Thermal extraction and characterization of pectin from semi-solid by-products of the olive oil industry

Research Article Received: 2 January 2025 Revised: 28 March 2025 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/jsfa.14316 Thermal extraction and characterization of pectin from semi-solid by-products of the olive oil industry Africa Fernandez-Prior,a,b * Luna Barrera-Chamorro,a,b Elvira Marquez-Paradas,a,b Carol López-de-Dicastillo,c Maria C. Millan-Linares,dAlvaro Villanueva-Lazodand Sergio Montserrat-de la Paza,b Abstract BACKGROUND: The valorization of agro-industrial by-products is crucial for promoting sustainability and circular economy. Olive mill semi-solid by-products (OMSbP), also known as alperujo, contain valuable bioactive compounds, including pectin, which can be extracted and used in food applications. However, the structural properties an antioxidant potential of these pectin require further characterization to assess their potential as functional ingredients. RESULTS: In the present study, pectin was extracted from OMSbP using a citric acid-based thermal extraction process. The yield varied depending on the olive variety and ripeness degree, with higher extraction efficiency in more mature samples (48.2 g kg −1 ). Structural analysis by attenuated total reflectance/Fourier transform-infrared spectroscopy confirmed a high homogalacturonan content (∼500 g kg −1 ) and a degree of methyl esterification of approximately 60%. The monosaccharide profile indicated enrichment in arabinose, rhamnose, galactose and mannose. Antioxidant activity, assessed through DPPH (i.e. 2,2-diphenyl-1-picrylhydrazyl) radical scavenging, showed a strong correlation with mannose content (r=−0.9967). Additionally, pectin-based films demonstrated thermal stability comparable to commercial pectin used in food packaging. CONCLUSION: This study highlights the potential of OMSbP-derived pectin as a biofunctional ingredient with antioxidant activity and thermal stability. The extraction method ensures a sustainable approach for utilizing olive industry by-products. Future research should explore its bioavailability and application in edible films and coatings. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. Keywords: olive oil semi-solid by-products; Olea europaea; structural characterization; antioxidant activity; food packaging INTRODUCTION The increasing environmental challenges humanity faces today are driving the development of more sustainable production and consumption systems. Biomass resources, as a result of their abundance, represent a valuable opportunity for the creation of high-value products. 1 In the olive oil sector, the two-phase extraction system generates a semi-solid by-product known as olive mill semi-solid by-product (OMSbP) or alperujo. 2-4 This by-product, primarily composed of olive peel, pulp, seeds and pit fragments, contains valuable bioactive compounds such as polyphenols and carbohydrates, including cellulose, hemicellulose and pectin. 5 Since the 1990s, OMSbP has played a role in reducing the environmental impact of olive oil extraction by minimizing liquid waste accumulation. However, its high moisture content presents challenges for efficient management. Recent industrial thermal pre-treatments have enhanced the extraction of bioactive *Correspondence to: A Fernandez-Prior, Department of Medical Biochemistry, Molecular Biology, and Immunology, School of Medicine, University of Seville, Av. Sanchez Pizjuan s/n, 41009 Seville, Spain, E-mail: mfer[email protected] (Fernandez-Prior) aDepartment of Medical Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Seville, Seville, Spain bInstituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Seville, Spain cPackaging Group, Instituto de Agroquimica y Tecnologia de los Alimentos, Spanish National Research Council (IATA-CSIC), Paterna, Spain dDepartment of Food & Health, Instituto de la Grasa, Spanish National Research Council (IG-CSIC), Seville, Spain © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 1 compounds, including hydroxytyrosol and various carbohydrate fractions. This progress marks a significant step toward establishing an OMSbP biorefinery, where optimizing carbohydrate extraction and utilization remains a key objective. Pectin, a complex heteropolysaccharide, is composed mainly of homogalacturonan (HG) domains and rhamnogalacturonan I (RG-I) regions with a galacturonic acid (GalA) backbone. The GalA units may be substituted with methyl groups, as some of the C-6 carboxyl groups are methyl-esterified. This esterification influences the structural characteristics and physicochemical properties of pectin, thereby determining its functionality in food and other industrial applications. Found in the primary cell wall and middle lamella of plant tissues, pectin contributes significantly to plant structure and integrity. 