**3.2 Degree of esterification (DE): FTIR**

To characterize the different extracted pectin samples, three of them were tested on their degree of esterification: the two sample sets used for measuring extraction yield of the enzymatic and the microwave-assisted extraction methods, and pectin extracted by microwave in bulk amounts, used later for film production. EN pectin had a DE = 49.10 ± 2.66% and was significantly different from the Mi pectin (DE = 77.34 ± 0.88%) and exhibited a significant difference from the bulk Mi pectin (**Table 2**).

The Mi pectin was considered highly esterified in both cases since the degree of esterification was greater than 50%. The EN pectin could be described as moderately esterified because the resulted degree of esterification was located at the boundary of high- and low-esterified pectins.


*Data are presented as mean ± standard deviation (n = 3). Different subscript letters indicate significantly different values at p < 0.05 using Tukey's HSD.*

#### **Table 2.**

*DE of pectin extracted by different methods.*


*Data are presented as mean ± standard deviation (n = 3). Asterisks show significant differences between the two extraction methods at p < 0.05 using a two-sided t-test with the same variance.*

#### **Table 3.**

*Monosaccharide composition of pectin differing in the extraction method without glucose.*

#### **3.3 Pectin composition: ion-exchange chromatography**

To compare the monosaccharide compositions of the differently extracted pectin samples, ion-exchange chromatography was performed. Prior to this analysis, the pectin samples were digested with Driselase. The monosaccharide compositions of both pectin samples were comparable. For both extraction methods, the main monosaccharide was galacturonic acid, which did not differ significantly between the two extraction methods. Rhamnose, arabinose, and galactose differed significantly (**Table 3**).

The undigested pectin powders were also tested for their free glucose and fructose contents, using two different Megazyme kits. The K-GLUC kit measured only the free glucose and the K-FRUGL measured the free glucose as well as the free fructose. It showed that the pectin powder extracted from orange peel contained some free sugar such as fructose and glucose. The Mi pectin contained significantly more free sugars than EN pectin (**Table 4**).

#### **3.4 Size-exclusion chromatography**

The different pectin samples were analyzed by SEC to test the behavior of the molecules in aqueous solutions and to get information about their particle size. The molecular weight (Mw), the intrinsic viscosity ([ƞ]), the hydrodynamic radius (Rh), and the average conformation of the polymer (α) were determined. The chromatograms of the two pectin samples each showed two populations of molecules, which were quantified. The two peaks were overlapping in the chromatogram, leading to approximate integration.

The two populations of molecules from the two pectin samples analyzed differed significantly in their molecular weight. The molecules extracted using the microwaveassisted procedure were characterized by higher molecular weight than those of the other pectin sample (**Table 5**). The chromatogram showed that Mi pectin had higher dispersity in molecules than EN pectin. The intrinsic viscosity of the different peaks varied significantly. The first peak of the EN pectin had the highest intrinsic viscosity (8.73 ± 0.39 dl/g). The intrinsic viscosity was higher at the first peaks, implying a more open structure and a higher hydrodynamic radius. The average conformation of both molecule populations of the EN pectin was significantly higher than that of the other pectin samples (**Table 5**).

#### **3.5 Mechanical tests of the pectin films**

The mechanical properties of the six pectin-containing film prototypes (for preparation see **Table 1**, Section **2.6**) were determined in triplicates. The parameters from these


*Data are presented as mean ± standard deviation (n = 3). Asterisks show significant differences between the two extraction methods at p < 0.05 using a two-sided t-test with the same variance.*

#### **Table 4.**

*Free glucose and fructose measurement by two different Megazyme kits and UV/Vis spectroscopy.*

*Extraction of Pectin from Orange Peel Wastes as an Ingredient for Edible Films Containing… DOI: http://dx.doi.org/10.5772/intechopen.108625*


*Data are presented as mean ± standard deviation (n = 9). Asterisks show significant differences between the two extraction methods at p < 0.05 using a two-sided t-test with same variance.*

#### **Table 5.**

*Molecular weight, intrinsic viscosity, hydrodynamic radius, and average conformation of the two pectin samples analyzed by OMNISEC with PEO-24 K as a calibration standard.*

measurements, maximum strain and maximum stress, are shown in **Figures 1** and **2**, respectively. The maximum strain of the film Mi2.5No was 138.26 ± 13.97% and differed significantly from Mi1.5Millet, Mi2.5Millet, and EN1.5No, with considerably lower maximum strains than Mi2.5No. The films EN1.5Millet and Mi1.5No showed intermediate maximum strains and were not significantly different from the data of all other prototypes (**Figure 1**).

