*3.2. α- and β-Glucan Content*

The enzymatic method for β-glucan measurement is widely used due to its accuracy and reliability. In this approach, complete hydrolysis of glucans requires a controlled acid measured with GOPOD reagent. α-glucans were determined using specific enzymes for α-glycosidic bond hydrolysis under alkaline conditions, and β-glucans were calculated by the difference between total and α-glucan content. This method aimed to achieve complete hydrolysis of both α- and β-glucans to glucose, minimizing the loss of glucose through secondary reactions (Table 2).


the difference between total and α-glucan content. This method aimed to achieve complete hydrolysis of both α- and β-glucans to glucose, minimizing the loss of glucose

> **Total Glucans (%)**

husk 4.6 ± 0.2 11.3 ± 0.7 5.0 ± 0.2 6.3 ± 0.4

husk 3.74 ± 0.3 7.2 ± 0.4 3.6 ± 0.3 3.6 ± 0.5

**α-Glucans (%)**

**β-Glucans (%)**

**Table 2.** Recovered glucans (g/100 g DM) and total α- and β-glucans (%).

**(g/100 g DM)**

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 6 of 16

**Table 2.** Recovered glucans (g/100 g DM) and total α- and β-glucans (%)*.*

As can be seen from the data reported in the table, the percentage of total glucans determined by the enzymatic method confirmed that walnut husks from Montalto (11.3 ± 0.7%) had a higher content than walnut husks from Zumpano (7.2 ± 0.4%). Additionally, Montalto husks were richer in β-glucans (6.3 ± 0.4%) than in α-glucans (5.0 ± 0.2%), while Zumpano husks, compared to the Montalto husks, contained α- and β-glucans in equal percentages (3.6 ± 0.4%). Zumpano husks, compared to the Montalto husks, contained α- and β-glucans in equal percentages (3.6 ± 0.4%). *3.3. FT-IR Spectroscopic Analysis of Glucans* Infrared spectroscopic analysis of glucans provided information on the fundamental molecular vibrations of covalent bonds in the 4000–400 cm−1 IR region, showing the char-

#### *3.3. FT-IR Spectroscopic Analysis of Glucans* acteristic bands of the major functional groups (Figure 2). The comparison of glucan spec-

through secondary reactions (Table 2).

**Samples Recovered Glucans**

Montalto

Zumpano

Infrared spectroscopic analysis of glucans provided information on the fundamental molecular vibrations of covalent bonds in the 4000–400 cm−<sup>1</sup> IR region, showing the characteristic bands of the major functional groups (Figure 2). The comparison of glucan spectra from Montalto and Zumpano husks highlighted that the qualitative profile of the structures does not significantly change. The IR band centred around 3437 cm−<sup>1</sup> was generated by the symmetrical and asymmetric stretching of the OH groups present on the glucan backbone. The bands at 2920–2918 cm−<sup>1</sup> were due to CH<sup>2</sup> stretching of CH2OH groups. The presence of proteins in the sample linked to the glucan backbone by amide bonding provided the stretching of the CN and NH groups [31,32] generating the band at 1641 cm−<sup>1</sup> (the amide I). OH and CH bending from in-plane ring deformation generated two IR peaks at 1322 cm−<sup>1</sup> and 1261 cm−<sup>1</sup> , respectively. COC and CC stretching vibrations of the glucosides ring originated two bands in the region of 1090–1025 cm−<sup>1</sup> , indicating the presence of cyclic structures of monosaccharides [33]. The 950–780 cm−<sup>1</sup> region is called the "anomeric region", where it was possible to distinguish between the two anomeric glycosidic bond types of the glucopyranose rings which generated absorption bands at 860–830 cm−<sup>1</sup> for α-linkage and at 920–890 cm−<sup>1</sup> for β-linkage [19]. tra from Montalto and Zumpano husks highlighted that the qualitative profile of the structures does not significantly change. The IR band centred around 3437 cm−1 was generated by the symmetrical and asymmetric stretching of the OH groups present on the glucan backbone. The bands at 2920–2918 cm−1 were due to CH<sup>2</sup> stretching of CH2OH groups. The presence of proteins in the sample linked to the glucan backbone by amide bonding provided the stretching of the CN and NH groups [31,32] generating the band at 1641 cm−1 (the amide I). OH and CH bending from in-plane ring deformation generated two IR peaks at 1322 cm−1 and 1261 cm−1, respectively. COC and CC stretching vibrations of the glucosides ring originated two bands in the region of 1090–1025 cm−1 , indicating the presence of cyclic structures of monosaccharides [33]. The 950–780 cm−1 region is called the "anomeric region", where it was possible to distinguish between the two anomeric glycosidic bond types of the glucopyranose rings which generated absorption bands at 860–830 cm−1 for α-linkage and at 920–890 cm−1 for β-linkage [19].

**Figure 2.** FTIR spectra in the 400 to 4000 cm<sup>1</sup> region of walnut husk glucans from Montalto (red line) and from Zumpano (black line). **Figure 2.** FTIR spectra in the 400 to 4000 cm−<sup>1</sup> region of walnut husk glucans from Montalto (red line) and from Zumpano (black line).