6 OMSbP-derived pectin has exhibited notable biological activities, including antioxidant, antiproliferative and anti-inflammatory effects, highlighting its potential as a functional ingredient. 7-12 The increasing demand for natural food additives is driving interest in sustainable sources of pectin. 13 The present study investigates the extraction and characterization of pectin from OMSbP using a citric acid-based thermal method, analyzing its structural properties, antioxidant capacity and potential applications in food technology. MATERIALS AND METHODS Raw material OMSbP (i.e. alperujo), a semi-solid by-product composed of olive peel, pulp, seeds, and pit fragments, was obtained from Almazara de la Subbetica S.C.A. (Cordoba, Spain). OMSbP samples were collected from two different olive varieties: Arbequina and Hojiblanca during the early harvest season of 2022–2023 (designated as aeOMSbP and heOMSbP, respectively). Additionally, samples were taken from a mixture of these varieties at two different ripening stages during the same harvest season: an early-stage sample (meOMSbP) and a fully mature sample (mmOMSbP). Samples were collected directly at the end of the horizontal centrifuge in the two-phase extraction system and stored at −20 °C until further processing. Preparation of cell wall material and pectin extraction OMSbP samples were processed following the Renard method with modifications to obtain extracted pectin. 14 Briefly, 1 L of 800 g kg −1 ethanol was added to 1 kg of fresh OMSbP, and the mixture was incubated overnight under continuous shaking at room temperature. After centrifugation at 15000 ×gfor 20 min (RC5C; Sorvall, Mechelen, Belgium), the supernatant was discarded, and the remaining alcohol-insoluble residue (AIR) was dried at 40 °C for 24 h. Pectin extraction was performed by suspending 30 g of AIR in 300 mL of 30 g kg −1 citric acid solution, adjusting the pH to 3.0, and stirring at 95 °C for 95 min. 15 The mixture was then centrifuged at 3700 ×gat 4 °C for 15 min, and the supernatant was treated with ethanol (2:1 v/v) and stored at 4 °C for 16 h to precipitate pectin. After an additional centrifugation step at 3700 ×gfor 30 min at 4 °C, the precipitate was washed three times with anhydrous ethanol and freeze-dried. Chemical characterization of extracted pectin Pectin yield was calculated as the ratio of the dry extracted pectin to the original AIR weight, expressed as g kg −1 dry OMSbP. The neutral sugar content (NSC) was determined using the Anthrone method, as described by Dishe. 16 Briefly, 100 μLof sample (triplicate) was mixed with 200 μL of 0.2% (w/v) anthrone in concentrated sulfuric acid, heated at 100 °C for 5 min, and measured at 630 nm in a microplate reader (iMark; Bio-Rad, Hercules, CA, USA). Glucose was used as the standard (0.02–0.2 mg mL −1 ). Uronic acid content was quantified using the m-hydroxydiphenyl method, as described by Blumenkrantz and Asboe-Hansen. 17 Samples (200 μL, triplicate) was mixed with 1.2 mL of 0.0125 Msodium tetraborate in sulfuric acid, heated at 100 °C for 5 min, and reacted with 20 μL of 0.15% mhydroxybiphenyl in 0.5% NaOH. Absorbance was measured at 520 nm using a galacturonic acid standard. Glycosidic composition was analyzed by gas chromatography (GC, HP6890 Plus; Hewlett Packard, Palo Alto, CA, USA) after the derivatization into alditol acetates, with initial trifluoroacetic acid hydrolysis using inositol as the internal standard. 18 Quantification was performed using standard solutions of L-rhamnose (Rha), L-arabinose (Ara), Dgalactose (Gal), D-glucose (Glc), D-mannose (Man), D-Fucose (Fuc) and D-xylose (Xyl). The chromatographic conditions were described by Lama-Muñoz et al. 19 The total phenolic content (TPC) was determined using the Folin–Ciocalteu spectrophotometric method with slight modifications. 20 Briefly, 20 μL of each sample was mixed with 100 μL of Folin–Ciocalteu reagent and 80 μL of a 0.70 Msodium carbonate solution in water. The mixture was stirred and incubated for 10 min to allow colour development. Absorbance was measured at 655 nm. A calibration curve was established using gallic acid as the standard. The protein content of the samples was determined using a nitrogen elementary analyzer (LECO, St Joseph, MI, USA), based on internal combustion at 1000 °C followed by thermal conductivity detection. The protein content (g kg −1 of dry extract) was calculated using the conversion factor 6.25, in accordance with the ISO method. 