Synthesis and characterization of succinylated pectin hydrogels with enhanced swelling performances

Synthesis and characterization of succinylated pectin hydrogels with enhanced swelling performances Stefano Liotino a,b,1 , Stefania Cometa c,1 , Stefano Todisco d , Piero Mastrorilli d , Carlos Bengoechea e , Antonio Salomone a,b , Elvira De Giglio a,b,* a Department of Chemistry, University of Bari Aldo Moro, Via Orabona 4, 70126 Bari, Italy b INSTM, National Consortium of Materials Science and Technology, Via G. Giusti 9, 50121 Florence, Italy c Jaber Innovation s.r.l., Via Calcutta 8, 00100 Roma, Italy d Department of Civil, Environmental, Territorial, Construction and Chemical Engineering “DICATECh”, Polytechnic University of Bari, Via Orabona 4, 70125 Bari, Italy e Chemical Engineering Department, University of Seville, Calle Virgen de ´ Africa, 7, 41011 Sevilla, Spain ARTICLE INFO Keywords: Pectin Succinylated pectin Hydrogel film Crosslinking Swelling ABSTRACT A novel polymeric material was obtained through succinylation of pectin (S-Pec), resulting in greater stability, film-forming ability, transparency, swelling, and water retention capacity compared to native pectin (Pec). Spectroscopic techniques confirmed the success of the succinylation reaction performed on pectin, employing the reaction of galacturonic acid with succinic anhydride as a model reaction under similar experimental conditions. Moreover, fluorinated succinic anhydride was used to gain insight into the succinylation degree by X-ray Photoelectron Spectroscopy, and a different thermal behavior of S-Pec compared to Pec was confirmed through thermoanalytical characterization. Additionally, the effect of cross-linking either Pec or S-Pec in the presence of divalent cations (i.e., calcium or magnesium ions) on water retention capacity and stability was tested. A significant improvement in the ability to absorb and retain water or saline solution was found for magnesiumcrosslinked succinylated pectin, while the in vitro hydrogel stability was higher for the calcium-crosslinked one. The obtained polymer represents a promising substrate for the development of natural-based superabsorbent polymers. 1. Introduction Nowadays, there is a growing interest in the development of natural biopolymer-based materials, which would be useful for different applications, such as medicine, agriculture, environment, food, clothing, energy storage, sanitary sector, etc., aligning with the principles of Green Chemistry and the 2030 Agenda for Sustainable Development [1]. The natural origin of the feedstocks employed could revolutionize those sectors through the development of advanced materials with a better final product quality that overcome the non-biodegradability of the traditional fossil-based polymers [2]. Moreover, the use of biopolymers from agri-food waste aids in the management and valorization of such waste, avoiding also an undesirable competition with food supplies. Pectin is a natural heteropolysaccharide, mainly based on D-galacturonic acid units linked by α (1,4) glycosidic bonds, that may be extracted from several food waste biomass (i.e., mainly citrus peels, apple pomace, sugar beet pulp) [3]. Depending on the food source and extraction approach used, it can be highly or partially esterified with methanol [4]. Additionally, the main chain can be interrupted by α (1,2)- linked L-rhamnose units, which may possess neutral sugars attached as well. This natural polymer has already found many applications in food [5], pharmaceutical and cosmetic [6], nutraceuticals [7], drug delivery [8], tissue engineering [9], wound dressing [10], or packaging industries [11]. This wide applicability is mainly based on its characteristic properties (swellability, emulsifying and/or thickening capacity) and its ability to form either films, beads, nanoparticles or hydrogels, when conveniently processed. On the other hand, superabsorbent polymers (SAPs), widely employed in diapers [12], sanitary napkins [13], water reservoirs [14], or nutrient carriers in agriculture [15], must display excellent swelling * Corresponding author at: Department of Chemistry, University of Bari “Aldo Moro”, via E. Orabona 4, 70126 Bari, Italy. E-mail addresses: [email protected] (S. Liotino), [email protected] (S. Cometa), [email protected] (S. Todisco), [email protected] (P. Mastrorilli), [email protected] (C. Bengoechea), [email protected] (A. Salomone), [email protected] (E. De Giglio). 1 These authors equally contributed to the work. Contents lists available at ScienceDirect Reactive and Functional Polymers journal homepage: www.elsevier.com/locate/react https://doi.org/10.1016/j.reactfunctpolym.2025.106331 Received 17 February 2025; Received in revised form 28 April 2025; Accepted 7 May 2025 Reactive and Functional Polymers 214 (2025) 106331 Available online 8 May 2025 1381-5148/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). properties with superior liquid retention ability. In this specific application, pectin, similarly to other native carbohydrates, typically performs poorly compared to the traditional synthetic polymers (i.e., polyacrylic derivatives). Thus, traditional SAPs can absorb 10–1000 g of deionized water per gram of dry SAP, whereas the absorption capacity of common hydrogels is typically lower than 1 g/g [16]. Some interesting water absorption features were reported by Yoshimura et al. [17] for crosslinked pectin hydrogels, but these failed when the absorption of saline solution (NaCl 0.9 % w/w) was tested. Moreover, SAPs should retain a significant amount of absorbed liquid and absorb liquid under external pressure to be applied in real devices. As this is unlikely to be exhibited by unmodified carbohydrates, the development of bio-based SAPs where physical or chemical modifications