*3.7. FT-IR Spectroscopic Analysis of Pectins*

The FT-IR spectra of walnut husk pectins from Montalto and Zumpano, scanned at wave numbers ranging from 4000 to 400 cm−<sup>1</sup> and corrected against the background spectrum of air, are reported in Figure 5. The qualitative profile of the spectra of the samples from different pedoclimatic areas only showed differences in the absorption intensity of the characterizing bands. The 3600–2500 cm−<sup>1</sup> region presented two major peaks: the first one, centred around 3437 cm−<sup>1</sup> , generated by OH stretching, due to inter- and intra-molecular hydrogen bonding of the galacturonic acid backbone, and the second one, at 2915 cm−<sup>1</sup> , due to C–H absorption stretching. The 1800–1500 cm−<sup>1</sup> region revealed the presence of a first band at 1747–1746 cm−<sup>1</sup> , generated from ester carbonyl groups stretching, and a second one at 1633–1621 cm−<sup>1</sup> due to the carboxylate ion stretching [38]. These bands were

very important since it was possible to assess the degree of methoxylation of pectin by considering the corresponding intensities. Moreover, analysis of the spectra showed that in PM pectins (red line), the intensity of the band relating to the ester groups was greater than that relating to the carboxylate groups, whereas in PZ (black line), the intensities of the two bands are reversed. In the "fingerprint region" (1450 to 900 cm−<sup>1</sup> ) [39], Raman bands were evident at 1019–1017 cm−<sup>1</sup> , generated from COH deformation, at 1103–1106 cm−<sup>1</sup> due to C-C stretching and 1041–1040 cm−<sup>1</sup> arising from asymmetric COC stretching vibration. The presence of α-glycosidic linkage was highlighted by a weak band at 840 cm−<sup>1</sup> , generated by the ring vibration of [40]. very important since it was possible to assess the degree of methoxylation of pectin by considering the corresponding intensities. Moreover, analysis of the spectra showed that in PM pectins (red line), the intensity of the band relating to the ester groups was greater than that relating to the carboxylate groups, whereas in PZ (black line), the intensities of the two bands are reversed. In the "fingerprint region" (1450 to 900 cm−<sup>1</sup> ) [39], Raman bands were evident at 1019–1017 cm−<sup>1</sup> , generated from COH deformation, at 1103–1106 cm−<sup>1</sup> due to C-C stretching and 1041–1040 cm−<sup>1</sup> arising from asymmetric COC stretching vibration. The presence of α-glycosidic linkage was highlighted by a weak band at 840 cm−<sup>1</sup> , generated by the ring vibration of [40]. tively. The surface of crude glucans from Zumpano husks (GZ) showed the presence of aggregates in the form of microcrystals, whose dimensions were variable as it was more evident at high magnifications (5000× and 10,000×) (Figure 3a–d). The size distribution of the crystals was uneven: some crystals were significantly larger than others. The microcrystals observed were rectangular and needle-like in nature. The presence of particles probably glassy in nature which were incorporated into the matrix were also observed. Microcrystals were also observed in the glucans from Montalto hulls (GM), but unlike those present on the surface of glucans from Zumpano husks, they were larger compared to GZ, not aggregated and had a cubic form (Figure 3A–D).

The morphological investigation was carried out to highlight any different surface structures arising in the same type of fibre (glucans) from different soil and climate conditions. All the sample were analysed under the same conditions: four magnifications equal to 100×, 2500×, 5000× and 10,000 with a scale equal to 200, 10, 4 and 2 µm, respec-

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

*3.4. Scanning Electron Microscopy (SEM) of Glucans*

**Figure 3.** Scanning electron images of glucans from Zumpano (GZ, **a–d**) and Montalto (GM, **A–D**) (at four different magnifications and scales. (**a**) and (**A**) (100X, 200 µm); (**b**) and (**B**) (2500X, 10 μm); (**c**) and (**C**) (5000X,4 μm); (**d**) and (**D**) (10,000X, 2 μm). **Figure 3.** Scanning electron images of glucans from Zumpano (GZ, **a–d**) and Montalto (GM, (**A–D**)) (at four different magnifications and scales. (**a**,**A**) (100×, 200 µm); (**b**,**B**) (2500×, 10 µm); (**c**,**C**) (5000×, 4 µm); (**d**,**D**) (10,000×, 2 µm). **Figure 3.** Scanning electron images of glucans from Zumpano (GZ, **a–d**) and Montalto (GM, (**A–D**)) (at four different magnifications and scales. (**a**,**A**) (100×, 200 µm); (**b**,**B**) (2500×, 10 µm); (**c**,**C**) (5000×, 4 µm); (**d**,**D**) (10,000×, 2 µm).