21 DPPH radical scavenging capacity Lyophilized pectin samples were dissolved in water at 10 mg mL −1 and filtered through a 0.45-μm nylon filter. The antioxidant activity was assessed using the 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging method, 22 where 5μL of each extract was added to 195 μL of 3.8 mg per 50 mL DPPH solution in methanol, incubated for 30 min at room temperature, and measured at 490 nm. The results were expressed as EC 50 values (mg mL −1 ), representing the concentration required to reduce radical activity by 50%. Degree of methyl esterification by ATR/FT-IR and 1 H NMR spectroscopy ATR/FT-IR spectroscopy was conducted to determine the degree of methyl esterification of the extracted pectin. Three replicates of each sample were analyzed, and peak intensity was used for measurement. This method provides a reliable qualitative and semi-quantitative assessment of pectin esterification, as reported by Muñoz-Almagro et al. 23 The samples were ground in an agate mortar for proper homogenization before analysis. Spectra were recorded using a FT/IR-4700 spectrophotometer (Jasco, Tokyo, Japan) across the range 400–4000 cm −1 . Degree of methyl esterification (DM) was calculated using the ratio of the absorption bands at 1730 and 1620 cm −1 :DM=A 1730 /(A 1730 +A 1620 ). 1D 1 H NMR spectroscopy of meOMSbP and mmOMSbP samples was also carried out to determine their structural characteristics. Each pectin samples was dissolved in D 2 O, and the 1 H NMR spectra were recorded at 25 °C using an Avance NEO 500 MHz spectrometer (Bruker, Billerica, MA, USA) equipped with a Onebay www.soci.org A Fernandez-Prior et al. wileyonlinelibrary.com/jsfa © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. J Sci Food Agric 2025 2 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License console featuring three radiofrequency channels and a BBFO 5 mm dual resonance Z-gradient probe. Surface morphology analysis by scanning electron microscopy (SEM) Lyophilized pectin samples were analyzed by SEM to examine surface morphology. The samples were sputter-coated with a thin layer of gold–palladium under vacuum using an EM ACE600 instrument (Leica Microsystems, Wetzlar, Germany) to improve conductivity. Imaging was performed with a Zeiss Crossbeam 550 scanning electron microscope (Zeiss, Madrid, Spain) at an accelerating voltage of 2.00 kV and different magnifications. 24 Thermogravimetric analysis of pectin films Pectin films from meOMSbP and mmOMSbP samples were obtained via the casting technique, where 20 g kg −1 pectin dispersions in distilled water were poured into Petri dishes and dried at 37 °C overnight. The resulting films (thickness: 45–55 μm) were analyzed for thermal stability using thermogravimetric analysis (TGA) Q-500 instrument (TA Instruments, New Castle, DE, USA). Approximately 5–8 mg of each sample was placed in alumina capsules and subjected to a temperature range of 30–800 °Cat a heating rate of 10 °C min −1 under nitrogen atmosphere (50 mL min −1 ). The maximum degradation temperatures (T d ) was determined. Statistical analysis Pearson's correlation coefficients were calculated to dentify significant relationships between pectin yield, chemical components, molecular features and antioxidant activity. Correlation heat maps were generated using the ggplot2 package (Wickham H, ggplot2: Elegant Graphics for Data Analysis. Springer-Ver-lag, New York (2016)). (v3.4.2). https://link.springer. com/book/10.1007/978-3-319-24277-4. Linear regression models were used to evaluate the predictive power of specific variables for antioxidant activity, with the coefficient of determination (R 2 ) used to assess model fit. All values are expressed as the mean ±SD. The number of replicates used for each measurement is explicitly stated to ensure transparency and reproducibility. Statistical analyses were performed using Prism, version 8.01 (GraphPad Software Inc., San Diego, CA, USA). Significance between sample groups was determined using a paired t-test for equal variances. P<0.05 was considered statistically significant. RESULTS AND DISCUSSION Chemical characterization of extracted pectin from OMSbP Extracted pectin from OMSbP using citric acid at 95 °C from different olive varieties (i.e., aeOMSbP versus heOMSbP) and ripeness stages (i.e., meOMSbP versus mmOMSbP) were fully characterized for its chemical composition, as shown in Table 1. Freezing the Table 1. Yield, chemical composition and molecular features of OMSbP pectin from two different olive varieties (aeOMSbP and heOMSbP) and two different ripeness stages (meOMSbP and mmOMSbP) Extracted pectin from olive mill semi-solid by-product (OMSbP) aeOMSbP heOMSbP P-value meOMSbP mmOMSbP P-value Yield (g kg −1 ) a,b 24.80 ±1.90 34.90 ±2.90 0.007 40.00 ±3.10 48.20 ±3.90 0.046 Proteins b 27.40 ±1.90 33.10 ±0.40 0.007 18.50 ±1.50 12.20 ±0.10 0.002 Phenols b 7.80 ±0.10 6.40 ±0.60 0.016 07.20 ±0.20 5.30 ±0.10 <0.001 Total carbohydrates b 715.40 ±9.70 570.10 ±7.10 <0.001 570.70 ±20.60 504.90 ±0.90 0.005 Monosaccharide composition c Galacturonic acid (GalA) 62.48 ±0.17 52.20 ±0.07 <0.001 51.63 ±0.29 50.03 ±0.16 0.001 Rhamnose (Rha) 1.59 ±0.10 