are carried out to improve their overall performance is pursued to reduce the existing gap between biopolymers and petrochemical materials. Chemical modifications of pectin involve predominantly the carboxylic acid found at the C6 position or the hydroxyl groups of pectin, producing different functional groups, such as ester [18], amide [19], acyl [20] or alkyl bonds [21]. To improve the swelling performance of polymers, a high quantity of ionizable groups is desirable, as the resulting higher electrostatic repulsion will tend to push the chains apart, leading to an increase in swelling. Among the ionizable groups that could be attached to the pectin molecule (e.g., carboxylate, phosphate groups, amine/amidic), carboxylates were selected considering sodium polyacrylate as model. Moreover, previous studies on phosphorylation of pectin did not supply hydrogels [22]. As far as amidated pectin is concerned, Feng et al. [23] demonstrated that the higher the degree of amidation, the lower the swelling of amidated pectin. On the other hand, succinylation of pectin, represented in Fig. 1, is a reaction that can increase significantly the number of carboxyl COO − groups in pectin chains, rather than to convert the COO − groups already present in pectin into other functional groups: In the literature, there are several examples of succinylation of different polysaccharides such as cellulose [24], starch [25], chitin [26], guar gum [27] or agar [28] but, to the best of our knowledge, no data were reported on succinylation of pectin. Once the reaction was performed, the obtained product was comprehensively characterized through different techniques (Fourier Transform Infrared Spectroscopy in Attenuated Total Reflectance mode (FT-IR/ATR), X-ray Photoelectron Spectroscopy (XPS), solid state and solution state Nuclear Magnetic Resonance (NMR)) to confirm the chemical modification of pectin. Additionally, the reaction between galacturonic acid sodium salt and succinic anhydride was carried out, as model reaction, in the same experimental conditions, to furtherly confirm the succinylation. Moreover, fluorinated succinic anhydride was used to gain information on the succinylation degree using XPS analysis. The use of fluorinated compounds is a well-known strategy that exploits the F1s signal as a marker element in XPS analysis to obtain the quantification of the marked groups on a polymer [29]. Once the succinylation was checked and confirmed, thermal properties of the product were assessed by Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). The swelling behavior of the succinylated pectin was also studied through free swelling capacity (FSC) and centrifuge retention capacity (CRC), and the results were compared to those of native pectin. Moreover, the effect of cross-linking was assessed with the techniques described for both the unmodified pectin and the succinylated product employing CaCl 2 or MgCl 2 . The resulting swelling and water retention capabilities were evaluated, extending the measurements up to 10 days, to evaluate the hydrogel stability. Viscoelastic properties of succinylated pectin and succinylated pectin crosslinked with magnesium were also evaluated. In conclusion, this study reports a valuable strategy to synthesize novel succinylated pectin-based hydrogels with improved gel characteristics, such as high transparency, film ability, stability and interesting swelling performances. 2. Materials and methods 2.1. Materials Pectin (viscosity 30.5 to 36.5, degree of esterification 70 %, Cat. 416,862,500, Thermo Scientific, Waltham, Massachusetts, USA) – coded as Pec – succinic anhydride (CAS number 108–30-5), 4-(dimethylamino) pyridine (CAS number 1122-58-3), D-Galacturonic acid sodium salt (CAS number 14984–39-5, purity >95.0 %), tetrafluorosuccinic anhydride (CAS number 699–30-9, purity >98 %) calcium and magnesium chloride (CAS numbers 10,043–52-4 and 7786-30-3, respectively) were purchased from Sigma-Aldrich (Merck, Milan, Italy). Hydrogel films were prepared with ultrapure water, obtained through a Milli-Q® distillation system (Millipore-Merck, Darmstadt, Germany). All solvents and reagents were purchased from Sigma Aldrich (Merck, Milan, Italy), unless otherwise specified. For polymer purification, dialysis tubing cellulose membranes with molecular weight cut-off equal to 14,000 were used. 2.2. Synthesis of succinylated pectin Pectin (0.5000 g) was dissolved in 50 mL of water under magnetic stirring. Then, DMAP (1.2557 g) and succinic anhydride (1.0351 g) were added to the mixture. The reaction was carried out 24 h at room temperature. The reaction mixture was basified to pH =8.20 with NaOH 9 % w/V. The product was precipitated with ethanol (50 mL) and filtered; the precipitate was washed with ethanol. The product was dissolved in water (10 mL) and dialyzed for 3 days. The product was dried at 50 ◦C for a few days to isolate the succinylated pectin (S-Pec) [30]. These reaction conditions were selected after optimization of the reaction conditions, reported in the Supplementary Data (Table S1 and Fig. S1). For the model reaction, D-Galacturonic acid sodium salt (0.5000 g) was dissolved in 50 mL of water under magnetic stirring. Then, DMAP (1.2557 g) and succinic anhydride (1.0351 g) were added to the mixture. The reaction was carried out 24 h at room temperature. Finally, succinylation with a fluorinated succinic anhydride was carried out following this protocol: pectin (0.0600 g) and DMAP (0.1160 g) in anhydrous CH 2 Cl 2 (3.0 mL) were cooled to 0 ◦C in an icewater bath. Then tetrafluorosuccinic anhydride (0.10 mL) was added to the solution dropwise and mixed for 1 h. The reaction was carried for another 12 h at room temperature under nitrogen. The solution was filtered and washed with ethanol to obtain the tetrafluorosuccinylated Fig. 1. Scheme of the succinylation reaction of pectin. S. Liotino et al. Reactive and Functional Polymers 214 (2025) 106331 2 pectin (FS-Pec) [31]. 