An Acid-Free Alternative to Pectin Production from the Cell Walls of Olive Oil Waste and Different Fruits Using Choline Chloride

Citation: Bermúdez-Oria, A.; Castejón, M.L.; Fernández-Prior, Á.; Rodríguez-Gutiérrez, G.; Fernández-Bolaños, J. An Acid-Free Alternative to Pectin Production from the Cell Walls of Olive Oil Waste and Different Fruits Using Choline Chloride. Foods 2023,12, 4166. https://doi.org/10.3390/ foods12224166 Academic Editors: Bruno Medronho and María JoséAliaño-González Received: 17 October 2023 Revised: 14 November 2023 Accepted: 15 November 2023 Published: 17 November 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article An Acid-Free Alternative to Pectin Production from the Cell Walls of Olive Oil Waste and Different Fruits Using Choline Chloride Alejandra Bermúdez-Oria †, María Luisa Castejón†,África Fernández-Prior , Guillermo Rodríguez-Gutiérrez and Juan Fernández-Bolaños * Department of Food Phytochemistry, Instituto de la Grasa (Spanish National Research Council, CSIC), Pablo Olavide University, Building 46, Ctra de Utrera km 1, 41013 Seville, Spain; [email protected] (A.B.-O.); [email protected] (M.L.C.); [email protected] (Á.F.-P.); [email protected] (G.R.-G.) *Correspondence: [email protected] †These authors contributed equally to this work. Abstract: The pectin from the cell walls of olive waste (alperujo) and apple, orange and strawberry fruits was extracted using choline chloride (ChCl) and the yield and chemical and structural compositions were compared to pectin extracted using citric acid (CA) and ammonium oxalate/oxalic acid (AOOA). According to the results, the alperujo pectin extracted using ChCl from alcohol-insoluble residue (AIR) showed a higher yield (2.20–2.88% on the basis of dry weight of AIR) than using CA (0.65–1.22%) but lower than using AOOA (3.92–5.42%). For fruit pectin, the highest yield was obtained using CA (8.81–16%), followed by AOOA (5.4–6.63%), although for apple pectin, ChCl gave a similar yield (5.36%) to AOOA. The uronic acid contents in all ChCl pectins (45.9–70.6% dry basis AIR) were higher or similar to that of the other extracting agents (30.6–65.2%), although a lower level of neutral sugar side chains was detected, with a lower degree of branching and degree of methylation. The NMR and FT-IR spectroscopy of the pectin isolated using ChCl confirmed its slightly different structural composition with respect to CA and AOOA pectin. Therefore, depending on the source material and functionality, pectin isolated using ChCl could be an acid-free alternative to pectin production. Keywords: pectin; choline chloride; alcohol-insoluble residue (AIR); cell wall; NMR and IR spectroscopy; deep eutectic solvents (DES) 1. Introduction Pectin is a heteropolysaccharide located in the middle lamella complex of the primary cell walls of fruits and vegetables [ 1 ]. It consists of galacturonic-acid-based polysaccharides, homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II and xylogalacturonan are the main types commonly isolated and purified [ 2 ]. HG is a linear structure of α -1,4-D-galacturonic acid units with poor solubility which can be partially methyland acetyl-esterified [ 3 ]. RG-I contains a backbone of alternating rhamnose and galacturonic acid residues, which can be branched by several neutral polymers such as arabinans, galactans, and arabinogalactans [ 4 ]. Pectin is widely used for its technological properties in the food industry as a gelling and stabilizer agent, thickener, and emulsifier, but it also presents functional properties associated with reducing the blood cholesterol level, improving immunity, and showing anti-tumor activity [ 5 ]. However, the chemical and macromolecular characteristics of pectin and its functional properties and potential uses depend mainly on the structural properties of the source of the native pectin plant and on the impact of the extraction method [ 6 ]. High-methoxyl (HM) pectin is widely used as a texturing agent for the production of jams and jellies, while low-methoxyl (LM) pectin can be used as a fat replacer in spreads, ice cream, emulsified meat, or low-calorie products, among others [7]. Foods 2023,12, 4166. https://doi.org/10.3390/foods12224166 