1.71 ±0.04 0.126 1.95 ±0.24 0.69 ±0.03 0.001 Fucose (Fuc) 0.34 ±0.02 0.65 ±0.07 0.002 0.58 ±0.06 0.54 ±0.18 0.734 Arabinose (Ara) 7.55 ±0.48 9.05 ±1.41 0.156 10.30 ±0.28 13.58 ±3.04 0.136 Xylose (Xyl) 4.70 ±0.37 4.37 ±1.03 0.629 4.75 ±0.83 3.68 ±0.58 0.141 Mannose (Man) 2.81 ±0.14 3.89 ±0.34 0.007 4.24 ±0.24 4.79 ±1.49 0.562 Galactose (Gal) 10.71 ±0.96 9.81 ±1.62 0.454 12.97 ±2.10 13.04 ±1.25 0.963 Glucose (Glc) 9.82 ±0.67 10.62 ±1.67 0.484 13.58 ±1.07 13.65 ±1.20 0.943 Molar ratio d Homogalacturonan (HG) 60.89 ±0.10 50.49 ±0.04 <0.001 49.68 ±0.24 49.34 ±0.03 0.072 Rhamnogalacturonan I (RGI) 21.45 ±1.39 22.27 ±3.15 0.701 27.18 ±2.84 27.99 ±4.31 0.799 R1, linearity 1.80 ±0.11 1.47 ±0.24 0.096 1.18 ±0.12 1.11 ±0.11 0.498 R2, RG contribution 0.03 ±0.00 0.03 ±0.00 >0.999 0.04 ±0.00 0.01 ±0.00 <0.001 R3, length of side chains 11.48 ±0.17 11.01 ±1.57 0.633 11.93 ±0.40 38.59 ±5.97 0.002 DM (%) e 58.67 ±2.08 54.67 ±1.53 0.055 61.33 ±1.53 59.00 ±0.33 0.061 Data are expressed as the mean ±SD (n=3). a Based on AIR. b Expressed as g kg −1 of dry extract. c Relative %. d Molar ratios: exhibiting the primary structural properties of pectin molecules; HG =GalA –Rha, homogalacturonan; RGI =2(Rha) + Ara + Gal, R1 =GalA/(Fuc + Rha + Glu + Ara + Gal+Xyl), the linearity of pectin; R2 =Rha/GalA, the contribution of RG to pectin population; R3 =(Gal+Ara)/ Rha, the length of side chains attached to RGI. e Degree of methyl esterification (DM) determined by ATR/FTIR. Characterization of pectin from by-products of the olive oil industry www.soci.org J Sci Food Agric 2025 © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. wileyonlinelibrary.com/jsfa 3 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License raw material before citric acid extraction was crucial to prevent pectinolytic enzyme activity. The pectin extraction yield ranged from 24.8 g kg −1 (aeOMSbP) to 48.2 g kg −1 (mmOMSbP). A significantly higher yield was obtained from mmOMSbP (48.2 g kg −1 ) compared to meOMSbP (40.0 g kg −1 ), representing a 20% decrease in extraction efficiency at an earlier ripeness stage. However, these yield were lower than those reported for pectin from orange peel (approximately 200 g kg −1 ), which is the main agroindustrial waste used for the commercial production of pectin, 25 pomegranate peel (110 g kg −1 ), 26 young apples (80 g kg −1 ) 27 and sugar beet (160 g kg −1 ). 28 The protein content of the extracted pectin was within the range reported for commercial apple pectin (approximately 30 g kg −1 ) 29 and higher than that found in coffee pulp (approximately 10 g kg −1 ). 30 Additionally, the phenolic content of OMSbP pectin was higher than that of citrus pectin (1.5–1.8 g kg −1 ), but the similar to values reported for apple pectin (up to 6 g kg −1 ). 31 To assess the carbohydrate composition and degree of branching, the monosaccharide content was determined using gas chromatography. Significant differences were observed between different ripeness stages, with early-harvested eOMSbP samples showing a higher content of both uronic acids and neutral sugars. This resulted in a preferential loss of total carbohydrates from the polymeric cell wall in late-harvested samples, particularly Rha and Xyl, which contribute to arabinoxylan structures in the cell wall. As shown in Table 1, OMSbP samples contained seven primary monosaccharides: Rha, Ara, Gal, Glu, Xyl, Fuc and Man, in varying proportions. GalA was the dominant monosaccharide, with levels ranging from 500.3 g kg −1 (mmOMSbP) to 624.8 g kg −1 (aeOMSbP), confirming its high HG content. The GalA ratio exceeded 50% in all analyzed pectin samples, which is higher than the 35% reported by previous studies. 32 Rha, Gal and Ara together accounted for over half of the total neutral sugars, suggesting a high contribution of RG-I to the pectin structure. The HG content (49.34–60.43%) was greater than that of RG-I (21.45–27.99%), indicating that the linear backbone structure predominated over side chains. Pectin containing more than 74% GalA has been reported to influence gut microbiota composition and its microbial metabolites, 33 particularly through the activity of RG-I, which plays a role in modulating immune response. 