2.3. Ionic crosslinked hydrogel film preparation To obtain a crosslinked system, during the precipitation, CaCl 2 or MgCl 2 solution (10 % w/V) in 50 mL of ethanol were added. The product was dissolved in water (10 mL) and dialyzed for 3 days. The product was then dried at 50 ◦C for a few days. 2.4. Film production Pec, S-Pec, Caand Mg-crosslinked S-Pec films were fabricated by dissolving the polymers in water, casting in Petri dishes (diameter 5 cm) and drying in an oven at 50 ◦C for 3 days. The obtained films were used for physico-chemical characterizations as well as for swelling/retention and stability tests. 2.5. Fourier transform-infrared spectroscopy in attenuated Total reflectance mode (FT-IR/ATR) Films were analyzed using FT-IR/ATR analyses through a Spectrum Two PE instrument (Perkin Elmer, Milan Italy), endowed with a universal ATR accessory (UATR, Single Reflection Diamond/ZnSe). Spectra were recorded from 400 to 4000 cm −1 with a 4 cm −1 resolution. 2.6. X-ray photoelectron spectroscopy (XPS) The prepared dried films were examined using XPS (PHI 5000 VersaProbe II, MN, USA). Analyses were performed in high-power mode with an AlK α X-ray radiation source and an instrument base pressure of 10 −9 mbar. Wide scans and high-resolution scans have been recorded in fixed analyzer transmission (pass energy of 117.4 eV and 29.35 eV, respectively). The MultiPak software (v.9.9.0.8) was used for data mining, setting the reference charge to 284.8 eV (hydrocarbon peak). 2.7. Nuclear magnetic resonance (NMR) An NMR spectrometer Bruker Avance I 400 was used to record solution and solid state NMR spectra. The solution state spectra were recorded with a 5 mm inverse broadband (BBI) probe and were calibrated setting 3-(Trimethylsilyl)propionic-2,2,3,3-d 4 acid sodium salt (TSP-d 4 ) singlet signal at ™ =0.00 ppm. Water pre-saturation (to remove residual signal of water) was accomplished in 1D and 2D experiments. 1D proton spectra ( 1 H NOESY) were acquired with a noesygppr1d pulse program (size of fid (TD) =128 K datapoints; spectral width (SW) =20 ppm; transmitter offset =4.70 ppm; dummy scans (ds) =4; number of scans (ns) =64; acquisition time =8.12 s; mixing time (d8) =0.01 s; recycle delay (d1) =30 s). 2D 1 H COSY experiments were acquired with a cosygpprqf pulse program (size of fid (TD) =4096 datapoints ×256 increments; spectral width (SW) =10 ppm; transmitter offset =4.70 ppm; number of scans (ns) =4; recycle delay (d1) = 30 s). Solid-state NMR experiments were carried out using a 4.0 mm HX MAS probe at 298 K with samples packed in zirconia rotors. 1 H – 13 C CP/ MAS NMR experiments were performed using 3.25 μ s proton π /2 pulse length, ν CP of 55.0 kHz, contact time of 1.0 s, ν dec of 76.9 kHz and recycle delay of 5.0 s. A two-pulse phase modulation (TPPM) decoupling scheme was used for the 1 H decoupling. Chemical shifts for 13 C were referenced to the methylene signal of adamantane (δ 38.48). 2.8. Thermal analyses by thermo-gravimetric analysis (TGA) and differential scanning calorimetry (DSC) The thermal behavior of the dehydrated hydrogels was assessed by a PerkinElmer TGA-400 instrument (Perkin Elmer, Milan, Italy), heating 5–10 mg of the samples in the range 30–800 ◦C. The analyses were performed on nitrogen, with a gas flow set at 20 ml/min. Data were recorded using the TGA Pyris software (version 13.3.1.0014). Thermal proprieties were also investigated by a Perkin Elmer – DSC 4000 calorimeter. The nitrogen flow was 20 mL/min and the heating rate of 10 ◦C/ min. The samples were heated from 30 ◦C to a maximum temperature of 170 ◦C. Samples for DSC measurements were prepared from the polymer film to a mass of about 2 mg. 2.9. Swelling measurements A tea bag was employed in this test, following the EDANA recommended method (i.e., EDANA Recommended test method: Polyacrylate Superabsorbent Powders – Determination of the Free Swell Capacity in Saline by Gravimetric Measurement (NWSP 240.0.R2 (15)) with slight modifications [32]. Briefly, a weighed dried SAP (0.0500 ±0.010 g) was tested at room temperature placed into a nonwoven tea bag, and successively sealed. The tea bag was put in a large pan, containing distilled water or saline solution (NaCl 0.9 % w/v) and it was allowed to freely swell for 30 ′ . The Free Swell Capacity (FSC) was expressed as (1): FSC (g/g) = (W2▬W1)/WSAP (1) where W 1 was the initial tea bag +SAP weight, W 2 the tea bag +SAP after swelling, and W SAP was the SAP weight. Successively, the tea bag containing the swollen SAP was placed in a centrifuge, equipped with a basket rotor at 1400 rpm for 3 ′ . Therefore, the tea bag was weighed to determine the CRC by applying the following formula (2): CRC (g/g) = (W2▬W1)/WSAP (2) where W 1 was the initial tea bag +SAP weight, W 2 was the tea bag + SAP after centrifugation, and W SAP was the SAP weight. A blank test was performed to ascertain the contribution of water absorption relevant to the tea bag. Each measurement was repeated in triplicate, and results were reported as mean ±standard deviation. 2.10. Viscoelastic and tensile properties Frequency sweep tests from 0.1 to 1 Hz were carried out for the casted films using a Discovery DHR3 rheometer (TA Instruments, USA) with a rectangular geometry in torsion mode. A constant strain within the linear viscoelastic range was always employed. At least two replicates of each measurement were performed, and typical parameters are reported as averages and standard deviations. Tensile mechanical properties were measured with an RSA3 rheometer (TA Instruments, USA) using a rectangular geometry in tension mode for the casted films at a constant extension rate of 0.01 mm/s until failure. At least three replicates were performed on samples, and mechanical properties are reported as means and standard deviations. 