https://www.mdpi.com/journal/foods Foods 2023,12, 4166 2 of 17 The interaction between pectin chains and pectin with other cell wall polysaccharides (cellulose and hemicelluloses) and proteins depends on various crosslinks. These include ionic bridges between HG chains via divalent ions (Ca++), covalent bonds between uronic acid and the hydroxyl group of the neighboring polysaccharide, hydrophobic interactions between methoxyl groups, and hydrogen bonds between the carboxyl and alcohol groups [ 8 ]. Therefore, pectin extraction is a complex process in which the molecular interaction conditions the hydrolysis and solubilization of different types of pectin from the cell wall, and is influenced by different factors such as solvent, temperature, pH, time, and material/solvent ratio [ 9 ]. Depending on the solvent used, hot water, chelating agents, dilute mineral acids (HCl, H 2 SO 4 , HNO 3 ), or organic acid (citric acid), the extractable pectin has specific structural domains which imply specific properties both from a technological and functional point of view [ 10 ]. Conventional extraction methods using dilute mineral acids, currently replaced by organic acids with a high acidity and high temperature, could influence the purity and quality of the extracted pectin. The use of chelators such as citric acid and oxalic/oxalate under acidic conditions has been considered a good pectin extracting agent with respect to the yield and physicochemical properties of the pectin [ 7 , 11 , 12 ]. However, the industry currently avoids the use of organic acid in pectin extraction because it is a more expensive process than mineral acid since organic acid requires a higher amount of acid to reach a certain pH. The process must be sustainable and economical [ 13 ]. For this reason, the search for an acid-free extraction process, in addition to hot water, with good yields and good physicochemical and bioactive properties is an ambitious challenge. Emerging technologies such as ultrasound, microwaves, supercritical water extraction, or enzymatic treatments have been developed to extract pectin, although their scale-up is problematic due to high investment costs [ 10 ]. In addition, new solvents with low-cost and environmentally friendly properties, such as deep eutectic solvents (DES), have been tested as potential solvents for pectin extraction. These solvents are eutectic mixtures composed of at least two components in a certain molar ratio, a hydrogen bond acceptor (HBA), most predominantly quaternary ammonium salt, and hydrogen bond donors (HBD) from a wide range of compounds such as sugars, organic acids, and/or alcohols. The hydrogen bonds between them are responsible for the formation of DES. These DES provide an additional molecular interaction with the wall material and deconstruct it, helping to extract pectin [ 14 , 15 ]. Although DES are cheap and relatively easy to prepare, they require mixing of the compounds in order to reach the eutectic point. It could be thought that these DES are safe and their toxicity and biodegradability is low. However, there is some controversy surrounding the greater negative effects depending on the composition, viscosity, and concentration in the eutectic mixture than in the individual components [ 16 ]. In addition, the high viscosity of DES and the fact that it is not easy to separate them from the final product can make their use difficult [ 9 , 10 ]. In the case of carboxylic acid– choline chloride (ChCl)-based DES, the most commonly used for pectin extraction, their applicability is compromised because they are degraded by the esterification reaction between the carboxylic acid and the alcohol moiety of the ChCl [ 17 ]. Also, some authors suggest the possibility of using an aqueous solution of choline chloride to fractionate the lignocellulosic biomass, extracting lignin and improving the hemicellulose-derived stream, instead of pre-treatments using DES comprising ChCl and carboxylic acids [18,19]. In this study, the possibility of using quaternary ammonium salt ChCl as an inexpensive, available, non-toxic, low-viscosity agent with a good hydrogen bonding capacity in the isolation of pectin is examined. We investigated the use of ChCl alone, and