34 In the present study, the R1 values of OMSbP pectin samples (1.11–1.80) were found lower than those reported for grapefruit peel pectin (2.52) 35 and pistachio green hull pectin (1.97), 36 indicating that OMSbP pectin has a higher degree of branching. However, the R1 value of aeOMSbP pectin (1.80) was higher than of sweet lemon peel (1.53), 37 highlighting variations in structural complexity among different sources. The R2 and R3 values further supported that the major contributions to pectin structure were from RG-I domains, with mmOMSbP exhibiting the highest degree of braching (38.59) compared to other samples (approximately 11). To better understand the relationship between the structural components and pectin properties, Pearson correlation coefficients were calculated (Fig. 1). Strong correlations (>0.95) were observed between multiple variables, including yield and total carbohydrates (−0.96), yield and arabinose (0.97), yield and mannose (0.99), yield and R1 (−0.97), and yield and DPPH (−0.98). Additionally, significant correlations were found between protein content and galactose (−0.96), phenols and xylose (0.95), total carbohydrates and galacturonic acid (0.98), total carbohydrate and mannose (−0.98), total carbohydrate and glucose (0.96), total carbohydrate and DPPH (0.99), rhamnose and R2 (0.99), rhamnose and R3 (−0.96), xylose and R2 (0.95), mannose and R1 (−0.97), glucose and rhamnogalacturonan I (0.99), glucose and R1 (−0.96), homogalacturonan and DPPH (0.96), and R1 and DPPH (0.97). The structural complexity and functionality of pectin are closely related. 38 Recent studies have modeled the correlation between pectin structure and its techno-functional properties. Xie et al. 39 established relationships between pectin characteristics and functional attributes using Pearson correlation coefficients, highlighting properties such as rheology, chewiness, adhesion, freeze–thaw stability and hardness. These results reinforced the notion that pectin conformation, monosaccharide composition, and molecular weight are the primary factors influencing its physicochemical behavior. Further studies have demonstrated the impact of pectin structure and its DM on gelatinization properties. 40 A study by Luo et al. 41 validated a multiple regression model demonstrating how variations in GalA content, molecular weight, DM and amidation degree influence gelatinization characteristics such as peak and valley viscosity, as well as setback and breakdown values. These findings underscore the significance of structural parameters in determining pectin functionality, further supporting the complex interplay between chemical composition and techno-functional attributes. Antioxidant activity and correlation with structural components Figure 2A presents the antioxidant activity of the extracted pectin, measured through DPPH radical scavenging. The EC 50 values indicate that meOMSbP exhibited a 6% higher free radical scavenging ability compared to mmOMSbP. This increase is directly proportional to the 7% higher phenolic content found in meOMSbP, confirming that the antioxidant activity of these extracts is primarily attributed to their phenolic compounds rather than the pectin structure itself. Phenols readily bind to sugars, transferring their antioxidant properties to these molecules. 7 The EC 50 values obtained for the extracted pectin derived from olive pomace by-products were lower than those reported for Spirulina sp., a microalga known for its notable antioxidant capacity. The most effective concentration was 2.5 mg mL −1 , and this activity has been mainly attributed to the presence of bioactive compounds such as phenolic acids, phycocyanins and tocopherols. 42 Sugars can also enhance the bioavailability of phenols, creating a synergistic functional relationship between the two compound groups. 43 This interaction is of high nutritional interest because it makes this extracted pectin from OMSbP more valuable for the formulation of health-oriented food products compared to commercial pectin that lack phenolic associations. To further investigate whether specific characteristics of the OMSbP-derived pectin, such as yield, chemical composition and molecular structure, influence its antioxidant capacity, Pearson correlation coefficients were calculated. Figure 2B demonstrates that DPPH exhibited strong correlations (>±0.5) with multiple variables, both positive and negative. The strongest correlation was found between DPPH and mannose content (r=−0.9967). To assess the significance of this relationship, a simple linear regression model was applied, yielding the equation y=2.3805–0.1991x, where ‘y’represents antioxidant activity and ‘x’represents mannose concentration. The model confirmed that mannose had a significant impact (P<0.05) on DPPH, explaining 99% of the variance in the data (R 2 =0.9901). This suggest that increasing mannose content in extracted pectin could enhance their antioxidant properties. Previous studies have shown that the antioxidant activity of oligogalacturonides is closely related www.soci.org A Fernandez-Prior et al. wileyonlinelibrary.com/jsfa © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. J Sci Food Agric 2025 4 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License to their structural attributes, including monosaccharide compositions and molecular weight. 44-47 The antioxidant mechanisms of pectin are intricate, and further research is needed to explore the relationship between polysaccharide structure and antioxidant function in more depth. 