1. Introduction
Apples are some of the most widespread fruits, with multiple benefits for the consumer’s health. In the Republic of Moldova, in 2017, apple production reached 430,000 tons from a total orchard area of 56,000 hectares. It was forecasted that the cultivated area for apple trees would increase by 52,400 hectares during the period of 2017–2027, resulting in a total apple production of 793,000 tons [
1]. Significant amounts of apple pomace (globally, 4 million tons/year) are produced as a byproduct during the processing of apples for jams, juices, and fermented products. Despite the fact that apple pomace is primarily used as animal feed or fertilizer, it is a substantial source of functional components such as carbohydrates, dietary fibers (including pectin), phenolic compounds, and others [
2]. The pectin derived from apple pomace is used in the pharmaceutical, food, cosmetic, and other industries, where it serves as a biopolymer, preservative, antioxidant, anticorrosive agent, protective agent for diverse surfaces, etc. [
2,
3]. Fibers obtained from fruits offer an advantage over cereal fibers due to their superior solubility, lower phytic acid content, and the presence of bioactive molecules associated with antioxidant activity [
4]. Pectin is industrially obtained from apple pomace through conventional extraction (CE) methods, such as using hot acidified water with either mineral acids (sulfuric, hydrochloric, nitric) or organic acids (citric, malic, oxalic) from pH 1.5 to 3.0 and temperatures ranging from 60 to 100 °C for 0.5 to 6 h, followed by alcoholic precipitation [
2,
5,
6]. The cost-effectiveness and optimization of pectin extraction can be improved through application of unconventional extraction techniques such as the microwave-assisted extraction (MAE) [
7], ultrasound-assisted extraction (UAE) [
8,
9], pulsed electric field extraction [
10], subcritical water extraction [
11], enzyme-assisted extraction [
12], as well as combinations of different extraction methods [
13,
14,
15]. The sustainability of unconventional methods such as UAE and MAE were proved, as the methods exhibit reduced energy and reagent consumption, shorter processing times (15–30 min as opposed to 1–3 h), and improved quality and yield of the final product compared to conventional methods [
16,
17,
18].
Several studies have confirmed that the antioxidant activity (AA) of pectin is influenced by the structure and composition of its chains, as well as by the presence of co-extracted contaminants in the polysaccharide matrix, which are associated with polyphenols, proteins, and other antioxidants [
19,
20,
21,
22]. Apple pectin, depending on its concentration, exhibited an approximately 5-fold greater DPPH radical-scavenging effect compared to other polysaccharides [
23].
The pectin extraction method also influences AA. Pectin obtained by unconventional methods from various sources, with lower degree of esterification (DE) and higher anhydrogalacturonic acid (AUA) content, exhibits a higher AA compared to pectin extracted through CE [
11,
24,
25]. Wang et al. [
26] reported that pectic polysaccharides extracted with hot-compressed water from apple pomace showed in vitro AA and an inhibitory effect on free radicals. The IC
50 values of such pectin oscillated between 1.4–3.5 mg/mL for 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and about 1 mg/mL for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).
Recent studies have shown that modified pectin obtained through unconventional methods displays different structural features [
26,
27,
28]. It contains a higher amount of galactoside residues compared to xylan and arabinan [
28] and shows more advanced radical-scavenging and anticancer activities compared to native pectin and pectin extracted through conventional methods [
29,
30]. Hydrolytic degradation of polysaccharides during unconventional extraction is accompanied by the formation of several reducing terminal groups, respectively, by the improvement of the antioxidant potential of pectin [
29,
30].
The DE of pectin also influences its AA due to its ability to chelate heavy metal ions [
31,
32]. A study on the influence of modified citrus pectin on the oxidative stability of flaxseed/sunflower emulsions confirmed that low-methoxyl pectin (DE of 33%) exhibited a higher lipid antioxidant potential compared to high-methoxyl pectin (DE of 58%) [
33].