not in the eutectic mixture, as well as whether any interaction between the ChCl and certain components of the complex cell wall via hydrogen bonds, as is the case with eutectic solvents, could favor pectin solubilization. In preliminary assays, the application of ChCl to olive waste (alperujo) revealed the extraction of pectic polysaccharide, which presented bound phenols [ 20 ]. Since the polyphenols attached to pectin can influence, especially in the case of alperujo, the purity and quality of the extracted pectin, we investigated in this work the use of ChCl for the Foods 2023,12, 4166 3 of 17 isolation of pectin from the cell wall without interference. We tested the extraction of pectin from the cell walls as alcohol-insoluble residue (AIR), which reduces the content of extractives, removing part of the phenols, pigments, and minerals, among others. To achieve this objective, taking into account significant variation among olive waste, three different olive waste samples (ALP-1, ALP-2, ALP-3) were used. In addition, in order to compare this behavior of ChCl in the extraction of pectin from alperujo with other plant sources, the ChCl pectin extraction from three different types of fruit (apples, oranges, and strawberries) was also assessed. In all cases, the yield and the chemical and structural compositions of the ChCl pectin extracted, as a measure of the quality of the pectin, was compared to two commonly used pectin extraction agents such as citric acid (CA) and ammonium oxalate/oxalic acid (AOOA) [ 7 , 11 , 21 ]. Therefore, our aim was to confirm these preliminary results and evaluate whether ChCl is a potential extracting agent of cell wall pectin which improves the selectivity and purity of the extracts. 2. Materials and Methods 2.1. Raw Material Two different fresh samples of olive waste (alperujo) (a semi-solid residue composed of olive peel, pulp, seeds, and ground stones) were collected, at the beginning (ALP-1) and in the middle (ALP-2) of the harvesting season, directly from the end of the horizontal centrifuge of the two-stage extraction system from an experimental virgin oil mill of the Instituto de la Grasa (CSIC) (Seville, Spain). The third sample of alperujo (ALP-3) was supplied by a pomace oil extraction factory (Marchena, Seville, Spain) after a certain period (unknown) of storing the olive paste. In all three cases, the samples were stored at 4 ◦ C until used. The oranges, apples, and strawberries were purchased from a local market (Seville, Spain) and were used immediately. 2.2. Chemicals The choline chloride was purchased from Thermo Scientific (Kandel, Germany). The citric acid was obtained from Merck (Darmstadt, Germany). The oxalic acid was purchased from Acros Organics (Morris Plains, NJ, USA) and the ammonium oxalate, galacturonic acid, m-hydroxydiphenyl, trifluoroacetic acid, rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, dialysis bags of cellulose with a molecular cut-off of 12,000 Da, and alcohol oxidase were purchased from Sigma-Aldrich-Fluka (St. Louis, MO, USA). 2.3. Preparation of Cell Wall Material Alcohol-insoluble residue (AIR) from the fruits and the three samples of alperujo was obtained using the cold alcohol-insoluble residue method of Renard (2005) [ 22 ]. Briefly, the apples and orange were peeled and the calix or green hull was removed from the strawberries. All the fruits were cut into 2–3 pieces and the alperujo samples (1.0–1.5 kg) were suspended in 2–4 L of 96 mL/100 mL of ethanol solution (volume fraction), depending on the moisture of the sample, at room temperature and directly ground in a domestic blender for 60 s and filtered through nylon cloth. The resulting paste was suspended in 500 mL of 70 mL/100 mL ethanol solution and ground for bursts of 15 s and filtered and washed with additional ethanol (70 mL/100 mL) on a nylon cloth. This process was repeated until the filtrate had no color. This solid was dried using solvent exchange with 96% ethanol three times, and pure acetone three times, and kept overnight in an oven at 40 ◦C. 2.4. Extraction of Pectin from the Cell Wall The extraction methods were carried out using choline chloride (ChCl), citric acid (CA), and ammonium oxalate/oxalic acid (AOOA). For the latter two, the conditions used in the literature were followed [9,11]. In all cases, the treatments were carried out in duplicate. The AIR of three different olive waste (alperujo) samples and the apple, orange, and strawberry fruits were macerated using ChCl at 60 ◦ C for 18 h in order to ensure good Foods 2023,12, 4166 4 of 17 contact between the ChCl and the cell wall component (pectin), with the formation of possible DES or at least to form hydrogen bonds that facilitate their extraction. In the second step, pectin extraction from the ChCl samples was carried at 80 ◦ C for 2 h and compared to two commonly used methods for pectin extraction using organic acids of a chelating nature, such as citric acid (CA) and ammonium oxalate/oxalic acid (AOOA) in the same conditions. 2.4.1. Pectin Extraction with Choline Chloride (ChCl) Approximately 25 g of AIR from the alperujo samples (ALP-1, ALP-2, ALP-3) and 25 g of ChCl were mixed in 250 mL of deionized water and kept in maceration at 60 ◦ C with continuous agitation in a rotatory evaporator, without a vacuum, for 18 h. The AIR samples obtained from fruit (apple, orange, and strawberry) were processed in a similar way. The procedure was followed according to the preparation of natural deep eutectic solvent (NADES) using the heating method described by Dai, van Spronsen, Witkamp, Verpoorte, and Choi. (2013) [ 23 ], although with some modifications. In this case, we prolonged the maceration time, with gentle stirring and rotational agitation in a round-bottomed flask, in order to ensure good contact between molecules. Subsequently, the mixture was treated at 80 ◦ C for 2 h in a shaking water bath. After the extraction solutions were centrifuged at 3000 × gfor 15 min, the liquids obtained were precipitated with ethanol to a final concentration of 80%, and the mixture was kept at 4 ◦ C overnight. The samples were then centrifuged at 3000 × gfor 10 min. The precipitated pectins were redissolved in deionized water and subsequently dialyzed for removing phenols, pigments, monosaccharides, and other low-molecular-mass components. This dialysis was carried out against deionized water for 48 h, using dialysis bags of cellulose with a molecular cut-off of 12,000 Da, with water changed every 12 h. Finally, the pectins were lyophilized at a reduced pressure of 0.3 mbar and at − 50 ◦ C. The yield was determined gravimetrically and expressed as the ratio of pectin mass obtained to the initial mass of AIR. 2.4.2. Pectin Extraction Using Citric Acid (CA) and Ammonium Oxalate/Oxalic Acid (AOOA) Approximately, 25 g of AIR from the alperujo samples (ALP-1, ALP-2, ALP-3) was added to 1 L of deionized water, corresponding to the ratio 1:40 (AIR: water), and then the pH was adjusted to 2.2 using 1.9 g of citric acid as the extracting agent or the pH adjusted to 3.4 via the addition of 16 g of ammonium oxalate and 4 g of oxalic acid as extracting agents. The same procedure was used for the AIR obtained from the fruit samples (apple, orange, and strawberry). The extraction process was carried out at 80 ◦ C for 2 h in a shaking water bath. The supernatants were obtained using centrifugation and the precipitated pectins as previously described in Section 2.4.1. 2.5. Chemical Characterization of Pectin Extracts An estimation of the galacturonic acid (Gal A) content was obtained via hydrolysis of the pectin with concentrated sulfuric acid, followed by quantification of the Gal A in a standard solution according to the spectrophotometric method described by Blumenkrantz and Asboe-Han for uronic acids [ 24 ]. It is worth noting the very high proportion of Gal A with respect to the glucuronic acid found in alperujo samples, which has been described in previous work [ 25 ]. Also, for the other pectins from the fruit samples, which are composed mainly of galacturonic polymers, analysis of the uronic acid was the method to estimate the galacturonic acid present in the samples. To determine the individual neutral sugar composition, the pectin samples were hydrolyzed using 2 N trifluoroacetic acid (TFA) at 121 ◦ C for 1 h and then further converted into alditol acetates (reduction and acetylation). The resulting alditol acetates were analyzed using