2.11. Statistical analysis For swelling and retention tests, both in saline solution and in water, the results of three independent experiments were statistically analyzed using the one-way ANOVA, followed by Tukey’s test of significance, to identify the groups that were significantly different (p <0.05). 3. Results and discussion 3.1. Film production In Fig. S2, the photographs of the obtained films were reported (see Supplementary Data). It was interesting to observe that S-Pec (Fig. S2b) produced transparent films differently from unmodified pectin (Fig. S2a). The addition of metal ions increased the film opacity, and this feature was much more pronounced in S-Pec Ca (Fig. S2d) than in S-Pec S. Liotino et al. Reactive and Functional Polymers 214 (2025) 106331 3 Mg (Fig. S2c). Moreover, a more in-depth morphological characterization, using SEM (experimental details were reported in paragraph S1 of Supplementary Data), of the S-Pec films is reported in Fig. S3 (see Supplementary Data). Both the Mgand Ca-crosslinked films exhibited a surface structure with small aggregates or cavities, even if this heterogeneous morphology was more evident for S-Pec Ca, while the S-Pec Mg film surface appeared smoother and more similar to that of the uncrosslinked S-Pec film. 3.2. FT-IR/ATR characterization In Fig. 2 the FT-IR/ATR spectra of Pec and S-Pec were reported. The broad peak at 3382 cm −1 referred to O – H stretching, while the signal at 2920 cm −1 was C – H stretching. The stronger signals at 1733 cm −1 and 1650 cm −1 indicated the ester carbonyl and carboxylate ion stretching bands. The region between 1300 and 800 cm −1 represented the “fingerprint”. [33] The IR spectrum shows an increase in signal intensity at 1650 cm −1 after the succinylation reaction (red line). This experimental evidence supports the binding of the succinic portion to the hydroxyl groups of pectin. Since the added succinic portion contains an equal number of ester and carboxylate carbonyl groups and, given that carboxylate ions were a minority (30 %) in the native pectin, the relative increase in the percentage of these groups is more pronounced. 3.3. XPS results XPS analysis of succinylated pectin, as well as the pristine polymer, was carried out to verify the changes in surface chemical composition of the reaction product, in particular when succinylation was carried out using tetrafluorosuccinic anhydride. In Fig. 3, C1s spectra and relevant curve fittings of pectin (a), succinylated pectin (b), and tetrafluorosuccinylated pectin (c) were reported. The C1s curve fitting of pectin was carried out using four peaks relevant to C-C/CH, C-OR(H), O-C-O, and COOR(H) contributions. Peak percentages were reported in Table 1. The calculated C-OR(H)/O-C-O ratio was equal to 2.8:1, and the COOR(H)/O-C-O one was equal to 0.3:1. As far as the S-Pec sample is concerned, an additional peak, falling at 285.5 eV and relevant both to carbon in α -position to carboxylic groups present in succinic moieties, in addition to carbon bonded to nitrogen of traces of residual DMAP, was detected. In the case of FS-Pec, the peak at 285.5 eV was only related to C – N, while the CF 2 groups in α -position to carboxylic groups of succinic moieties fell at higher BEs, i. e. at 289.9 eV [34]. The calculated C-OR(H)/O-C-O ratios in S-Pec and FS-Pec were 2.6:1 and 2.7:1, respectively, while the calculated COOR(H)/O-C-O ratios were equal to 0.5:1 for both the succinylated samples. The increase of the latter ratio with respect to that recorded for pristine pectin evidenced the presence of additional COOR(H) groups, due to succinylation. More clearly, the success of the succinylation reaction was evident when tetrafluorosuccinic anhydride was employed. In this case, it was possible to distinguish the COOR(H) relevant to pectin from those derived from succinic moieties. Considering that CF 2 /COOR(H) succ ratio should be 1:1, the difference between the COOR(H) and CF 2 peak areas supplied an indication of COOR(H) Pec , i.e., 3.6 %. The fact that the ratio COOR(H)Pec/O-C-O obtained (0.3:1) was in agreement with that recorded in the pristine polymer supports the methodology followed. Concluding, the ratio between half of the CF 2 peak (considering that each succinic unit contains two CF 2 groups) and the O-C-O peak (indicative of the monomeric unit of pectin), allowed for an estimation of the succinylation degree, which was equal to 0.14. Finally, XPS analysis of Caand Mg-crosslinked S-Pec evidenced almost similar surface composition, with additional calcium and magnesium signals, respectively, with a Mg1s/COOR(H) ratio equal to 0.35:1 and Ca2p/COOR(H) ratio equal to 0.44:1. No significant changes in C1s curve fittings of the crosslinked samples were observed with respect to uncrosslinked succinylated pectin. 3.4. NMR characterization Fig. 4 shows the comparison between 1 H– 13 C CP/MAS spectra of succinylated pectin and pristine pectin. It is apparent that the 1 H – 13 C CP/MAS spectrum of succinylated pectin shows two new signals at ™ 176 and ™ 39 ascribable to carboxylic and methylenic carbons of the succinic moiety, respectively, thus confirming the derivatization of pectin with succinic anhydride. The corresponding signals for succinylated cellulose were found around ™ 180 (carboxylic C) and ™ 33 (methylene C) [35] or, in another study, at ™ 175 and ™ 30. [36] A close inspection of the 1 H – 13 C CP/MAS spectra shown in Fig. 4 indicates that the intensity of the C4 signal of pectin decreases upon succinylation. This may suggest that the succinyl moiety binds preferentially on O – C4. Moreover, partial hydrolysis of the COOMe groups originally present in pristine pectin is indicated by the decrease in intensity of the signals at ™ 53 (COOCH 3 ) and ™ 171 (COOCH 3 ) of the methyl galacturonate moiety [37]. Solution 1 H NMR at 323 K of succinylated pectin (Fig. 5a) showed two signals that can be tentatively ascribed to the unequivalent methylenes of HO 2 C–CH 2 –CH 2 –COO–Pectin. Such signals fell at ™ 2.61 and ™ 2.76 and were demonstrated to be mutually (scalarly) coupled ( 1 H COSY, Fig. 5b). For comparison, the corresponding methylene signals of succinylated galacturonic acid (succinylated glucose), prepared using the same procedure followed for succinylated pectin and easily solubilized in water, fell at ™ 2.62 and ™ 2.78 (™ 2.58 and ™ 2.71). 