Multiple research studies have shown the positive influence of pectin on health status [
2,
3,
6,
27]. Pectin has probiotic properties and contributes to the proper functioning of the intestine, as it retains water and various waste substances, facilitating the elimination of toxins and protecting the colon’s mucus membrane [
31,
34]. Pectin can bind and remove heavy metals from the body [
31,
32,
34] and lower the cholesterol level [
34,
35] and serum glucose level [
36]. Additionally, it has the capacity to capture free radicals and reduce the risk of cancer [
37]. Pectic polysaccharides reduce inflammation, have antibacterial properties [
38], and stimulate the immune response [
39].
Pectin is used in the food industry as a thickening additive; it acts as a protective and stabilizing colloid in food and beverages. Pectin with a DE > 50% (high-methoxyl pectin—HM) forms a gel in solutions with a high concentration of sugar or solid substances, at a pH lower than 3.5. This is applied in the production of jams and jellies, fruit fillings, desserts, etc. [
3]. Pectin with a DE less than 50% (low-methoxyl pectin—LM) forms a gel in a wide pH range (2.0–6.0), in the presence of calcium ions or other multivalent cations. It is used in the production of dietetic dairy products, soy-based products, etc. [
40]. Other properties associated with pectin are: protein stabilization, softness in texture, increase in volume, and syneresis control in low-calorie foods [
4,
6,
41].
Studies conducted in recent years have highlighted the sustainability of using pectin for the formulation and preservation of functional foods and encapsulation of bioactive compounds [
42]. The production of edible coatings based on pectin and other biodegradable polymers is encouraged by the United States Environmental Protection Agency (US EPA) program to minimize packaging waste. Pectin-based nanoemulsions play an important role in creating a new generation of active packaging with health benefits [
43]. Pectin-based films are biodegradable and possess excellent mechanical properties, providing the possibility to extend the shelf life of packaged foods [
44,
45], control moisture loss, and reduce the degradation of bioactive compounds during storage [
46,
47].
The objective of this study was to examine the impact of unconventional extraction methods, such as ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), on the yield, properties, and antioxidant activity of raw pectin extracted from apple pomace. The study also aimed to assess the potential of pectin as a binding and coating agent in the production of dried fruit bars; the protective effect of pectin films on functional products during storage was also investigated.
2. Materials and Methods
2.1. Materials
Golden Delicious apples were harvested in the autumn of 2021 at “AgroProduct” SRL, located in the village of Colicăuți, Briceni, Republic of Moldova (48°18′36″ N 27°8′54″ E). They were stored in a refrigerator for 10 months (Briceni) until the spring of 2022 at a temperature of 2 ± 1 °C and a relative humidity of the air of 87 ± 1%. For the production of the fruit bars, dried fruits were used: diced apples, seedless cherries, and prunes (purchased from Cazantip SRL, Chisinau, Republic of Moldova), as well as dried rosehips (purchased from Rose Line SRL, Taul, Donduseni, Republic of Moldova). The dried fruits were obtained by dehydrating fresh fruits, without any added sugar or other artificial flavors. The rosehip pulp powder was obtained from dried seedless rosehip pulp, ground to a particle size of 60 ± 10 µm.
6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (purity ≥ 97%) and 1,1-diphenyl-2-picrylhydrazyl-hydrate (DPPH) (≥95%) were provided by Alpha Aesar (Haverhill, MA, USA). Aluminum chloride hexahydrate (≥98%) and the standard compounds: β-carotene (≥95%), gallic acid (GA) (≥97%), rutin (≥94%), and quercetin (≥95%), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Folin–Ciocâlteu phenol reagent (2.1 N) was purchased from Chem-Lab NV (Zedelgem, Belgium). Citric acid monohydrate, ethanol, n-hexane, methanol, diethyl ether, acetonitrile, chloroform, sodium carbonate, sodium chloride, chlorhydric acid, and sodium hydroxide were purchased from Chemapol (Prague, Czech Republic). All reagents used in this study were of analytical or chromatographic grade. All spectrophotometric determinations were performed on a UV-1900 spectrophotometer (Shimadzu, Tokyo, Japan).