At the maximum stress, a significant difference between films produced with and without the whole-grain kabog millet flour was visible (**Figure 2**). The maximum stress of the film Mi2.5Millet was 3.50 ± 0.33 MPa, and the one from the film Mi2.5No was 8.38 ± 0.61 MPa. The only difference in the composition of these two films was the addition of 2 g whole-grain kabog millet flour to Mi2.5Millet, resulting in more brittle film properties. This observation was also significant between EN1.5No and EN1.5Millet and between Mi1.5No and Mi1.5Millet (**Figure 2**).

To determine the hydrophobicity of the created films, the static water contact angle was measured on the surface of the films. The films showed significant differences in

#### **Figure 1.**

*The maximum strain of selected films. The different codes stand for different film prototypes compositions: EN = enzymatic extraction of pectin; Mi = microwave-assisted extraction of pectin; 1.5 = contains 1.5 g extracted pectin; 2.5 = contains 2.5 g extracted pectin; No = without whole-grain kabog millet flour; Millet = with wholegrain kabog millet flour. Data are presented as mean ± standard deviation (n = 3). Different subscript letters indicate significantly different values at p < 0.05 using Tukey's HSD.*

#### **Figure 2.**

*The maximum stress of selected films. The different codes stand for different film prototypes compositions: EN = enzymatic extraction of pectin; Mi = microwave-assisted extraction of pectin; 1.5 = contains 1.5 g extracted pectin; 2.5 = contains 2.5 g extracted pectin; No = without whole-grain kabog millet flour; and Millet = with whole-grain kabog millet flour. Data are presented as mean ± standard deviation (n = 3). Different subscript letters indicate significantly different values at p < 0.05 using Tukey's HSD.*

#### **Figure 3.**

*Water contact angle left and right of selected films. The different codes stand for different film prototypes compositions: EN = enzymatic extraction of pectin; Mi = microwave-assisted extraction of pectin; 1.5 = contains 1.5 g extracted pectin; 2.5 = contains 2.5 g extracted pectin; No = without whole-grain kabog millet flour; and Millet = with whole-grain kabog millet flour. Data are presented as mean ± standard deviation (n = 3). Different subscript letters indicate significantly different values at p < 0.05 using Tukey's HSD.*

contact angle as a function of the extraction method of pectin, the pectin concentration, and the presence of whole-grain kabog millet flour (**Figure 3**). Based solely on the extraction method of the pectin, it was observed that films produced with EN pectin showed significantly higher water contact angle than films produced from Mi pectin. This could be observed by comparing the mean contact angle of EN1.5No (76.94° ± 4.90) and Mi1.5No (27.68° ± 8.15). Biopolymers produced from EN pectin were characterized by higher hydrophobicity compared to biopolymers made from Mi pectin.

Comparing the difference in water contact angle between Mi2.5No/Millet and Mi1.5No/Millet, it could be observed that the change in the contact angle by adding the whole-grain kabog millet flour was decreased. With increasing pectin content, the effect of the whole-grain kabog millet flour on hydrophobicity was significantly reduced, and overall hydrophilicity was enhanced significantly. Comparing the water contact angle of Mi1.5Millet at 71.48° ± 4.05 and of Mi1.5No at 27.68 ± 8.15,

*Extraction of Pectin from Orange Peel Wastes as an Ingredient for Edible Films Containing… DOI: http://dx.doi.org/10.5772/intechopen.108625*

#### **Figure 4.**

*Water droplet on the surface of a) Mi1.5Millet b) EN1.5No c) Mi1.5No. The different codes stand for different film prototypes compositions: EN = enzymatic extraction of pectin; Mi = microwave-assisted extraction of pectin; 1.5 = contains 1.5 g extracted pectin; No = without whole-grain kabog millet flour; Millet = with whole-grain kabog millet flour.*

a significant increase in contact angle, and therefore in hydrophobicity could be observed with adding whole-grain kabog millet flour to the biopolymer. In comparison with the other two pairs of films, there was no significant effect observed from the addition of whole-grain kabog millet flour to the film. It is visible that the water contact angle was significantly lower for Mi1.5No compared to the films with enzymatically extracted pectin (EN1.5No) or added kabog millet flour (Mi1.5Millet) (**Figure 4a**–**c**).

