insoluble dietary fibers (%
w/
w) of BSG powders obtained through Processes A and B. Dietary fibers can represent up to 70% (
w/
w) of BSG [
49] and are classified according to their solubility in water. Soluble dietary fibers include β-glucans, pectic polysaccharides, arabinogalactans, highly branched arabinoxylans, and xyloglucans, while insoluble dietary fibers include cellulose, hemicellulose, and lignin [
48,
50]. Barley (
Hordeum vulgare) and its by-products, such as BSG, are notable as sustainable sources of dietary fibers, offering a mix of soluble and insoluble fibers with numerous health benefits [
51], thus allowing for the development of fiber-enriched food products.
Soluble fibers have prebiotic activity, selectively enhancing the growth and/or activity of beneficial bacteria in the colon, thereby contributing to the improvement of intestinal flora and overall host health. Additionally, soluble fibers form colloidal solutions in the intestine, contributing to the reduction in blood sugar and cholesterol levels and aiding in the prevention of cardiovascular diseases, type 2 diabetes, cancer, obesity, and other conditions [
52,
53]. Insoluble fibers, on the other hand, help in the formation of fecal bolus, accelerate intestinal transit, and play a crucial role in the body’s detoxification [
53]. However, they are not fermentable by intestinal flora, which reduces their potential as a functional ingredient. Food processing techniques can be used to modify the structural properties and, consequently, the functional and nutritional roles of insoluble fibers [
54].
There were significant differences (
p < 0.05) in soluble dietary fiber content between the two groups of powders (A and B). The percentage of soluble dietary fiber in powders B1 and B2 exceeded that in A1 and A2, indicating that ultrasound positively affected the soluble fiber content of BSG. Ultrasound treatment has been used for structural modification of BSG and other biological materials [
9,
16,
28,
31]. β-glucan can be extracted from BSG and used as a food supplement. Recently, it was demonstrated that BSG β-glucan improved the color and increased the stability of orange juice by reducing pulp precipitation and exhibited higher lightness and color values [
55].
Table 5 also shows that both Processes A and B, especially the application of ultrasound and the particle size (Fractions 1 and 2, above and below 2.36 mm, respectively), directly influenced the content of insoluble dietary fiber (
p < 0.05). Cellulose is a common insoluble dietary fiber found in barley cell walls, as is hemicellulose, which is composed of various sugar monomers, including pentoses like xylose and arabinose, and hexoses like glucose and mannose [
51]. According to Lynch et al. [
1], hemicelluloses, composed mainly of arabinoxylan, are the main constituent of BSG (up to 40% dry weight), followed by cellulose.
Several factors affect the proximate composition of powder or flour obtained from BSG, including the ingredients used in the beer formulation and the brewing process itself [
1,
12,
27]. Jin et al. [
56] presented a detailed proximate analysis of various craft BSGs, comparing them with those from industrial breweries. It was observed that craft BSG has significant differences in its proximate components compared to industrial BSG. Therefore, based on the proximate analysis, it may be possible to determine the origin of the BSG, whether craft or industrial, and further studies should be conducted with different BSG samples to confirm our findings.
3.2.4. Powder Particles Sieve Size Distribution
Table 6 shows the particle sieve size distribution of BSG powders obtained by Processes A and B after milling (
Figure 1) using an ultracentrifugal mill equipped with an 80 µm sieve at 12,000 rpm.
In
Table 6, the effects of ultrasound applied in Process B and the fraction separation (
Figure 1) applied in both processes are evident with respect to the different particle size distributions of the powders. For A1, the highest proportion of particles (36.3%) falls between 90 and 75 µm, for A2 (31.8%) between 75 and 45 µm, for B1 (38.3%) between 125 and 90 µm, and for B2 (27.9%) between 180 and 125 µm. In both A1 (93.8%) and B1 (92.4%), the particles are primarily distributed between 125 and 75 µm, while in A2 (98.4%) and B2 (96.4%), the particles are mainly distributed between 125 and 45 µm. This indicates that the separation of dried (D) and sieved (S) BSG into two fractions (coarse and fine) before milling (
Figure 1) had a significant effect on the particle size distribution.
Statistically significant differences (
p < 0.05) were observed in the percentage of particles retained in the sieves with nominal sizes of 125, 90, 75, and 45 µm. For these three sizes (90, 75, and 45 µm), the fraction separation (
Figure 1) into coarse (1: equal to or larger than 2.36 mm) and fine (2: smaller than 2.36 mm) particles affected the percentage of particles between A1 and A2 or B1 and B2. Process B, which included the ultrasound operation, produced a higher proportion (
p < 0.05) of particles in the 125 and 75 µm range for Fraction 2 compared to Process A. However, for the 90 and 45 µm sizes, A2 showed a higher proportion (
p < 0.05) of particles than B2. When analyzing Fraction 1, B1 had a higher proportion (
p < 0.05) of particles at 90 µm than A1, whereas A1 had a higher proportion (
p < 0.05) of particles at 75 µm than B1.
It was used an 80 µm sieve to mill Fractions 1 and 2 of the four powders obtained. Particles larger than this sieve size were found in all powders, indicating that the milling equipment used in this study was not fully efficient in reducing the particle size below 80 µm. However, particles below 125 µm are considered suitable for many food applications [
5,
10,
12,
35,
36].