2.2. Productions of Apple Pomace (AP)
Apple pomace was obtained after squeezing the juice from Golden Delicious apples. After juice extraction, the pomace was blanched in a 0.2% (anhydrous) citric acid solution for 10 min to inhibit enzyme activity and oxidative processes that could alter the properties of the pomace. The pomace was further pressed at a temperature of 25 ± 1 °C and dried using forced convection in a SLW 115 SMART laboratory oven (Pol-Eco Aparatura, Wodzisław Śląski, Poland) at a temperature of 70 ± 1 °C, until reaching a final moisture content of 12.0 ± 0.13%, and then was finely ground to a particle size of 140 ± 10 µm.
2.3. Characterizations of Apple Pomace Powder
The following physicalchemical indicators of the apple pomace (AP) were determined: titratable acidity [
48]; moisture and ash content [
49]; fat content using Soxhlet extraction [
50]; protein content [
51]; total dietary fiber [
52]; and insoluble dietary fiber [
53]. The content of soluble substances was determined with a Kruss DR 201-95 digital refractometer (Kruss, Hamburg, Germany).
2.4. Methods of Extractions and Purifications of Pectin from Apple Pomace
Based on the results of preliminary attempts to obtain pectin by non-conventional UAE and MAE methods, the optimal parameters of the extraction regime were selected, conditions under which the quality and yield of pectin were superior. Samples of AP (60 ± 1 g) were placed in glass containers along with the aqueous solution of citric acid. By adjusting the acid concentration in the extraction mixture to pH 1.5, 2, and 2.5 and the liquid-to-solid ratio (LSR) to 10, 15, and 20 (v/w), the samples were prepared in triplicate.
The extraction of pectin using the ultrasound-assisted extraction (UAE) method (ISOLAB Laborgeräte GmbH, Germany) was performed at a frequency of 37 kHz for 15 and 30 min at a temperature of 60 ± 1 °C.
The extraction of pectin using the microwave-assisted extraction (MAE) method was performed in a microwave oven (MS23F301TAS Samsung, Zhongshan, China) with a magnetron power of 450 W for 5 and 10 min.
The extracts obtained through UAE and MAE were cooled to room temperature, subjected to centrifugation at 4000 rpm for 10 min, and the supernatants were collected. The pectin was sedimented by adding a volume of 96% ethyl alcohol, in a ratio of 1:1 (v/v). The suspensions were placed in a refrigerator at a temperature of 4 ± 1 °C for 12 h, after which the pectin was separated by filtration through a cotton cloth. The sediment was washed twice with a 60% ethyl alcohol solution and subjected to drying in a convection dryer (SLW 115 SMART, Pol-Eco Aparatura, Wodzisław Ślaski, Poland) at a temperature of 55 ± 1 °C until reaching a moisture content of 4.8 ± 0.1%. Finally, 54 UAE pectin samples (27 samples extracted for 15 min and 27 samples extracted for 30 min) and 54 MAE pectin samples (27 extracted for 5 min and 27 extracted for 10 min) were obtained. Subsequently, the extraction yield of pectin was calculated.
2.5. Pectin Characterization
The equivalent weight (EW), methoxyl group (MeO) content, anhydrogalacturonic acid (AUA) content, and degree of esterification (DE) of the extracted pectin were determined according to the methods described in the literature [
54,
55].
2.5.1. The Preparation of Pectin Solutions for Titrimetric and Spectroscopic Analysis
First, 0.5 g of pectin was wetted with 5 mL of 96% ethanol, followed by the addition of 1.0 g of sodium chloride and carbon dioxide-free distilled water up to a volume of 50 mL. The mixture was thoroughly stirred for 10 min, then brought up to a volume of 100 mL with distilled water and left at room temperature for 30 min [
54,
55].
2.5.2. The Determination of Equivalent Weight (EW)
After 30 min, 25 mL of the pectin solution was taken and 2–3 drops of phenolphthalein were added. This was followed by the titration with NaOH solution—0.1 N—until a constant pink color was obtained for 30 s. The EW of pectin was calculated according to the method and formula proposed by Ranaganna [
54].