### **4. Discussion**

#### **4.1 Extraction yield**

The extraction yield differed significantly between the two extraction methods. The yield from enzymatic extraction using Celluclast® 1.5 L (~3%) was significantly lower than other studies using a similar protocol for apple pomace [19]. Orange peels contain 20.9% pectin [20] and apple pomace, 19–20% [21]. Therefore, the source of the pectin was not the main reason for the difference in yield. A possible explanation for the low yield in the current study is the incubation time of 3 h, which was shorter than 18 h used by Ref. [19]. It can be assumed that more cell wall components are digested with longer incubation time, and more pectin can be extracted [19]. Yield could be increased in further experiments by using longer incubation time or increasing the enzyme concentration. The yield from microwave-assisted extraction (~15%) was comparable to other studies [22]. The presence of free sugar, especially in the Mi pectin powder, can contribute to errors in the gravimetric measurement of the yield. A possible source of error could be also the precipitation of other polymers, which can bind to pectin, such as cellulose. Cellulose can also be extracted from plant material using microwave-assisted extraction and ethanol as solvent [23]. Therefore, it is possible that other polymers present in plant cell walls can precipitate and affect the gravimetric measurement of the pectin extraction yield.

#### **4.2 Degree of esterification**

The DE differed significantly between the two extraction methods. EN pectin can be described as moderately esterified because its DE was just at the boundary between high- and low-esterified. The obtained DE (~49%) was comparable to DE values using the same enzyme for extracting pectin [15]. It may be possible that only pectin with a lower degree of esterification can be released from the plant cell by enzymatic treatment. The DE of the microwave-assisted extracted pectin could be considered as highly esterified (~77%). The result was comparable to that of microwave-assisted extracted orange peel pectin (~71%) from [24], from lime peels (~71–92%) [25], and apple pomace pectin extracted by microwave (~74%) [9].

#### **4.3 Pectin composition**

As expected, the monosaccharide compositions of both pectin samples were comparable because the pectin was extracted from the same source and should therefore have a similar composition. The main component was galacturonic acid, which is the backbone molecule of pectin [4]. The galacturonic acid contents from both Mi pectin and EN pectin did not differ significantly. It was comparable to the galacturonic acid content in apple pectin [19] and orange peel pectin [24, 26]. The other monosaccharides except for rhamnose, galactose, and arabinose were also similar. The three significantly differing monosaccharides could be converted during extraction. The free glucose test with the K-GLUT assay was significantly higher in the Mi pectin powder than in the enzymatically extracted one. Due to the differences in the free glucose testing between the two applied kits, it can be assumed that the results had some uncertainties. These could be explained by the approximation of the reaction endpoint used in the K-FRUGL kit. Another reasonable explanation would be the presence of cellulose in the extracted pectin powder. (Nano) cellulose fibrils can be extracted using microwave-assisted methods [23]. Considering that orange peel has a cellulose content of approximately 50%, it is reasonable that the pectin powder also contained nanocellulose molecules [27]. Since microwave-assisted extraction is more vigorous and can increase the solubility of certain cellular compounds, it can be explained that more free glucose and nanocellulose fibrils were extracted from the cell wall and the glucose content, therefore, was higher in the Mi pectin sample than in the EN pectin powder.

### **4.4 Pectin molecular properties**

The observed molecular weights of the first peak of each pectin sample from enzymatic extraction and microwave-assisted extraction were ~ 211 ± 18 kDa and ~ 485 ± 20 kDa, respectively. These values were comparable to the molecular weights of orange peel pectins, which had a molecular weight of 120 ± 10 kDa to 360 ± 20 kDa [28]. The dispersion of particle size and, therefore, of the molecular weight was broader in microwave-assisted extraction due to the rapid increase in temperature and internal pressure [16]. The second peaks could depict cleaved pieces of pectin or other dissolved cell components like cellulose. There are cellulose molecules or cellulose derivatives that would fit in the range of the molecular weight of the second peaks [29]. The intrinsic viscosity values from enzymatic extraction (~8.7 dl/g) and microwave-assisted extraction (~6.5 dl/g) fit within the range of 4.8–10.8 dl/g for the intrinsic viscosity of orange peel pectin [28]. The Mi pectin was characterized by a significantly lower intrinsic viscosity than the EN pectin. A lower intrinsic viscosity