*3.5. Differential Scanning Calorimetry (DSC) of Glucans*

150 and 220 °C (Figure 4).

**Figure 4.** DSC records of walnut husks' glucans from Montalto (red line) and from Zumpano (black-**Figure 4.** DSC records of walnut husks' glucans from Montalto (red line) and from Zumpano (blackline). vibration. The presence of α-glycosidic linkage was highlighted by a weak band at 840 cm−1 , generated by the ring vibration of [40].

The calorimetric curves recorded for the glucan samples are very similar. Both mainly presented an endothermic peak (1) near 125 °C and a glass transition (TG) between

mental impact and it was cheaper [37]. The obtained results showed that the pectin content of husks from Montalto (PM, **Figure 5.** FTIR spectra in the 400 to 4000 cm<sup>1</sup> region of walnut husk pectins from Montalto (red line) and from Zumpano (black line). **Figure 5.** FTIR spectra in the 400 to 4000 cm−<sup>1</sup> region of walnut husk pectins from Montalto (red line) and from Zumpano (black line).

#### 69.8 ± 0.1 g/100 g DM) was about three times higher than that extracted from Zumpano *3.8. Determination of Esterification Degree 3.8. Determination of Esterification Degree*

hulls (PZ, 27.1 ± 0.3 g/100 g DM), which indicated that different pedoclimatic areas influenced fibre content. *3.7. FT-IR Spectroscopic Analysis of Pectins* The FT-IR spectra of walnut husk pectins from Montalto and Zumpano, scanned at wave numbers ranging from 4000 to 400 cm−1 and corrected against the background spectrum of air, are reported in Figure 5. The qualitative profile of the spectra of the samples from different pedoclimatic areas only showed differences in the absorption intensity of the characterizing bands. The 3600–2500 cm−1 region presented two major peaks: the first Pectin is a complex polysaccharide composed of at least five different sugar moieties, with 80–90% of its dry weight being galacturonic acid (Gal*A*). A major percentage of the Gal*A* is present in homogalacturonan (HG) regions of pectin as unbranched chains in which a variable proportion of the Gal*A* contains a methyl ester at the C6 position [41]. The functional properties of pectins in foods, such as gelling capacity, and their reactivity towards calcium and other cations, are largely dependent on the amount of methylated Gal*A* subunits. Thus, degree of methylation (DM) is an important parameter for characterisation of food pectins [42]. DE was used to classify the pectins into high-methoxyl (HM) form, when the esterified group content was higher than 50%, and low-methoxyl Pectin is a complex polysaccharide composed of at least five different sugar moieties, with 80–90% of its dry weight being galacturonic acid (Gal*A*). A major percentage of the Gal*A* is present in homogalacturonan (HG) regions of pectin as unbranched chains in which a variable proportion of the Gal*A* contains a methyl ester at the C6 position [41]. The functional properties of pectins in foods, such as gelling capacity, and their reactivity towards calcium and other cations, are largely dependent on the amount of methylated Gal*A* subunits. Thus, degree of methylation (DM) is an important parameter for characterisation of food pectins [42]. DE was used to classify the pectins into high-methoxyl (HM) form, when the esterified group content was higher than 50%, and low-methoxyl (LM) form, if

(LM) form, if the content was lower than 50%. The esterification degree of PM and PZ was

titrimetric and instrumental methods and the results were reported as means ± standard

the content was lower than 50%. The esterification degree of PM and PZ was evaluated by titrimetric method and indicated as DE, while DM was the instrumental FTIR value (Figure 6). The analyses on each sample were performed in triplicate by both titrimetric and instrumental methods and the results were reported as means ± standard deviation. The results showed that titrimetric values were slightly higher than those obtained by the instrumental method, and that the esterification degree, regardless of the method used, of pectin from Montalto was significantly higher than that of pectins from Zumpano. The titrimetric percentage values for DE of pectins from Montalto and Zumpano were 65.9 ± 0.8 and 39.4 ± 0.6, respectively, while the corresponding %DM were 56.3 ± 1.1 and 37.2 ± 0.5. deviation. The results showed that titrimetric values were slightly higher than those obtained by the instrumental method, and that the esterification degree, regardless of the method used, of pectin from Montalto was significantly higher than that of pectins from Zumpano. The titrimetric percentage values for DE of pectins from Montalto and Zumpano were 65.9 ± 0.8 and 39.4 ± 0.6, respectively, while the corresponding %DM were 56.3 ± 1.1 and 37.2 ± 0.5.