gas chromatography (GC) [ 26 ], inositol as internal standard, and an HP-5890 series II gas chromatograph fitted with a 30 m ×0.25 mm fused silica capillary column (SP-2330 from Supelco, Bellefonte, PA, USA). The chromatographic conditions of GC were described by Lama-Muñoz, Rodríguez-Gutiérrez, Rubio-Senent, and Foods 2023,12, 4166 5 of 17 Fernández-Bolaños, (2012) [ 25 ]. The molar constituent composition of each pectin extract was used to calculate the proportion of the different pectin structural domains. The pectin backbone is composed of HG (100% Gal A) and RG I ((Rha): GalA of 1:1). Therefore, the relative molar ratio of Rha and GalA inferred that HG = GalA − Rha and RGI = GalA − HG + Rha [10]. The total sugar content was determined as the sum of rhamnose (Rha), fucose (fuc), arabinose (ara), xylose (xyl), mannose (man), galactose (gal), and glucose (glc) on a weight basis. The total phenolic content was estimated using the Folin–Ciocalteu spectrophotometric method and expressed as grams of gallic acid equivalents [ 27 ]. The total protein content was determined using the Bradford method [ 28 ], using the Coomassie Brilliant Blue G-250 reagent. Absorbance was measured at 595 nm. Bovine serum albumin was used as the standard. All the colorimetric measurements were carried out using the iMark ™ Microplate Absorbance Reader (Bio-Rad Laboratories, Madrid, Spain). The degree of methylation (DM) was measured via the estimated methanol released by the saponification of pectins with 2M NaOH at 20 ◦ C for 60 min [ 29 ]. The amount of methanol released was measured enzymatically using alcohol oxidase (0.01 unit/ µ L in 100 mmol/L citrate buffer, pH = 6.5) and a spectrophotometric method, as described previously by Galanakis, Tornberg, and Gekas, (2010) [ 30 ], using a standard solution containing methanol for calibration. The DM was calculated as the moles of methanol measured per mole of uronic acid. The molecular characteristics were determined using high-performance size-exclusion chromatography (HPSEC), using Jasco equipment (LC Net II ADC, Kyoto, Japan) equipped with a refractive index detector (Jasco RI-1530) and a TSKgel GMPWXL (dextran MW < 50,000 KDa) column (300 × 7.8 mm i.d., Tosoh Bioscience LLC, King of Prussia, PA, USA). The samples were eluted using distilled water at a flow rate of 0.5 mL/min containing 0.01M trifluoroacetic acid. The system was calibrated with dextrans of 252, 110, 70, and 40, 6 kDa, and glucose. 2.6. Spectroscopic Characterization of Pectin Extracts The infrared spectroscopic spectra (FT-IR) of the pectin were determined using a Bruker Invenio spectrophotometer using a platinum attenuated total reflectance (ATR) accessory, equipped with a diamond crystal (Bruker Optics GmbH & Co.KG, Ettllingen, Germany). The spectra were acquired by scanning from the frequency range of 400–4000 cm−1, with a resolution of 4 cm−1resolution and an average of 16 scans per sample. 1H NMR spectroscopy was performed at 25 ◦ C and pH 4.5–4.8 for the ChCl pectin extract, using a Bruker Avance NEO 500 spectrometer equipped with a BBFO 5 mm dual resonance Z-gradient probe and using D2O as the solvent. Chemical shifts were expressed in δ units (ppm) relative to the solvent peak (D2O) assigned at 4.79 ppm. The conditions used for the experiments were 500 MHz; pulse power 90 ◦ ; relaxation delay 10 s; number of scans 32; number of dummy scans 4; and acquisition time (AQ) 3.41 s. 2.7. Statistical Analysis The STATGRAPHICS ® plus 4.0 software was used for statistical analysis. The results were expressed as mean values of analytical triplicates ± standard deviation. Analysis of variance (ANOVA) as well as the least significant difference (LSD) method were applied to compare the means. Results were statistically significant at a p-value < 0.05 calculated at a confidence level of 95%. 