*Extraction of Pectin from Orange Peel Wastes as an Ingredient for Edible Films Containing… DOI: http://dx.doi.org/10.5772/intechopen.108625*

describes a more condensed structure and higher molecular density [30]. In contrast to the intrinsic viscosity, the Mi pectin had a significantly higher hydrodynamic radius than the EN pectin, indicating a looser conformation. Due to this contradictory relationship, it can be assumed that the condensation of the pectin structures in water is similar. The average conformation of the two pectin samples differed. The EN pectin had values nearly twice as high as the Mi pectin. The Mi pectin had a conformation that can be described as a semi-flexible random coil-like structure due to its α value between 0.5 and 0.8 [30]. On the other hand, the EN pectin had both populations of particles with an average conformation higher than 0.8. They tend to be in a more rigid and rod-like structure.

#### **4.5 Mechanical testing of the created films**

The differences in maximum strain showed the trend that the elongation could be enhanced by increasing the pectin content. The maximum strain of Mi1.5No was 102.25 ± 17.93%, and if increasing the pectin content by 1 g/100 ml, the maximum strain reached 138.26 ± 13. 97% (Mi2.5No). The maximum stress was therefore increased by approximately 36%, which can be explained by increased film thickness and consequently, increased cross-sectional area; thus, more cross-linkages that need to be broken [18]. The film microstructure was altered with changing pectin content by analyzing with scanning electron microscopy [18]. With 2.5 g pectin per 100 ml film-forming solution, the film structure was the smoothest [18]. By increasing or decreasing the pectin content, the microstructure was disturbed. A significant effect of the different extraction procedures on the maximum strain of the film could not be observed. A significant change in maximum strain by adding whole-grain kabog millet flour could only be observed between the prototypes Mi2.5No (138.26 ± 13.97%) and Mi2.5Millet (69.45 ± 5.50%). The higher maximum strain of Mi2.5No cannot be explained by increasing the cross-sectional area because the films containing wholegrain kabog millet flour were characterized by the increased film thickness. It could be assumed that the internal arrangement was disturbed by adding the kabog millet flour; therefore, the elongation properties decreased. Compared to other biodegradable films based on polymers, the produced films tended to be rigid and inelastic. There were formulations for biodegradable films characterized by a maximum strain between 300% and 700% [13]. Regarding the maximum mechanical stress to break, films with kabog millet flour broke with the application of approximately half of the force per cross-sectional area than films without kabog millet flour. This leads to the conclusion that adding kabog millet flour changed the properties of the films toward higher brittleness. No significant differences between the maximum stress data of the films produced from different pectin samples could be observed. Therefore, the extraction method did not influence the mechanical properties of the produced pectin edible films.

#### **4.6 Water contact angle**

The water contact angles on the surface of the produced films provided information about the hydrophobic characteristics of the different film prototypes. A low water contact angle characterizes more hydrophilic properties of the edible pectin film [13]. In the application of pectin films for covering food products, a certain hydrophobicity would be desired to stabilize the protection layer against water. It could be observed that the films produced from EN pectin resulted in higher water

contact angles than films from the Mi pectin sample. This is contradictory to the results of the degree of esterification because the EN pectin showed a greater number of free carboxy groups due to its lower degree of esterification and, therefore, should be more hydrophilic [31]. A possible explanation for the lower contact angles observed for the Mi pectin could be its higher hydrodynamic radius. The higher hydrodynamic radius indicates higher hydrophilicity. Furthermore, the rod-like conformation of the EN pectin does not allow as many interactions with the surrounding water because only a part of the functional groups is exposed on the surface of the rod and therefore is characterized as more hydrophobic. The addition of the whole-grain kabog millet flour showed a significant increase in water contact angle between the films Mi1.5Millet and Mi1.5No, while between the other pairings, a difference could be observed but it was less pronounced. The addition of the kabog millet flour increased the hydrophobicity, which could be explained by its wholegrain character. In whole-grain products, lipids are present, including some lipophilic compounds such as carotenoids and tocopherols [11]. These lipophilic structures may shift and enhance the hydrophobic properties of the films. During the measurements of the water contact angle, some properties of the film were observed, which could be interesting for further research. The pectin films, especially the ones with kabog millet, showed volume expansion upon the addition of water, and the droplet of water turned white over time, so it can be assumed that some hydrophilic film compounds migrated into the water droplet.