**Figure 6.** Effects of the two pedoclimatic areas (M and Z) on esterification degree of walnut husks' pectins (P) evaluated by titrimetric method (DE) and instrumental methods (DM). Error bars indicated standard deviation (*n* = 3). Asterisks on the dashes indicate significant differences among the two methods used for esterification degree evaluation of pectins from the same agroclimatic area **Figure 6.** Effects of the two pedoclimatic areas (M and Z) on esterification degree of walnut husks' pectins (P) evaluated by titrimetric method (DE) and instrumental methods (DM). Error bars indicated standard deviation (*n* = 3). Asterisks on the dashes indicate significant differences among the two methods used for esterification degree evaluation of pectins from the same agroclimatic area (\*\*\*\* *p* < 0.0001).

#### (\*\*\*\* *p* < 0.0001). *3.9. Scanning Electron Microscopy (SEM) of Pectins*

ure 7A–C).

*3.9. Scanning Electron Microscopy (SEM) of Pectins* All the pectins were studied for morphological investigation under the same conditions: three magnifications equal to 100×, 2500×, and 5000× with a scale equal to 200, 10, All the pectins were studied for morphological investigation under the same conditions: three magnifications equal to 100×, 2500×, and 5000× with a scale equal to 200, 10, and 4 µm, respectively. SEM images showed different surface structures depending on the pedoclimatic area of provenance of the walnut husks. The surface of PZ showed a leafy appearance and the presence of crystals which were incorporated in a spherical matrix. Filaments with large vesicles were also present (Figure 7a–c).

and 4 µm, respectively. SEM images showed different surface structures depending on the pedoclimatic area of provenance of the walnut husks. The surface of PZ showed a leafy appearance and the presence of crystals which were incorporated in a spherical ma-The surface of PM exhibited a uniform structure, characterized by a very smooth lamellar appearance. This lamella also showed microcrystals incorporated in a matrix (Figure 7A–C).

trix. Filaments with large vesicles were also present (Figure 7a–c).

The surface of PM exhibited a uniform structure, characterized by a very smooth la-

mellar appearance. This lamella also showed microcrystals incorporated in a matrix (Fig-

**Figure 7.** Scanning electron images of pectins from Zumpano (PZ, **a**–**c**) and Montalto (PM, **A**–**C**) at three different magnifications and scales. (**a**) and (**A**) (100×, 200 µm); (**b**) and (**B**) (2500×, 10 μm); (**c**) and (**C**) (5000×,4 μm). **Figure 7.** Scanning electron images of pectins from Zumpano (PZ, (**a**–**c**)) and Montalto (PM, (**A**–**C**)) at three different magnifications and scales. (**a**,**A**) (100×, 200 µm); (**b**,**B**) (2500×, 10 µm); (**c**,**C**) (5000×, 4 µm).

#### *3.10. Differential Scanning Calorimetry (DSC) of Pectins 3.10. Differential Scanning Calorimetry (DSC) of Pectins*

The calorimetric curves recorded for the PM (red line) and PZ (black line) were similar, but unlike the calorimetric curves for glucans, they showed two peaks, one of which was endothermic and the other exothermic (Figure 8). Peak one, which was very similar to that observed for glucan calorimetric curves, was endothermic and was located between 130 and 135 °C. It had a higher enthalpy for the PM samples (E = 326.8 J/g) than for the PZ samples (E = 212.4 J/g). Peak two, between 240 and 250 °C, was exothermic, and it showed similar enthalpy values for both samples (E = −90.6 J/g; E = −77.1 J/g). The calorimetric curves recorded for the PM (red line) and PZ (black line) were similar, but unlike the calorimetric curves for glucans, they showed two peaks, one of which was endothermic and the other exothermic (Figure 8). Peak one, which was very similar to that observed for glucan calorimetric curves, was endothermic and was located between 130 and 135 ◦C. It had a higher enthalpy for the PM samples (E = 326.8 J/g) than for the PZ samples (E = 212.4 J/g). Peak two, between 240 and 250 ◦C, was exothermic, and it showed similar enthalpy values for both samples (E = −90.6 J/g; E = −77.1 J/g).