3. Results and Discussion 3.1. Extraction Yields and Chemical Characterization of Pectin Extracted Using Choline Chloride (ChCl) Tables 1a and 1b show the yield and chemical composition of the extracted pectins. The yields on AIR dry weight basis of the three alperujo pectins obtained using ChCl ranged from 2.2 to 2.9. kg/100 kg, and showed a 30 to 70% higher yield than using CA. Foods 2023,12, 4166 6 of 17 However, the highest yield found (3.9–5.4 kg/100 kg) was obtained using AOOA, which is 45 to 60% higher than ChCl extraction. The efficiency of chelating agents for pectin extractions depends on the Ca 2+ content and the distribution of free acid groups in the HG chain [ 7 ]. The extraction efficiency in the case of apple, orange, and strawberry pectin was different among them and with respect to olive waste. In these three cases, the percentages of pectin obtained using CA were the highest, and exceeded 40% for apple and strawberry and almost 70% for orange with respect to ChCl extraction. The strong acid condition improves the release and dissolution of pectin from the cell wall [ 31 ]. However, the yield of pectin extracted from apples using ChCl was even slightly higher than that of the AOOA agent (p< 0.05). These results indicate that ChCl interacts with the cell wall, which is a priori a good pectin extracting agent and is influenced by the cell wall of the plant source. The chemical composition of the extract obtained using organic acids (CA and AOOA) from various sources together with that of ChCl for comparison are also shown in Tables 1a and 1b. The main components in all pectin samples were the uronic acid (UA) and neutral sugar (NS) contents, and, to a lesser extent, the protein and phenolic compounds. The value of UA in ChCl extraction ranged from 45.9 to 53.5 kg/100 kg (dry basis) (S.I.) for the olive waste pectin, and 70.6 kg/100 kg for apple, 61.7% for orange, and 68.6 kg/100 kg for strawberry pectins, which showed the highest proportion compared to those found by the two commonly used agents for pectin extraction. Statistical analysis of these UA values indicated that ChCl has a similar or significant effect (p< 0.05) with respect to the other extraction agents. Their high contents in UA and lower concentration of different organic compounds indicate the great purity of the pectin extracted using ChCl. In addition, although in some cases, the yields of the ChCl pectin decreased, the pectin quality was in accordance with the lower content of NS, which occurred in alperujo pectin as well as in the strawberry pectin, where, in this last case, the contents of phenolic compounds and proteins were also lower. The contents of UA and NS were very similar using the three extracting agents in the case of orange pectin, while the content of UA and NS in apple pectin extracted using ChCl was highest in comparison to the other organic acid agents. Therefore, the results show that this greater or lesser purity of the pectin not only depends on the type of starting material but is also influenced by the extraction agent employed [7,32]. Although there are differences among the compositions of NS of all the pectic material, they all have arabinose as the main neutral sugar residue, except for strawberry pectin, with the galactose at a similar level or slightly higher than arabinose. In addition, arabinose together with other NS including rhamnose, fucose, xylose, and galactose, which comprise the branched region of pectin, showed a much lower content in the pectin obtained using ChCl than the one obtained using CA, except for orange pectin, which was very similar in both cases. Likewise, except for orange pectin, in all the other samples, the pectin obtained using ChCl contained a higher homogalacturonan (HG) proportion than the pectins obtained using CA and AOOA. Pectin with a higher proportion of arabinose and galactose was obtained from olive waste (alperujo) rather than from fruits, which could be part of the RG-I as arabinan, galactan, or arabinogalactan chains [ 33 , 34 ]. In addition, the presence of glucose and mannose in the alperujo pectins (ALP-1, ALP-2 and ALP-3) obtained using CA was higher than in the ones extracted using AOOA and ChCl, possibly indicating the extraction of non-pectic polysaccharides such as hemicelluloses together with the pectin molecule, or the presence of RG-II, although this RG-II has not been described in olive pomace pectin [33]. Foods 2023,12, 4166 7 of 17 Table 1. ( a ) Yield (kg/100 kg AIR), chemical composition (kg/100 kg extract), and neutral monosaccharide and uronic acid (UA) compositions (% molar ratio) of