3.5. TGA and DSC analyses To analyze the thermal properties of succinylated pectin, TGA and differential TGA (DTGA) analyses were carried out under a nitrogen atmosphere to examine its thermal stability and weight loss or decomposition with respect to pristine pectin. The TGA thermograms of pectin and succinylated pectin, as well as the DTGA plots derived from them, were reported in Fig. 6a, the thermogram of the pristine pectin revealed three distinct stages of weight loss. In the first stage, starting from the initial temperature, there was a weight loss of 7 % within the range of 30–200 ◦C, attributed to dehydration resulting from the absorbed moisture in the pectin [38]. In the second stage, a rapid mass loss (45 %) occurred between 200 and 300 ◦C (T peak =254 ◦C). Finally, a slow mass loss was recorded between 300 and 800 ◦C, with a final residue equal to 2 %. These thermal events could be attributed to the pyrolytic decomposition of pectin, which involves decarboxylation, bond cracking and Fig. 2. FT-IR/ATR spectra of Pec and S-Pec films. S. Liotino et al. Reactive and Functional Polymers 214 (2025) 106331 4 chain breaking, as reported in the literature [39]. As far as succinylated pectin is concerned, an initial weight loss in the temperature range 30–200 ◦C of 15 % was recorded, indicating a higher moisture absorption capacity of the product after succinylation. The second stage consisted of two partially overlapped events, falling at T peak1 =243 ◦C and T peak2 =266 ◦C. The onset of degradation for succinylated pectin occurred at a lower temperature, probably due to the rupture of new ester groups introduced by the reaction, as previously argued by Bridson et al. for succinylated lignins [40]. A final residue of 21 % indicated the presence of sodium ions in the product, as expected. Finally, as far as the Mgor Ca-crosslinked succinylated systems are concerned, significant thermal stabilization was detected, since T peak was found to fall at 273 ◦C (Fig. S4). This thermal stabilization may be related to the crosslinking effect, as previously reported by Zhang et al. [41]. Another interesting feature was that, after crosslinking, especially for S-Pec Mg sample, the water/volatiles content significantly increased, reaching 24 % in the temperature range 30–200 ◦C. In Fig. 6b, the DSC traces of the pectin and succinylated pectin were reported. The endothermic peak at 139 ◦C is relative to the melting temperature of the pectin [42]. This peak shifted to the lower value of 129 ◦C in the succinylated pectin. As reported for the succinylation of alginate [43], this phenomenon could be attributed to lower intermolecular forces between polymer chains and lower molecular packing ordering. The presence of succinyl groups breaks hydrogen bonds originally occurring in the pectin. 3.6. Liquid uptake/retention and stability results Water uptake and retention represent key properties for polymers aimed at being substrates for SAP production. The swelling capacity was prevalently linked to the presence of polar groups, while the liquid retention under external forces was related to the crosslink density of the network and the strength of the crosslinking agent. Usually, the swelling capacity decreases with crosslinking. This is due to a decrease in the space between the copolymer chains, as crosslinker concentration and/ or strength increases. On the other hand, more rigid systems, obtained by the presence of a crosslinked network, can retain more liquid. The liquid retention was also related to the ability of ionizable groups to bind water. Additionally, swelling performances were related to the ion strength of the swelling medium; i.e., swelling was significantly lower in saline solution than in distilled water. Considering these preliminary considerations, swelling and retention tests were carried out both on unmodified and succinylated pectin films, as well as on the two crosslinked unmodified and succinylated ones. Results were reported in Fig. 7. First of all, evidence of the significant improvement of FSC, both in saline solution and distilled water, is observed when the succinylated polymer (22.5 ±0.7 and 47 ±2 g/g, in NaCl 0.9 % and water, respectively) was tested in comparison to unmodified Pec film (13.5 ±0.7 and 22.0 ±1.4 g/g, in NaCl 0.9 % and water, respectively). This represents a twofold increase in swelling compared to the starting material. These results could be related to the introduction in the polymer matrix of polar groups, which significantly increased its liquid absorbency. In addition, the ability to retain the absorbed water after centrifugation was hugely improved by succinylation. Indeed, Pec films showed low CRC values (2.5 ±0.7 and 5.0 ±1.4 g/g, in NaCl 0.9 % and water, respectively), which indicated a poor ability of pectin to be used as such as SAP. Conversely, S-Pec films retained higher liquid amounts after centrifugation (6.5 ±0.6 and 12.5 ±0.7 g/g, in NaCl 0.9 % and water, respectively), thus confirming that the absorbed liquid remained in the S-Pec network after extensive centrifugation thanks to the interaction of water with polar groups. However, to obtain more performant SAPs, polymer crosslinking was necessary. In this work, an ionic crosslinking Fig. 3. C1s spectra and relevant curve fittings of (a) Pec, (b) S-Pec, and (c) FS-Pec. Table 1 Peak attributions and binding energies (BE) relevant to Pec, S-Pec, and FS-Pec. Uncertainty in BE peaks positions was ±0.2 eV.. Sample Peak BE(eV) C-C/C-H CH 2 α / C-N C-OR(H) O-C-O COOR(H)/ COO − Na + CF 2 Pec 284.8 –286.3 287.6 289.0 – S-Pec 284.8 285.5 286.4 287.8 288.9 – FS-Pec 284.8 285.5 286.4 287.8 288.8 289.9 Fig. 4. 