**2.2 Chemicals**

Hydrochloric acid (HCl) (≥37%), absolute ethanol (>99.8%), acetic acid (>99.8%), methanol (>99.9%), calcium chloride (CaCl2) (≥ 97%, granulated), sodium acetate (NaOAc) (>99.0%), and Driselase (protein ≥10% from *Basidiomycetes* sp.) were purchased from Sigma-Aldrich GmbH (Steinheim, Germany). The Celluclast® 1.5 L enzymes were ordered from Novozymes (Bagsværd, Denmark). Glycerol (85%) was obtained from Hänsler AG (Herisau, Switzerland).

#### **2.3 Microwave-assisted pectin extraction**

The microwave-assisted extraction of pectin was performed according to Ref. [14] with a household microwave oven (Mio Star MW 01). Ten grams of sample powder was weighed in triplicates. Milli-Q water (pH 1.5) was added to the samples to reach an optimal liquid-to-solid ratio of 20 ml/g. The suspensions were microwaved at a power of 450 W and irradiation time of 20 min, and stirred every 5 min. The suspensions were then cooled down and transferred into 50-ml tubes. The tubes were centrifuged (Eppendorf 5810 R; Hamburg, Germany) at room temperature for 10 min at 4000 rpm and the supernatants were collected. The pectin was precipitated for 1.5 h at room temperature using an equal volume of 95% ethanol. The dispersion was filtered and washed three times with 95% ethanol at room temperature. The extracted pectin was dried at 50°C overnight.

#### **2.4 Enzymatic extraction of pectin**

The enzymatic extraction of pectin was performed according to [15], except that the duration was reduced from 18 h to 3 h. For extracting the pectin, 10 g of the orange peel powder was weighed in triplicates. To get a liquid-to-solid ratio of 15:1, 150 ml of Milli-Q water (pH 4.5) was used. For the suspension, 500 μl of Celluclast® 1.5 L enzymes was added (Novozymes, Bagsværd, Denmark) and the samples incubated for 3 h in a water bath at 50°C. The resulting solution was cooled to room temperature and transferred into 50-ml tubes. The tubes were centrifuged (Eppendorf 5810 R; Hamburg, Germany) at 4°C for 10 min at 4000 rpm. The supernatants were collected, and 96% ethanol was added at 4°C to a final ethanol concentration of 70%. The suspensions were precipitated for 1 h and transferred into 50-ml tubes, which were then centrifuged at 4000 rpm for 20 min at room temperature. The precipitate was washed with 70% ethanol and centrifuged again for 20 min under the same conditions. The combined precipitates were dried for 24 h at 60°C.

#### **2.5 Characterization of the extracted pectins**

#### *2.5.1 Fourier-transform infrared spectroscopy (FTIR)*

To determine the degree of esterification (DE) of the extracted pectin, FTIR analysis was performed according to Ref. [16], using an FTIR spectrometer (Varian 640 with golden gate-diamond ATR). To obtain a dense surface of pectin powder on the surface of the ATR diamond, three droplets of 100% methanol were added to the pectin powder to increase the density. After evaporation of the solvent, the absorption was measured several times to get the optimal spectrum with a definite pectin signal and a smaller signal of the remaining methanol. The DE could be calculated then with Eq. (1) using the absorbance (Abs) at the wavelengths at 1630 cm−1 and 1745 cm−1, which are known as the fingerprint regions of pectin [16].

$$DE(\text{@}) = \left\lfloor \text{Abs}\_{1745} / \left( \text{Abs}\_{1745} + \text{Abs}\_{1630} \right) \right\rfloor \times \mathbf{100} \tag{1}$$

#### *2.5.2 Ion-exchange chromatography*

To analyze the pectin sample by ion-exchange chromatography, the pectin was digested with Driselase (Sigma-Aldrich) using a method of Ref. [17]. Five milligrams *Extraction of Pectin from Orange Peel Wastes as an Ingredient for Edible Films Containing… DOI: http://dx.doi.org/10.5772/intechopen.108625*

of the pectin samples was weighed in triplicates. For ion-exchange chromatography, a Thermo Scientific™ Dionex™ ICS-5000 (Thermo Fisher Scientific™, U.S.) was used with a PA1 Dionex CarboPac™ BioLc™ 4 x 50 mm column. The sampler had a 1 ml/ min rate, and the reference electrode was AgCl. The eluting solvents were A: 200 mM NaOH, B: 100 mM NaOH +1 M NaOAc, and C: water. The gradient system was 7.6% A and 92.4% C for 0–26 min running time: 50% A and 50% C for 26–33 min: 47.2% A, 6% B, and 46.8% C for 33–45 min: 35.2% A, 30% B, and 34.8% C for 45–78 min: 100% B for 78–91 min: 50% A and 50% C for 91–99 min: and 7.6% A and 92.4% C for 99–105 min.