the ChCl, AOOA, and CA pectins obtained from three different alperujo samples (ALP-1, ALP-2, ALP-3) and apples, oranges, and strawberries. ( b ) Yield (kg/100 kg AIR), chemical composition (kg/100 kg extract), and neutral monosaccharide and uronic acid (UA) compositions (% molar ratio) of the ChCl, AOOA, and CA pectins obtained from apples, oranges, and strawberries. (a) ALP-1 ALP-2 ALP-3 CA AOOA ChCl CA AOOA ChCl CA AOOA ChCl Yield 1.22 ±0.01 a 3.92 ±0.07 b 2.20 ±0.08 c 0.95 ±0.001 a 5.24 ±0.21 c 2.88 ±0.39 b 0.65 ±0.01 a 5.42 ±0.18 c 2.28 ±0.09 b Uronic Acid 30.6 ±6.41 a 40.8 ±5.71 b 45.9 ±6.32 b 39.3 ±6.01 a 47.7 ±5.18 b 53.5 ±3.34 c 34.6 ±1.48 a 47.5 ±1.47 b 49.5 ±9.08 b Phenols 2.21 ±0.16 a 1.76 ±0.26 a 2.64 ±0.22 a 2.84 ±0.66 b 2.03 ±0.13 b 1.659 ±0.15 a 4.89 ±1.16 b 3.54 ±0.16 a 6.46 ±0.95 b Neutral Sugar 37.13 ±0.91 b 31.84 ±1.39 a 29.89 ±5.06 a 28.69 ±1.5 b 26.66 ±1.63 b 17.13 ±0.65 a 30.4 ±1.70 c 26.32 ±0.70 b 23.56 ±0.53 a Protein 0.31 ±0.005 a 1.21 ±0.022 b 1.56 ±0.013b 1.33 ±0.03 c 0.75 ±0.015 b 0.31 ±0.00 a 1.01 ±0.01 a 3.59 ±0.03 b 0.79 ±0.025 a Ash 7.01 ±0.02 b 5.47 ±0.08 a 4.3 ±0.03 a 5.58 ±0.07 b 3.56 ±0.02 a 5.05 ±0.02 b - - - Total 77.2 81.1 83.8 77.7 80.7 77.6 70.9 80.9 80.3 Total Carbohydrates 67.7 72.6 75.3 68.0 74.4 70.6 65.0 73.8 73.1 Rhamnose (Rha) 1.68 1.71 1.85 1.98 1.86 1.40 3.11 2.78 2.58 Fucose (Fuc) N.D N.D 0.03 0.03 N.D N.D N.D N.D N.D Arabinose (Ara) 29.9 28.0 18.5 18.1 22.5 13.8 23.6 22.7 21.3 Xylose (Xyl) 2.53 1.74 5.86 2.52 1.56 1.16 2.12 1.91 1.91 Mannose (Man) 1.73 1.20 1.47 1.98 1.02 0.94 1.58 1.29 1.08 Galactose (Gal) 16.4 9.78 9.95 13.3 7.46 5.89 13.9 13.6 7.85 Glucose (Glc) 2.59 1.37 2.03 4.20 1.50 1.41 2.54 2.42 1.29 Galacturonic acid (GalA) 45.1 56.2 60.3 57.8 64.1 75.7 53.2 64.3 67.7 DM * 71.2 42.4 40.9 85 67.2 50.85 59.8 43.75 33.7 HG (GalA-Rha) 43.4 54.4 58.4 55.8 62.3 74.3 50.1 61.6 65.2 RG I (GalA-HG + Rha) 3.36 3.42 3.7 3.96 3.72 2.8 6.22 5.56 5.16 Linearity (GalA/Rha + Fuc + Ara + Xyl + Gal) 0.89 1.36 1.66 1.61 1.92 3.40 1.25 1.57 2.01 Branching (Rha + Gal/GalA) 1.03 0.67 0.47 0.54 0.47 0.26 0.70 0.56 0.43 Foods 2023,12, 4166 8 of 17 Table 1. Cont. (b) Apple Orange Strawberry CA AOOA ChCl CA AOOA ChCl CA ChCl Yield 11.4 ±0.4 c 6.63 ±0.05 b 5.36 ±0.1 a 8.81 ±0.12 c 5.35 ±0.01 b 2.82 ±0.21 a 16.0 ±0.95 b 9.1 5 ±0.39 a Uronic Acid 42.5 ±5.5 a 52.0 ±6.0b 70.6 ±7.7 c 64.5 ±5.6 b 65.2 ±8.3 b 61.7 ±5.4 a 63.3 ±3.04 a 68.6 ±2.1 b Phenols N.D N.D N.D N.D N.D N.D 9.16 ±0.029 b 6.26 ±0.039 a Neutral Sugar 14.2 ±1.16 b 11.1 ±0.50 a 16.4 ±0.42 c 19.9 ±0.98 b 16.4 ±0.90 a 23.2 ±0.18 c 7.56 ±0.21 a 6.84 ±1.58 a Protein 1.04 ±0.15 a 1.23 ±0.10 a 1.74 ±0.03 b 0.94 ±0.04 a 2.24 ±0.23 b 2.20 ±0.43 b 3.66 ±0.44 b 0.69 ±0.21 a Ash 1.47 ±0.02 b 1.02 ±0.01 a 3.21 ±0.14 c 7.68 ±0.28 b 1.05 ±0.02 a 7.1 ±0.32 b 0.55 ±0.02 a 1.32 ±0.01 b Total 59.2 65.4 89.1 93.0 84.9 94.2 84.2 83.6 Total Carbohydrates 56.7 63.1 84.1 84.3 81.6 84.9 86.5 75.3 Rhamnose (Rha) 0.90 0.78 0.74 0.73 0.73 0.83 0.73 0.61 Fucose (Fuc) 0.45 0.43 0.49 0.35 0.25 0.43 0.22 0.28 Arabinose (Ara) 15.30 10.4 12.3 14.1 11.7 14.7 3.1 2.2 Xylose (Xyl) 2.32 1.51 1.45 0.91 0.53 0.93 1.03 1.58 Mannose (Man) 0.40 0.50 0.31 0.78 0.48 1.63 0.44 1.66 Galactose (Gal) 4.96 3.46 3.48 6.1 6.01 7.59 4.4 1.93 Glucose (Glc) 0.72 0.59 0.74 0.60 0.43 1.19 0.76 0.79 Galacturonic acid (GalA) 74.9 82.4 80.5 76.4 79.9 72.7 89.3 90.9 DM * 87.1 72.2 68.5 52.4 39.9 73.3 59.7 HG (GalA-Rha) 74.0 81.6 79.8 75.7 79.1 71.9 88.6 90.3 RG I (GalA-HG + Rha) 1.8 1.56 1.48 1.46 1.46 1.66 1.46 1.22 Linearity (GalA/Rha + Fuc + Ara + Xyl + Gal) 3.13 4.98 4.36 3.44 4.15 2.97 9.42 13.70 Branching (Rha + Gal/GalA) 0.27 0.17 0.20 0.26 0.22 0.31 0.08 0.05 * Degree of methylation (DM) was expressed in a mol MeOH/mol GalA. The data shown are mean ± standard deviation. Different letters indicate significant differences between each of extracting agents by each alperujo sample and by each fruit sample, while similar letters indicate no significant difference at a p-value < 0.05 calculated at a confidence level of 95%. Foods 2023,12, 4166 9 of 17 Taking into account the molar ratio of UA to NS, as a measure of the linearity of pectin [ 35 ], and the molar ratio of the sum of arabinose and galactose to UA as a measure of the