1 H– 13 C CP/MAS spectra of succinylated pectin (bottom) and pristine pectin (top). 100.6 MHz, 298 K; MAS rate: 9 kHz. Fig. 5. a) portion of 1 H NMR of the crude obtained from the reaction of pectin succinylation (400 MHz, D 2 O, 323 K). Asterisked peak is due to DMAP; b) portion of 1 H COSY spectrum of the same solution (400 MHz, D 2 O, 323 K). S. Liotino et al. Reactive and Functional Polymers 214 (2025) 106331 5 was carried out, producing two different films, i.e., an opaque and stiff Ca-crosslinked film and a smoother and more flexible Mg-crosslinked film. To observe an improvement in swelling performances on the crosslinked systems, unmodified pectin films crosslinked by Ca and Mg were also produced and tested. In particular, Pec Ca films showed FSC values not statistically different from Pec films (12 ±2 and 17.0 ±1.4 g/g in water and saline solution, respectively), while CRC values statistically similar to Pec sample (4.5 ±0.7 and 7.5 ±0.6 g/g in water and saline solution, respectively). Pec Mg films evidenced a slight improvement of all the swelling/retention performances, even if not statistically superior to the S-Pec sample in terms of FSC (FSC: 15.0 ± 1.3 and 22 ±2 g/g in water and saline solution, respectively). For this system, an appreciable increase in CRC values was detected (CRC: 6.7 ± 0.6 and 10 ±3 g/g in water and saline solution, respectively). As far as the S-Pec systems are concerned, the Mg-crosslinked S-Pec film provided significantly better swelling/retention performances. This latter film showed FSC values not statistically different from the un-crosslinked system in saline solution (23 ±2 g/g) while statistically lower in water (40 ±3 g/g). On the other hand, CRC values benefited from the presence of Mg-crosslinking in both the swelling media tested (13 ±4 and 20 ±2 g/g, in NaCl 0.9 % and water, respectively). This result showed the strong synergy effect obtained by first succinylating pectin and subsequently crosslinking it with magnesium ions. The overall decrease in FSC and CRC values in saline solution compared to water can be related to the lower osmotic pressure due to the sodium ions present in the swelling medium [44]. Finally, Ca-crosslinked S-Pec resulted in a poorly swellable system. FSC values (15 ±3 and 21 ±2 g/g, in NaCl 0.9 % and water, respectively) were significantly lower than those of un-crosslinked S-Pec; as regards CRCs, no statistically significant variations were found, displaying CRC equal to 6.3 ±1.1 and 10.5 ±0.7 g/g, in NaCl 0.9 % and water, respectively. Despite these uninspiring performances, this system was particularly stable. Indeed, Pec, S-Pec, and the relevant Mgand Cacrosslinked S-Pec films were tested in terms of stability in aqueous medium for 1, 2, and 10 days. All systems, except Ca-crosslinked S-Pec, swollen in water for long times, appeared not stable, losing mass already after 1 h, and were totally dissolved after 24 h. In the long-term test, succinylated pectin crosslinked with Ca 2+ showed good resistance to retain almost intact its structure up to 10 days, with negligible weight losses. The main ionic crosslinking of pectin was exploited by calcium ions. The crosslinking process of pectin by Ca 2+ was described by the physical entrapment of calcium ions between non-methyl esterified galacturonate units from pectin, where the junction zones are formed in the so-called “egg box” model [45]. Moreover, Wellner and co-workers argued that ionic crosslinking did not involve only physical Fig. 6. TGA and DTGA traces (solid and dotted lines, respectively) of Pec and S-Pec films (a). DSC traces of Pec and S-pec films (b). Fig. 7. FSC (panels a and b) and CRC (panels c and d) values, for un-crosslinked and Mg 2+ or Ca 2+ crosslinked Pec and S-Pec films, both in NaCl 0.9 % w/V (panels a and c) and in distilled water (panels c and d). The results were obtained as the mean over three replicates (error bars are the associated standard errors). Similar letters above the error bars indicate no statistically significant difference (p >0.05). S. Liotino et al. Reactive and Functional Polymers 214 (2025) 106331 6 entrapment but also the interaction of calcium ions with the oxygen atom from the carboxylate group of pectin, making stable and thermoirreversible the three-dimensional network formed [46]. On the other hand, Wellner found that, among the different divalent ions tested, magnesium ions interacted the least with pectin. In this respect, we can conclude that, although magnesium ions produced hydrogels not stable over time, the effect exerted by magnesium crosslinking on S-Pec returned a material highly performant in terms of absorption and retention of liquids. Based on these observations, a critical issue could be linked to the biodegradability of the developed systems, especially those crosslinked with calcium. Yoshimura and co-workers studied the biodegradability of pectin-based hydrogels, chemically or ionically crosslinked, in activated sludge at 25 ◦C for 18 days and observed that only chemically crosslinked systems displayed a hindered biodegradability [17]. Moreover, the same authors carried out succinylation on many different polysaccharides, evidencing a minimal impact on biodegradability [24–27]. Biodegradability studies of our S-Pec based films will be performed in future studies to fully understand how the succinylation and/or the crosslinking influence the biodegradation kinetics. The S-Pec films developed in the present study could be considered as a green substitute for flexible porous absorbent pads. Thus, the FSC and CRC of a commercial absorbent foam layer used in feminine sanitary pads were tested, displaying a FSC value of 20.7 ±0.4 g/g and a CRC equal to 9 ±3 g/g. Therefore, the materials developed have saline absorption and retention values very close to those of the commercial products currently available in the market within the sector of interest. 