48 Degree of methyl esterification of extracted pectin from OMSbP The ATR/FT-IR spectra were analyzed to determine whether the lyophilized OMSbP pectin extracted from different olive varieties and ripeness stages affected functional groups and bonding configurations (Fig. 3). Overall, slight variations were observed in the spectral profiles of extracted pectin from the Arbequina variety (aeOMSbP) (Fig. 3A, green line) and the Hojiblanca variety (heOMSbP) (Fig. 3A, orange line). Additionally, differences were noted between early-season (meOMSbP) (Fig. 3B, pink line) and lateseason (mmOMSbP) (Fig. 3B, blue line) pectin samples. The broad absorption band at 3400 cm −1 , corresponding to O H stretching vibration of hydroxyl groups, appeared sharper in mmOMSbP compared to meOMSbP, likely because of a lower phenolic content (Table 1). 49 No significant differences were detected between the two olive varieties, aligning with precious findings, and potentially attributed to the high dissociation constant of the citric acid used during extraction. 50 The absorption band at 2925 cm −1 attributed to C H(—CH, —CH 2 ,—CH 3 ), remained consistent across all pectin samples, suggesting that ripeness did not influence these functional groups. However, aeOMSbP exhibited a higher intensity in this region compared to heOMSbP. Notably, extracted pectin from late-season (mmOMSbP) and Hojiblanca olives (heOMSbP) displayed a decrease in the band near 1730 cm −1 , corresponding to C O stretching of the methylesterified carboxyl groups (COO-R), whereas there was an increase at 1620 cm −1 , associated with carboxylate ion (COO − ) stretching, indicating slight variations in the degree of DM, as reflected in Table 1. The absorption bands at 1146 cm −1 and 1405 cm −1 , associated with C—OH stretching in side groups and C—O—C glycosidic bond vibration, respectively, remained unchanged across all samples, indicating consistency in glycosidic linkages. 51,52 Similarly, characteristic bands at 1250 cm −1 ,(—CH 3 CO stretching), 957 cm −1 (C O bending), 912 cm −1 (rocking mode of CH 3 ) and 820 cm −1 (—CCH and —COH bending at the C-6 position) were maintained, regardless of ripeness stage or olive variety, reinforcing the structural stability of extracted pectin. 1 H NMR analysis The 1 H NMR spectra (Fig. 4) of the extracted pectin from meOMSbP and mmOMSbP were acquired at 500 MHz to further Figure 1. Pearson correlation coefficient heatmap showing relationships among structural, conformational characteristics and antioxidant capacity of pectin. The color gradient represents the Pearson correlation coefficient, with values displayed within each square. Characterization of pectin from by-products of the olive oil industry www.soci.org J Sci Food Agric 2025 © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. wileyonlinelibrary.com/jsfa 5 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License corroborate and clarify their structures features. Spectra were recorded at 60 °C using samples dissolved in D 2 O, aiming to reduce sample viscosity. Measuring at this temperature also had the added benefit of shifting the residual water signal to approximately 4.4 ppm, avoiding overlap with relevant pectin signals. The most relevant signals appear in the 3.0–5.5 ppm region, corresponding to the skeletal and anomeric protons of the monosaccharides. Pectin samples with a high degree of methylation, such as those analyzed, typically display anomeric proton peaks around 5 ppm. In particular, anomeric H-1 and H-5 (COO − ) protons are observed at ⊐5.3 ppm and ⊐4.8 ppm, respectively, characteristics of GalA residues linked via ⊍-1,4 glycosidic bonds. Additionally, a peak at 4.6 ppm corresponds to H-4 protons, whereas signals at approximately 4.2 and 4.1 ppm are Figure 2. (A) DPPH radical scavenging activity (EC 50 ) of pectin samples. Values (mg mL −1 ) are expressed as the mean ±SD (n=3). Statistical significance between varieties and ripeness stages is indicated by P-value. (B) Correlation analysis between chemical composition and antioxidant capacity (EC 50 ,mgmL −1 ). The bar length represents the Pearson correlation coefficient, with values displayed with the bars. www.soci.org A Fernandez-Prior et al. wileyonlinelibrary.com/jsfa © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. J Sci Food Agric 2025 6 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Figure 3. Fourier transform-infrared spectroscopy (ATR/FT-IR) spectra of lyophilized OMSbP pectin extracted from (A) different olive varieties and (B) different ripeness stages. Arbequina variety (aeOMSbP, green line), hojiblanca variety (heOMSbP, orange line), early-stage ripeness (meOMSbP, pink line) and mature-stage ripeness (mmOMSbP, blue line). Figure 4. 