*4.1.4 Pectin extraction and purification*

For the highest pectin yield, pH was adjusted to 3.5 with sodium hydroxide. The solution was dispersed in ethanol, and the precipitates were obtained after it remained for a night at 4°C with centrifugation (4000 rpm, 20 min). Ethanol at a 70 and 96% (v/v) alcohol content was used for rinsing the separated solids. The mixture was centrifuged (4000 rpm, 30 min) to separate the pectin. The separated pectin was dissolved in deionized water, freeze-dried, and maintained at room temperature. The yield of pectin extraction was calculated using an equation (Bi and P as initial quantity in gram of substrate and pectin, respectively):

$$Y\_{pcc} \left(\text{\textquotedblleft}\right) = \frac{P}{B\_i} \times \mathbf{100} \tag{1}$$

<sup>2</sup> 101 stainless steel high pressure reactors.

#### *4.1.5 Analytical methods*

#### *4.1.5.1 Substrate characterization*

The TS, VS, and extractive contents of the orange wastes were measured according to the National Renewable Energy Laboratory (NREL) protocol. The morphology of the freeze-dried substrates was assayed using gold-coated procedure and scanning electron microscopy (15 kV) in order to observe the acid treatment effect.

#### *4.1.5.2 Pectin characterization*

Pectin characteristics (chemical structure, GalA content, and DE) were determined. The chemical structure was assessed by a Fourier transfer infrared spectrometer with a deuterated-triglycine sulfate detector in comparison with commercial pectin (resolution of 1 cm−1, 32 scans in 4400–400 cm−1).

The GalA content was quantified based on Ramos-Aguilar et al. [22]. Pectin (5 mg) was added to 2 mL of concentrated sulfuric acid (98%) and 1 mL of deionized water and adjusted to 10 mL. After an ice bath procedure (10 min), the centrifugation was performed at room temperature (2000 × g, 10 min). The liquid fraction (400 μL) was mixed with 2.4 mL of sodium tetraborate (75 mM in concentrated sulfuric acid) and 40 μL of 4 M potassium sulfamate solution (pH 1.6). The temperature of tubes decreased using indirect contact in an ice bath after insertion in boiling water for 20 min. The M-hydroxy di-phenyl solution in NaOH was added. The absorbance of samples was assayed at 525 nm using a UV-vis spectrophotometry.

The DE was obtained according to Santos et al. [23] as follows: the dry mass of pectin (0.1 g) was dissolved with 3 mL of ethanol 96% in 20 mL of distilled water at 40°C and 100 mL Erlenmeyer flasks were magnetically stirred up. The titration was performed with sodium hydroxide solution (V1 mL for the first neutralization step, 0.1 M) and phenolphthalein indicator for appearing a pale pink color. The sodium hydroxide solution (10 mL, 0.1 M) was added to this neutralized solution and stirred. The hydrochloric acid solution (0.1 M, 10 mL) was added and stirred in order to disappear the pink color completely. The sodium hydroxide solution (V2 mL for the second neutralization step, 0.1 M) was added to neutralize the excess of acid until the solution color changed to pink (V2). The DE of the pectin was determined using V1 and V2 as follows:

$$DE\left(\%\right) = \frac{V\_2}{V\_1 + V\_2} \times 100\tag{2}$$

#### *4.1.5.3 Statistical analysis*

Analysis of variance (ANOVA) was applied using the least significant difference (LSD) and Tukey's methods in SAS 9.1.3 software (p < 0.05).

#### **4.2 Case study No. 2**

High-quality pectin from cantaloupe waste: Eco-friendly extraction process, optimization, characterization, and bioactivity measurements—A case study by Kazemi et al. [17].

### *4.2.1 Extraction and optimization of pectin*

Fresh cantaloupe fruits were washed, peeled, cut, and placed in an oven (60°C, 48 h). The dried pieces were powdered and stored at 25°C. The MAE process was used to produce pectin from cantaloupe powder in a microwave oven according to Kazemi et al. [17]. pH levels were adjusted using organic citric acid. The response was expressed as the extraction yield (g kg−1). For optimization of the MAE process, MAE variables and their levels were selected for pectin extraction from cantaloupe rinds using BBD according to the literature. The effects of MAE-independent variables were optimized and investigated including microwave power (300–700 W), irradiation time (60–180 s), pH (1.5–3.0), and LSR (20–30 mL/g). In order to reduce systematic errors, all experiments were randomly performed, and then the results were put in a polynomial equation in order to predict the optimized condition for the MAE process.

## *4.2.2 Pectin characterization*

Pectin characteristics (physicochemical, structure, functional, and antioxidant assay) were determined. Three experiments were performed for each measurement, and the results were calculated and reported as mean value ± SD3 .