3.7. Viscoelastic and tensile properties To assess the effect of adding a cross-linking agent on the viscoelastic properties, succinylated pectin casted films either in the presence of Mg (S-Pec Mg) or in the absence of any divalent cation (S-Pec) were tested. Neither Pec nor S-Pec Ca could be characterized adequately as heterogeneous films were obtained. Both samples S-Pec and S-Pec Mg displayed a similar qualitative mechanical spectrum. Thus, the elastic modulus is well above the viscous one for the whole range of frequencies studied in torsional dynamic tests, not showing a great dependence on the frequency. However, the addition of Mg during processing resulted in a significant decrease in the loss tangent (tan δ =G”/G’) from 0.09 ± 0.01 (S-Pec) to 0.05 ±0.004 (S-Pec Mg) (Fig. S5a). Therefore, a reinforcement of the elastic behavior over the viscous one seems to be associated to the crosslinking that has been confirmed for S-Pec Mg. Crosslinking has already been related to a decrease in tan δ [47–49]. However, when mechanical properties were determined through uniaxial tensile tests, films from S-Pec displayed greater rigidity and strength compared to S-Pec Mg, despite the low reproducibility found for the latter. Thus, Young’s modulus and tensile strength for S-Pec were 9.6 ±0.4 MPa and 16.6 ±3.1 MPa, respectively, compared to 3.6 ±1.2 MPa and 6.7 ±2.4 MPa for S-Pec Mg (Fig. S5b). This may be related to the higher moisture content observed for S-Pec Mg (~25 %) in the TGA tests compared to S-Pec (~15 %) (Fig. S4). Moisture content is one of the most influential parameters on elasticity of biopolymer films, and that water removal from films has been previously used to increase their mechanical strength [50,51]. In any case, deformability before rupture was similar for both samples (2.9 ±1.3 %). 4. Conclusions Succinylation of pectin was carried out, for the first time, to supply a novel substrate to produce biodegradable superabsorbent hydrogels. A simple procedure (i.e., esterification of pectin by succinic anhydride followed by NaOH neutralization) was proposed, and a subsequent ionic crosslinking was carried out employing calcium or magnesium ions. FTIR analysis showed an increment of the carboxylate/ester groups ratio, relatable to the insertion of succinic moieties in the biopolymer molecule. XPS analysis further evidenced the success of succinylation when fluorinated anhydride was employed, resulting in an estimation of the succinylation degree of 0.14. Moreover, NMR analysis, both in solution and in the solid state, confirmed the succinylation of pectin. Regarding the thermal stability, TGA showed that the onset of degradation for S-Pec occurred at a lower temperature, due to the rupture of new ester groups introduced by the reaction, while DSC showed a decrease of the melting temperature in the succinylated pectin, probably due to the disruption of hydrogen bonds present in Pec due to the introduction of succinyl groups. S-Pec Mg showed a value of Young’s modulus and tensile strength lower than S-Pec, probably linked to the higher moisture content in the former. As far as the swelling performances of the tested films, the maximum performance in distilled water was obtained by uncrosslinked S-Pec (47 g of water per gram of dry film), while the maximum centrifugation retention capacity was achieved by Mg-crosslinked S-Pec (20 g of water per gram of dry film). Finally, the most stable hydrogel system was found to be the Cacrosslinked S-Pec film, which kept its integrity up to 10 days. An indepth study on how succinylation affects the degree of crosslinking, hydrophilicity, or morphological features of the system will be considered in future research. CRediT authorship contribution statement Stefano Liotino: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Stefania Cometa: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Stefano Todisco: Writing – original draft, Investigation, Formal analysis, Data curation. Piero Mastrorilli: Writing – review & editing, Writing – original draft, Investigation, Data curation. Carlos Bengoechea: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Antonio Salomone: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Elvira De Giglio: Writing – review & editing, Writing – original draft, Supervision, Investigation, Data curation, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors thank Dr. Giuseppe Iannaccone (Jaber Innovation s.r.l.) for the valuable discussions. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.reactfunctpolym.2025.106331. Data availability Data will be made available on request. References [1] https://sustainabledevelopment.un.org/post2015/transformingourworld, (n.d.). [2] M.J. Getahun, B.B. Kassie, T.S. Alemu, Recent advances in biopolymer synthesis, properties, & commercial applications: a review, Process Biochem. 