Two different kits from Megazyme were used to determine the free glucose and fructose in the pectin powder. The glucose kit K-GLUC and the glucose and fructose kit K-FRUGL were purchased from Megazyme Ltd. (Bray, Ireland), and the analyses were performed according to the manufacturer's protocols.

#### *2.5.3 Size-exclusion chromatography (SEC)*

For the sample preparation, 5 mg of the pectin samples was weighed in triplicates and dissolved in 5 ml of Milli-Q water. The solutions were kept for 1 h at 80°C while stirring in a water bath and afterwards at room temperature overnight while stirring. The transparent solutions were filtered through a nylon filter with pores of 0.45 μm (13 mm Syringe Filter, Nylon 66, 0.45 μm; BGB (Böckten, Switzerland)) into vials followed by analysis by size-exclusion chromatography (OMNISEC, Malvern Panalytical Ltd., Malvern, United Kingdom). The system consisted of an OMNISEC RESOLVE chromatography compartment combined with a pump, an autosampler and two A6000M columns in series (8.0 × 300 mm, OMNISEC REVEAL VISCOTEK, Malvern Panalytical Ltd., Malvern, United Kingdom). The OMNISEC RESOLVE detector compartment was equipped with a low and rightangle laser light scattering detector (LALS/RALS), a refractive index (RI), and a viscometer. The mobile phase was composed of a solution of 0.1 M NaNO3 and contained 0.02% of NaN3. Both columns were kept at 25°C, and the flow rate was 8.83 ml/min. The injection volume was 100 μl. A polyethyleneoxide (PEO-24 K, VISCOTEK, Malvern Panalytical Ltd., Malvern, United Kingdom) standard and a dextran standard (Dextran-T68K, American Polymer Standards Corporation, Mentor, US) were used for calibration. All vials were measured with three injections. The molecular weight (Mw), the intrinsic viscosity ([ƞ]), the hydrodynamic radius (Rh), and the average conformation of the polymer (α) were determined by the instrument software.

#### **2.6 Film preparation**

The pectin films were prepared according to Ref. [18]. Six different prototypes of films were produced (**Table 1**). They differed in the extraction method of the pectin, pectin content, and the presence of whole-grain kabog millet flour (Catmon Cebu, Philippines). The whole-grain kabog millet flour was prepared according to Ref. [11].

The pectin was added carefully to the Milli-Q water with stirring. Glycerol, CaCl2, and the whole-grain kabog millet flour (if present in the film formulation) were added. The suspensions were heated to 70°C, and 25 ml was poured into plastic weighing boats (Sigma-Aldrich GmbH, diameter = 13.5 cm). The films were dried overnight at room temperature and then in an oven at 29°C with 29% humidity until they were dry.

*Utilization of Pectin in the Food and Drug Industries*


#### **Table 1.**

*Ingredient composition of produced film prototypes (EN = enzymatic extraction, Mi = microwave-assisted extraction; No = without whole-grain kabog millet flour, millet = with whole-grain kabog millet flour).*

#### **2.7 Characterization of the pectin films**

#### *2.7.1 Mechanical tests*

Mechanical properties of the films were evaluated by measuring tensile length and elongation using a Z010 (ZwickRoell GmbH & Co. KG, Ulm, Germany) with a 10 N load cell. The experiments were performed at room temperature and in triplicates using film pieces of 0.5 mm × 2.5 cm. The maximum strain and the stress to break were calculated according to Eqs. (2) and (3) using the elongation (LF) from starting point of 1 cm, the breaking force (F) and the cross-sectional area (A).

$$\text{Maximum strain} \left( \text{\textquotedblleft} \right) = \left[ \left( \text{L}\_{\text{F}} - \text{1 cm} \right) / \text{1 cm} \right] \times \text{100} \tag{2}$$

$$\text{Maximum stress} \left( \mathbf{MPa} \right) = \mathbf{F} / \mathbf{A} \tag{3}$$

#### *2.7.2 Water contact angle measurement*

To analyze the water contact angle of the different film prototypes, a Drop Shape Analyzer-DSA 100E (A.KRÜSS Optronic GmbH, Hamburg, Germany) was used. The contact angle of a 10 μl Milli-Q water droplet was determined by analyzing the drop shape. Measurements were done in triplicates at room temperature.

#### **2.8 Statistical analysis**

Data obtained from FTIR, the mechanical tests, and water contact angle measurements were evaluated with Microsoft Excel. For statistical analysis, an ANOVA was used followed by a post hoc Tukey's HSD (p < 0.05). The data obtained from ionexchange chromatography, free sugar content analysis, and SEC were evaluated using IBM SPSS Statistics v. 28.0.0 (IBM, Armonk, United States) with a two-sided t-test, assuming homogenous variance (p < 0.05).

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