1D 1 HNMR spectra of extracted pectin from meOMSbP and mmOMSbP recorded at 500 MHz in D₂Oat60°C. Characterization of pectin from by-products of the olive oil industry www.soci.org J Sci Food Agric 2025 © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. wileyonlinelibrary.com/jsfa 7 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License attributed to H-3 and H-2 protons, respectively, in agreement with a previous study. 53 The region between 3.0 to 4 ppm shows overlapping signals assigned to galactose, arabinose and rhamnose ring protons, suggesting the presence of arabinogalactan structures or pectic side chains. Moreover, signals around 1.0–1.5 ppm are likely associated with the methyl groups of rhamnose, indicative of branched regions within the pectin structure. Notably, a series of doublet of doublets of doublets (ddd) signals observed between ⊐2.7 and ⊐2.9 ppm may be related to the binding of pectin to the citric acid used during extraction. These signals are attributed to methylene protons of citric acid, as previously suggested by Kermani et al. 54 This spectral profile confirms the complex composition of the extracted pectin, which comprise a GalA backbone with potential substitutions by neutral sugar. Surface morphology analysis by SEM The surface morphology of lyophilized OMSbP pectin extracted from different olive varieties (Fig. 5A–D) and at different ripeness stages (Fig. 5E–H) was analyzed using SEM (Fig. 5). The SEM images revealed structural differences between pectin samples. In the eOMSbP samples, degradation into smaller fragments was observed after the harvest season, leading to a reduction in the particle size under identical imaging conditions (Mag =50×and AV =2.0 kV/Mag =250×and AV =2.0 kV). Extracted pectin from aeOMSbP exhibited a smooth surface, likely because of its higher HG content and fewer structural disruptions. By contrast, mOMSbP pectin displayed a distinct structure with visible long cracks on the surface. This heterogeneous architecture, characterized by rough, compact and flaky structures, may be attributed to a lower content of acidic sugar side chains, which influences the organization and mechanical properties of the extracted pectin. 23 Thermal stability of OMSbP pectin-based films TGA and its derivative (DTGA) curves (Fig. 6A,B) provide critical insights into the thermal stability and decomposition behavior of pectin-based films derived from OMSbP, specifically from different ripeness stages. Understanding these thermal transitions is essential not only for evaluating processability under heat, but also for predicting their behavior in end-use applications such as food packaging, edible films or controlled-release systems. As shown in Fig. 6A, both pectin samples exhibited a similar thermal degradation profile, with minor differences more evident in the DTGA curves (Fig. 6B). The degradation followed a typical three-stage pattern, characteristic of polysaccharide-based materials. 55,56 The initial mass loss, observed around 93 °C, corresponded to the evaporation of free and bound moisture, reflecting the hygroscopic nature of pectin and the presence of hydroxyl groups capable of forming hydrogen bonds with water. 35 This step is particularly important in edible films because moisture retention can affect mechanical integrity and shelf-life performance. The second degradation stage, occurring at approximately 233 °C, was associated with the depolymerization and breakdown of the HG backbone, which is mainly composed of GalA units. Additional decomposition processed were related to the cleavage of glycosidic linkages and the decarboxylation of GalA. Thermal resistance in this phase may be influenced by the degree of esterification, molecular weight and presence of impurities or residual phenolic compounds coextracted from the olive by-product matrix. 57,58 A third major decomposition peak, observed around 443 °C, likely corresponds to the breakdown of more thermally stable fractions and the oxidative degradation of carbonaceous residues. The presence of a minor shoulder in the DTGA curves suggests partial carbonization and residual char formation, potentially as a result of lignocellulosic fragments or phenolic residues commonly retained in extracted pectin from agro-industrial residues. 58 These findings indicate that OMSbP pectin exhibit thermal stability comparable to that of commercial pectin, supporting its potential use in thermally processed applications such as low-temperature drying, extrusion or lamination. 