2.5.3. The Determination of the Methoxyl Groups (MeO)
To the neutral solution mentioned above, which was titrated for equivalent mass determination, 25 mL of 0.25 M NaOH was added. The mixture was thoroughly agitated and left for 30 min at room temperature in a stoppered flask. After that, 25 mL of 0.25 M HCl solution was added to the sample, and the excess acid was titrated with 0.1 N NaOH solution until a pink color appeared. For the calculation of MeO (%), the formula in [
54] was applied.
2.5.4. The Determination of the Content of the Anhydrogalacturonic Acid (AUA)
The volume of NaOH (mL) consumed for MeO and EW determinations was entered into the formula for AUA calculation [
55].
2.5.5. The Determination of the Degree of Esterification (DE)
The degree of esterification represents the content of esterified carboxylic groups in the pectin macromolecule. The values obtained above, for MeO (%) and DE (%), were entered into the formula in [
54] for calculating the AUA content.
2.6. The Production of Fruit Bars
For the production of fruit bars, apples, plums, and cherries were dried until reaching a moisture content of 11 ± 1% and then crushed. For 1000 g of fruit bars, the following quantities of dried fruits were used: apples—340 g, cherries—250 g, plums—200 g, rosehip powder—6 g, and apple pectin solution—150 g, from which one-third was used as a binding agent for the fruit mixture, while the remaining two-thirds were used for coating. For the production of bars, a sample of pectin extracted through MAE for 10 min, at pH~2, in the ratio LSR 20 (v/w) was selected. According to the results, the MAE method ensures a higher yield (13.54%). The selected pectin had optimal characteristics required for a binding and coating agent. The aqueous solution of pectin (4.0%) with the addition of citric acid (0.04%) was prepared by dissolving the pectin in distilled water at 40 ± 1 °C with continuous stirring until complete dissolution.
The composition was molded into rectangular bars with a mass of 35 ± 1 g and dimensions of 8.5 ± 2 cm in length, 3.0 ± 0.5 cm in width, and 1.3 ± 0.5 cm in height. The bars were subjected to three rounds of glazing. After each glazing, drying was carried out at a temperature of 82 ± 2 °C for 1 h. After the cooling stage, the glossy bars were packed in polyamide/polyethylene (PA/PE) vacuum pouches and stored in the dark, at a temperature of 18 ± 2 °C, for 360 days. The sensory, physicochemical, microbiological stability, and nutritional value analyses of the fruit bars were carried out on the 1st day, 90th day, 180th day, 270th day, and 360th day of storage.
2.6.1. Sensory Analysis of Fruit Bars
The sensory analysis of the fruit bars was conducted according to [
56], after every 3 months of storage, by 9 assessors using a 5-point scoring scale. The 5-point assessment system includes the following scores: 5—very good; 4—good; 3—satisfactory; 2—poor; 1—bad; and 0—very bad. Each evaluator was given a bar (35 g) and a sensory evaluation sheet with a list of descriptors. The appearance, shape, surface condition, consistency, color, smell, and taste were evaluated.
2.6.2. The Physicochemical Analysis of the Fruit Bars
During the storage period, every three months, the following physicochemical characteristics of the fruit bars were determined: pH value [
57], titratable acidity [
48], moisture content [
58], and water activity [
59]. The total polyphenols and flavonoids, as well as the DPPH antioxidant activity, were determined according to the methods described below.
2.6.3. The Microbiological Analysis of the Fruit Bars
For the microbiological analysis of the samples of fruit bars, the method for determining the count of aerobic mesophilic microorganisms and facultative anaerobes was used according to the standards in [
60,
61].
2.7. Preparation of Apple Pomace and Fruit Bar Extracts for Spectrophotometric Analysis
First, 0.5 g of sample (AP powder or ground bars) was placed into a volumetric flask and made up to a volume of 50 mL with a 50% (
v/v) aqueous solution of ethanol. The obtained samples were extracted using the ultrasound-assisted method (ISOLAB Laborgeräte GmbH, Eschau, Germany) at a frequency of 37 kHz and a temperature of 40 ± 1 °C, for a duration of 30 min. It was followed by centrifugation at 4000 rpm for 10 min, then separation and analysis of the supernatant (extract) [
62].