**Figure 8.** DSC records of walnut husks' pectins from Zumpano (red line) and from Montalto (black line).

#### **Figure 8.** DSC records of walnut husks' pectins from Zumpano (red line) and from Montalto (black **4. Discussion**

line). **4. Discussion** Glucans and pectins are nonstarch polysaccharides and fibre components, which possess a broad spectrum of biological activities. Glucans have hypocholesterolemic and hypoglycemic effects but also improve the defence of the immune system and induce defence mechanisms to respond to wounding [20,43,44]. Pectins are applied in the pharmaceutical field to treat human pathologies, such as cancers, liver damage, and inflammations [45]. Husk glucans, occurring in the bran of cereal grains (barley and oats and to a much lesser degree in rye and wheat, in amounts of about 7%, 5%, 2% and less than 1%, respectively) and many kinds of mushrooms, have been researched in order to valorise the waste from nut production. In this context, walnuts with green hulls were harvested from two locations of southern Italy, where walnut trees were widespread. Although the two areas were characterized by similar climatic conditions (annual raining, daily temperature and humidity average), the recovery of glucans was different for the hulls from the two areas. In fact, the glucan content from Montalto was 1.2 times higher than that of GZ, most likely due to the different soil characteristics. The glucan content from both Montalto and Zumpano husks is nevertheless significant compared to that of some cereals such as oat samples (0.71–5.06%) [46], broad bean pods (3.0–4.5%) and pomegranate *Akko* peels (2.5%) [12,13] The qualitative profile of the IR spectra of both GM and GZ glucans was similar. Furthermore, the observation of the anomeric region did not allow us to distinguish easily between the two glycosidic linkage types of the aldopyranoses. Despite this criticality, the use of the β-glucan assay kit allowed us to determine the α- and β-glucan content in both samples, showing that Montalto husks were richer in β-glucans while Zumpano husks contained α- and β-glucans in equal percentages. However, the β-glucan Glucans and pectins are nonstarch polysaccharides and fibre components, which possess a broad spectrum of biological activities. Glucans have hypocholesterolemic and hypoglycemic effects but also improve the defence of the immune system and induce defence mechanisms to respond to wounding [20,43,44]. Pectins are applied in the pharmaceutical field to treat human pathologies, such as cancers, liver damage, and inflammations [45]. Husk glucans, occurring in the bran of cereal grains (barley and oats and to a much lesser degree in rye and wheat, in amounts of about 7%, 5%, 2% and less than 1%, respectively) and many kinds of mushrooms, have been researched in order to valorise the waste from nut production. In this context, walnuts with green hulls were harvested from two locations of southern Italy, where walnut trees were widespread. Although the two areas were characterized by similar climatic conditions (annual raining, daily temperature and humidity average), the recovery of glucans was different for the hulls from the two areas. In fact, the glucan content from Montalto was 1.2 times higher than that of GZ, most likely due to the different soil characteristics. The glucan content from both Montalto and Zumpano husks is nevertheless significant compared to that of some cereals such as oat samples (0.71–5.06%) [46], broad bean pods (3.0–4.5%) and pomegranate *Akko* peels (2.5%) [12,13] The qualitative profile of the IR spectra of both GM and GZ glucans was similar. Furthermore, the observation of the anomeric region did not allow us to distinguish easily between the two glycosidic linkage types of the aldopyranoses. Despite this criticality, the use of the β-glucan assay kit allowed us to determine the α- and β-glucan content in both samples, showing that Montalto husks were richer in β-glucans while Zumpano husks contained α- and β-glucans in equal percentages. However, the β-glucan content in the Montalto husks was 1.75 times of that in Zumpano husks. The morphological investigation highlighted different surface structures for GM and GZ. The surface of GZ showed the presence of aggregates of microcrystals whose dimensions were variable, while the GM surface showed not aggregated microcrystals with a cubic form. The surface morphology of both GM and GZ was different from SEM images of barley and oat glucans [19,47]. Glucans, recovered from other type of biomasses such as *Vicia faba* L. pods using the same extraction method, showed different morphological surfaces, which exhibited agglomerates with a spongy appearance [13].

content in the Montalto husks was 1.75 times of that in Zumpano husks. The morphological investigation highlighted different surface structures for GM and GZ. The surface of Thermal analysis (DSC) confirmed the higher degree of crystallinity of GZ, since the corresponding melting enthalpy value was twice that recorded for the GM sample.