degree of branching [ 36 ] for all samples, except orange pectin, the ChCl pectin showed much more linearity and was less branched than the pectin obtained using CA, and more closely related to the one obtained using AOOA, such as apple pectin. The strawberry pectin extracted using ChCl was the most linear with the fewest neutral sugar side chains, showing ratios of 13.7 for linearity and 0.05 for branching, which means that it is mainly a HG region. Whereas the pectin with the lowest linearity was the one extracted using CA from ALP-1, presenting a pectin ratio of 0.89 and the highest degree of branching with a ratio of 1.03, its linearity increased using ChCl to up to 1.92, and the degree of branching decreased to 0.47. Interestingly, these results are contrary to those found previously when the alperujo was treated directly using ChCl under the same conditions of this study, with lower UA content, a short backbone of the HG domain, and a higher proportion of RG-I than the one treated with CA [ 20 ]. ChCl, as a good proton acceptor, seems to act differently within the alperujo by forming hydrogen bonds with compounds which are not present in the isolated cell wall. In both cases, the mechanism of the intermolecular interaction between ChCl and certain compounds present in the cell wall or alperujo matrix will have to be investigated. Furthermore, as shown in Tables 1a and 1b, all isolated pectin had a moderate to high degree of methylation (DM), which ranged from 33.7% for alperujo pectin to 87.1% for apple pectin. However, newly isolated pectin using ChCl for all samples showed a DM which was significantly lower than that recovered from all sources using CA and AOOA. Although the mechanism by which the ChCl acts is unknown, the results suggest an effective solubilization of the highly demethylated pectin region, possibly via the hydrogen bond between the free carboxyl group of the pectin molecule and the ChCl, which facilitates the disruption of the cell wall and its extraction. Since the initial pH in the extraction of the pectins using ChCl ranged from 4.5 to 4.8, possible demethylation by high pH was not discounted [37]. The molecular-sized distribution of the pectin extracts obtained for each extracting agent as determined using high-performance size exclusion chromatography (HPSEC) is shown in Figure 1. Due to different molecular shapes or hydrodynamic volumes and the density of the pectins, it is difficult to ascertain their molecular mass using HPSEC without using a specific detection such as light scattering or on-line viscosity [ 38 ]. However, a refractive index (RI) detector was used for rapid characterization of the pectin molecular size (MS). The RI profiles of the three samples of alperujo pectin obtained using the three extracting agents tested (Figure 1) appear quite similar and they are composed of 3–4 recognized zones of macromolecule populations, by which only the relative amounts of each zone varied. However, major differences in a greater proportion of smaller molecular sizes could be observed when the different alperujo samples were compared among themselves, possibly being influenced by the action of the enzyme during the different storage times of the olive waste or different degrees of ripening of the olive fruits [ 39 ]. The elution patterns showed a high proportion of high MS material at retention times of 11–12 min, which was only degraded in the ALP-3 sample pectin (Figure 1) obtained using ChCl, with the formation of fragments with lower MS between 13.5–15.5 min. In addition, a shoulder-shaped zone can be recognized at 15.5 min, which is in the samples obtained using AOOA, and much softer. It was followed by a zone with the lowest MS at 19–21.5 min or even at 22.5–23.5 min, which was the pectin fragment obtained using AOOA, which was somewhat more resistant to degradation, while the fragments obtained using CA or ChCl were similar. Foods 2023,12, 4166 16 of 17 P18-TP-616) and by the Ministry of Science and Innovation through the National Oriented Research Projects, Spain (PID2022-142731OB-C21). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data are contained within the article. 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