2.8. Total Polyphenols and Flavonoids by Folin–Ciocâlteu
The total polyphenol content (TPC) and flavonoid content (TFC) were determined spectrophotometrically using well-known methods [
63] with some modifications [
62].
The TPC was determined using the Folin–Ciocâlteu phenol reagent [
64] in relation to a calibration curve with gallic acid standard (0–500 mg/L, R
2 = 0.9977) and expressed in milligrams of gallic acid equivalents per 1 g of dried sample weight (mg GAE/g DW).
The TFC was determined with AlCl3·6H2O according to the quercetin (0–160 mg/L, R2 = 0.9972) calibration curve. The results were expressed in milligrams of quercetin equivalents per 1 g of sample DW (mg QE/g DW).
2.9. Total Tannins by Folin–Ciocâlteu
The total tannin content was determined by the Waterman and Mole method [
65], using the Folin–Ciocâlteu reagent. The results were calculated from a calibration curve using tannic acid (0–50 mg/L, R
2 = 0.9985) and expressed in milligrams of tannic acid equivalents per 1 g of DW of AP (mg TAE/g DW).
2.10. Total Carotenoids
For the determination of carotenoids, 2 g of the sample was extracted three times with 25 mL of a solution (1:1:1;
v:v:v, methanol: ethyl acetate: petroleum ether) using an ultrasound bath for 15 min. The filtrates were combined and analyzed spectrophotometrically according to the method described in [
66,
67].
2.11. Determination of DPPH Free Radical-Scavenging Activity
The method described by Paulpriya et al. [
68] was used to determine the antioxidant activity (AA). The DPPH radical-scavenging capacity was estimated for the hydroalcoholic or aqueous solutions of samples (AP powder, pectin, or ground bars) at a concentration of 5 mg/mL. The results were expressed in µmol Trolox equivalent (TE) per 1 g of dried weight of sample (µmol TE/g DW) from a calibration curve (0–500 µmol/L, R
2 = 0.9992) with Trolox.
2.12. Mathematical Modeling
To determine the influence of the pH of the ultrasonic and microwave extraction mediums on the pectin yield, the equivalent weight, the methoxyl content, the anhydrouronic acid content, the degree of esterification, the total polyphenol content, and the antioxidant activity, the MATLAB program (MathWorks, Inc., Natick, MA, USA) was used. The mutual information values, measured in bits. The more pronounced the influence of the pH of the medium in extracts on the investigated results, the higher the bit value [
69].
2.13. The Statistical Analysis of the Results
The calculations in this research were performed in triplicate and are presented as mean values ± standard error of the mean. The Microsoft Office Excel 2007 program (Microsoft, Redmond, WA, USA) was used. One-way analysis of variance (ANOVA) according to Tukey’s test at a significance level of p ≤ 0.05 was carried out with Staturphics software, Centurion XVI 16.1.17 (Statgraphics Technologies, Inc., The Plains, VA, USA).
4. Conclusions
The research results demonstrated that the non-conventional methods of UAE and MAE represent sustainable and easily controllable processes for obtaining pectin with anticipated properties for various applications.
Pectin obtained from Golden Delicious apple pomace showed different EW, MeO, AUA and DE values, depending on the extraction conditions. The MAE method provided a maximum pectin extraction efficiency of 19.88%, while the UAE method—9.91%.
The TPC in the raw pectin matrix obtained by non-conventional methods varied from 0.22–1.31%. The DPPH radical inhibition capacity of pectin aqueous solution showed values from 4.32 to 18.86 μmol TE/g DW, this being largely dependent on the TPC. The AA of pectins obtained by MAE for 10 min were associated with a higher content of AUA, with a lower DE and EW, as well as with the content of terminal reducing groups, released during the polysaccharide degradation process. MAE pectin, selected for bar production, had the optimal characteristics required for a binding and coating agent. The evaluation of the physicochemical and sensory parameters of the fruit bars every 3 months, over a period of 12 months, demonstrated the protective effect of pectin: reducing moisture loss, minimizing the degradation of bioactive compounds during storage, and maintaining the potential antioxidant activity of the product.