59 CONCLUSIONS The present study highlights the potential of extracted pectin from OMSbP as a functional and sustainable ingredient for food applications. Pectin obtained from early-stage ripeness OMSbP samples (eOMSbP) exhibited a higher GalA content and degree of methylation, along with distinct monosaccharide composition differences. Despite chemical variations among extracted pectin from different olive varieties and ripeness stages, they displayed significant antioxidant properties, likely attributed to their phenolic content and a strong correlation with mannose concentration. The thermal stability of OMSbP pectin-based films was comparable to that of commercial pectin, meeting key rheological Figure 5. Surface morphology of lyophilized OMSbP pectin extracted from different olive varieties and different ripeness stages. (A, B) Arbequina variety (aeOMSbP), (C, D) Hojiblanca variety (heOMSbP), (E, F) early-stage ripeness (meOMSbP) and (G, H) mature-stage ripeness (mmOMSbP), analyzed by SEM at two magnifications (Mag.). Upper SEM images were taken at Mag =50x and AV =2.0 kV (scale bar =100 μm), while lower SEM images were taken at Mag =250x and AV =2.0 kV (scale bar =30 μm). www.soci.org A Fernandez-Prior et al. wileyonlinelibrary.com/jsfa © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. J Sci Food Agric 2025 8 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License properties required in the food industry. The application of thermal treatment, similar to those being increasingly adopted by olive oil pomace processors, combined with citric acid extraction, enables the production of bioactive pectin with film-forming and heat-resistant properties. These characteristics support their potential use in food preservation and formulation. The findings of the present study confirm that thermal pre-treatments are an effective strategy for enhancing the extraction of bioactive compounds from olive pomace, contributing to the full valorization of this by-product. The pectin obtained demonstrated structural integrity, functional stability and antioxidant activity, reinforcing its suitability for incorporation into food products at the same time as promoting sustainability in the olive oil industry. Future research should focus on optimizing extraction conditions to further enhance pectin yield and bioactivity. Additionally, investigating the bioavailability and health effects of OMSbP-derived pectin in functional food applications could expand its potential in nutraceuticals and clean-label formulations. AUTHOR CONTRIBUTIONS SM-dlP was responsible for study conceptualization. AF-P, LB-C, CL-dD and AV-L were responsible for the methodology. AF-P, EM-P and SM-dlP were responsible for formal analysis. AF-P, CMM-L and SM-dlP were responsible for investigations. AF-P and SM-dlP were responsible for resources. AF-P and SM-dlP were responsible for writing the original draft. AF-P and SM-dlP were responsible for reviewing and editing. AF-P and SM-dlP were responsible for supervision. SM-dlP was responsible for funding acquisition. All authors have read and approved the final version of the manuscript submitted for publication. FUNDING This study was supported by the TED2021-130521A-I00 research project (Ministry of Science and Innovation, Government of Spain) into the Recovery, Transformation, and Resilience Plan funding by NextGenerationEU. ACKNOWLEDGEMENTS Africa Fernandez-Prior has the benefit of a Juan de la Cierva postdoctoral fellowship FJC2021-047485-I from the Spanish Ministry of Science and Innovation. Luna Barrera-Chamorro has the benefit of a PIF doctoral fellowship supported by the VII Program of Inner Initiative for Research and Transfer of University of Seville (VII-PPIT-US). Elvira Marquez-Paradas has the benefit of a FPU doctoral fellowship (FPU22/01097) from the Spanish Ministry of Science and Innovation. C. López de Dicastillo acknowledges the award of the Spanish government MCIU/AEI to the IATA-CSIC as Centre of Excellence Accreditation Severo Ochoa (CEX2021001189S/MCIU/AEI/10.13039/501100011033). CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest. DATA AVAILABILITY Data are available from the corresponding author upon reasonable request. REFERENCES 1 Reichembach LH, Guerrero P, de Oliveira Petkowicz CL and de la Caba K, Valorization of pectins from coffee wastes for the development of pectin-chitosan films. 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Food Bioprod Process 121:131–142 (2020). https://doi.org/10.1016/j.fbp. 2020.02.001. Figure 6. (A) Thermogravimetric analysis (TGA) curves and (B) their corresponding derivative TGA (DTGA) of OMSbP pectin-based films extracted from early-stage ripeness (meOMSbP, pink line) and mature-stage ripeness (mmOMSbP, blue line). Characterization of pectin from by-products of the olive oil industry www.soci.org J Sci Food Agric 2025 © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. wileyonlinelibrary.com/jsfa 9 10970010, 0, Downloaded from https://scijournals.onlinelibrary.wiley.com/doi/10.1002/jsfa.14316 by Readcube (Labtiva Inc.), Wiley Online Library on [17/06/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License