GZ showed the presence of aggregates of microcrystals whose dimensions were variable,

morphology of both GM and GZ was different from SEM images of barley and oat glucans [19,47]. Glucans, recovered from other type of biomasses such as *Vicia faba* L. pods using the same extraction method, showed different morphological surfaces, which exhibited

Thermal analysis (DSC) confirmed the higher degree of crystallinity of GZ, since the corresponding melting enthalpy value was twice that recorded for the GM sample. Pectins

agglomerates with a spongy appearance [13].

Pectins have also been investigated in walnut husks from Montalto and Zumpano for their valorisation, carrying out the extraction under acidic conditions at reflux temperature. The pectin content, as well as that of glucans, was found to be different in the two matrices: recovery of PM was 2.6 times that of PZ, confirming that soil and climate conditions influenced fibre content. It has been reported that extraction of pectins from other types of wastes or by-products, carried out using similar chemical conditions, yielded 5.2–12.2% from banana peels [48], 3.7–7.7% from passionfruit peel [49], 3.9–11.2% from pomegranate peels [50], and 7.3–19.1% pistachio green hull [51]. The percentage yield in PM was higher than that of other food wastes extracted under alkaline conditions such as leek leaves (12 ± 7%), endive roots (22 ± 8%), onion hulls (14 ± 0%), endive leaves (36 ± 8%), pumpkin kernel cake (29 ± 2.13%), tomato skins (29 ± 9.15%), and grape pomace (15 ± 3.07%) [52]. The comparison of our results with these data highlighted that the walnut husks from Montalto were a good source of pectins. Similarly to glucans, pectins were characterized by FTIR spectroscopy, surface morphological analysis (SEM) and thermal characteristics (DSC). In addition, the degree of methoxylation was determined, since it is an important structural factor influencing the functional properties of pectins. The FT-IR spectra of both PM and PZ did not show any significant differences in the characteristic bands of the structure, except for the absorption intensities of the peaks in the 1800–1500 cm−<sup>1</sup> region arising from ester carbonyl groups and carboxylate ion stretching. In fact, the IR spectra of PM showed that the intensity of the ester groups was greater than that relating to the carboxylate groups, whereas in PZ spectra the carboxylate ion band was stronger than the ester group band. These spectroscopic differences were reflected in the different degree of methoxylation (DM), calculated on the basis of the areas of the two bands between PM and PZ. The DM of PM was 1.5 times higher than PZ's. This was confirmed by the titrimetric method (DE), although the values obtained for DE are slightly higher than those obtained for DM. However, regardless of the method used, the esterification degree of PM was significantly higher than that of PZ, and considering that its percentage value (both DM and DE) was higher than 50, it is possible to classify PM as a high-methoxyl pectin, similarly to apple pectin, with a DM of 65.88% [53]. Additionally, pectins extracted from biomass (rind and peels) obtained from fruits with citric acid solution at high temperature, such as melon, kiwifruit, pomegranate and orange, were high-methoxyl pectins, having esterification degrees (DM) of 71.98%, 84.72%, 56.74% and 69.67%, respectively [54]. Morphological analysis of PM and PZ suggested different surface morphologies, exhibiting a very smooth lamellar appearance for PM with little pellets on it, comparable to the morphological characteristics of passion fruit pectin [25]. In contrast, the morphological structures of pectins from melon rind, kiwifruit, pomegranate and orange peels had some microfractures and hollow openings [54].

Calorimetric curves of PM and PZ were similar, showing two main peaks during the thermal analysis, one of which was endothermic and the other exothermic. The parameters associated with the two peaks were melting temperature and enthalpy (T<sup>m</sup> and ∆m, respectively), and degradation temperature and enthalpy (T<sup>d</sup> and ∆d, respectively). The first endothermic peak between 130 and 150 ◦C is ascribed to water evaporation: PM and PZ showed little differences for T<sup>m</sup> but ∆<sup>m</sup> of PM samples was higher than the PZ samples, which indicated that more energy was needed to absolutely remove water from PM, likely due to higher esterification degree. The second exothermic peak, between 240 and 250 ◦C, was caused by the degradation of pectin: PM and PZ showed little differences for T<sup>d</sup> and ∆<sup>m</sup> [55,56]. The above results indicated that geographical and climatic conditions in different regions could lead to significant differences both in the content of bioactive compounds and their morphological and thermal characteristics.