145 (2024) 261–287, https://doi.org/10.1016/j.procbio.2024.06.034. [3] M.C.N. Picot-Allain, B. Ramasawmy, M.N. Emmambux, Extraction, characterisation, and application of pectin from tropical and sub-tropical fruits: a review, Food Rev. Int. 38 (2022) 282–312, https://doi.org/10.1080/ 87559129.2020.1733008. S. Liotino et al. Reactive and Functional Polymers 214 (2025) 106331 7 [4] I. Frosi, A. Balduzzi, G. Moretto, R. Colombo, A. Papetti, Towards valorization of food-waste-derived pectin: recent advances on their characterization and application, Molecules 28 (2023) 6390, https://doi.org/10.3390/ molecules28176390. [5] N.S. Said, I.F. Olawuyi, W.Y. Lee, Pectin hydrogels: gel-forming behaviors, mechanisms, and food applications, Gels 9 (2023) 732, https://doi.org/10.3390/ gels9090732. [6] W.M. Kedir, E.M. Deresa, T.F. Diriba, Pharmaceutical and drug delivery applications of pectin and its modified nanocomposites, Heliyon 8 (2022) e10654, https://doi.org/10.1016/j.heliyon.2022.e10654. [7] P. Foti, F.V. Romeo, Citrus pectin as source to formulate a new nutraceutical complex, Food Biomacromol. 1 (2024) 26–27, https://doi.org/10.1002/ fob2.12009. [8] S.S. Han, S.M. Ji, M.J. Park, M. Suneetha, U.T. Uthappa, Pectin based hydrogels for drug delivery applications: a mini review, Gels 8 (2022) 834, https://doi.org/ 10.3390/gels8120834. [9] J. Koshy, D. Sangeetha, Recent progress and treatment strategy of pectin polysaccharide based tissue engineering scaffolds in cancer therapy, wound healing and cartilage regeneration, Int. J. Biol. Macromol. 257 (2024) 128594, https://doi. org/10.1016/j.ijbiomac.2023.128594. [10] B. Kocaa˘ ga, Y. ¨ Oztürk, H. Ceren Kurçin, ¨ O.Z. Güner-Yılmaz, O. Kurkcuoglu, M. Tatlier, ˙ I. ¨ Ozdemir, E. Kervancioglu Demirci, T. Kotil, S. Solako˘ glu, B. Aksu, S. Batirel, A. Bal-¨ Oztürk, F.S. Güner, Developing multifunctional pectin-based hydrogel for wound dressing: in silico, in vitro and in vivo evaluation, Eur. Polym. J. 216 (2024) 113280, https://doi.org/10.1016/j.eurpolymj.2024.113280. [11] S. Roy, R. Priyadarshi, Ł. Łopusiewicz, D. Biswas, V. Chandel, J.-W. Rhim, Recent progress in pectin extraction, characterization, and pectin-based films for active food packaging applications: a review, Int. J. Biol. Macromol. 239 (2023) 124248, https://doi.org/10.1016/j.ijbiomac.2023.124248. [12] T.S.B. Abd Manan, S. Beddu, D. Mohamad, N.L. Mohd Kamal, Z. Itam, T. Khan, H. Jusoh, N.A. Abdul Rahman, F. Mohamed Nazri, M.F.K. Mohd Yapandi, W.H. M. Wan Mohtar, M.H. Isa, Z. Che Muda, A. Ahmad, N. Wan Rasdi, Physicochemical properties of absorbent hydrogel polymers in disposable baby diapers, Chem. Phys. Lett. 774 (2021) 138605, https://doi.org/10.1016/j.cplett.2021.138605. [13] S. Ismaeilimoghadam, B. Azimi, M. Jonoobi, S. Danti, Highly performing polysaccharide hydrogels can replace acrylic acid-based superabsorbent polymers in sanitary napkins, Macro Materi. Eng 310 (2025) 2400278, https://doi.org/ 10.1002/mame.202400278. [14] A.M. AbdAllah, A.M. Mashaheet, K.O. Burkey, Super absorbent polymers mitigate drought stress in corn (Zea mays L.) grown under rainfed conditions, Agric. Water Manag. 254 (2021) 106946, https://doi.org/10.1016/j.agwat.2021.106946. [15] A.B. Ribeiro, H. Moreira, S.I.A. Pereira, M. Godinho, A.S.D.S. Sousa, P. Castro, C. F. Pereira, F. Casanova, R. Freixo, M.E. Pintado, ´ O.L. Ramos, Bio-based superabsorbent hydrogels for nutrient release, J. Environ. Chem. Eng. 12 (2024) 112031, https://doi.org/10.1016/j.jece.2024.112031. [16] F.L. Buchholz (Ed.), Modern Superabsorbent Polymer Technology, Wiley-VCH, New York Weinheim, 1998. [17] T. Yoshimura, K. Sengoku, R. Fujioka, Pectin-based surperabsorbent hydrogels crosslinked by some chemicals: synthesis and characterization, Polym. Bull. 55 (2005) 123–129, https://doi.org/10.1007/s00289-005-0422-1. [18] B. Shen, Z. Guo, B. Huang, G. Zhang, P. Fei, S. Hu, Preparation of hydrogels based on pectin with different esterification degrees and evaluation of their structure and adsorption properties, Int. J. Biol. Macromol. 202 (2022) 397–406, https://doi. org/10.1016/j.ijbiomac.2021.12.160. [19] P. Wang, Y. Gao, D. Wang, Z. Huang, P. Fei, Amidated pectin with amino acids: preparation, characterization and potential application in hydrocolloids, Food Hydrocoll. 129 (2022) 107662, https://doi.org/10.1016/j.foodhyd.2022.107662. [20] Y. Zhuang, Z. Guo, Q. Zhang, J. Liu, P. Fei, B. Huang, Preparation of functionalized pectin through acylation with alkyl gallates: experiments coupled with density functional theory, Int. J. Biol. Macromol. 202 (2022) 278–285, https://doi.org/ 10.1016/j.ijbiomac.2022.01.070. [21] H. Würfel, K. Geitel, H. Qi, T. Heinze, Chemical modification of pectin and polygalacturonic acid: a critical review, BioRes 16 (2021) 8457–8488, https://doi. org/10.15376/biores.16.4.Wurfel. [22] S. Qian, Z. Sheng, Q. Meng, L. Chen, D. Wu, J. Tang, Y. Liu, Z. Han, W. Hu, Evaluation of a novel phosphorylated red dragon fruit Peel pectin for enhancement of thermal stability and functional activity, Plant Foods Hum. Nutr. 77 (2022) 150–154, https://doi.org/10.1007/s11130-022-00958-3. [23] S. Feng, J. Yi, Y. Ma, J. Bi, The role of amide groups in the mechanism of acidinduced pectin gelation: a potential pH-sensitive hydrogel based on hydrogen bond interactions, Food Hydrocoll. 141 (2023) 108741, https://doi.org/10.1016/j. foodhyd.2023.108741. [24] T. Yoshimura, K. Matsuo, R. Fujioka, Novel biodegradable superabsorbent hydrogels derived from cotton cellulose and succinic anhydride: synthesis and characterization, J. Appl. Polym. Sci. 99 (2006) 3251–3256, https://doi.org/ 10.1002/app.22794. [25] T. Yoshimura, R. Yoshimura, C. Seki, R. Fujioka, Synthesis and characterization of biodegradable hydrogels based on starch and succinic anhydride, Carbohydr. Polym. 64 (2006) 345–349, https://doi.org/10.1016/j.carbpol.2005.12.023. [26] T. Yoshimura, I. Uchikoshi, Y. Yoshiura, R. Fujioka, Synthesis and characterization of novel biodegradable superabsorbent hydrogels based on chitin and succinic anhydride, Carbohydr. Polym. 61 (2005) 322–326, https://doi.org/10.1016/j. carbpol.2005.06.014. [27] R. Fujioka, Y. Tanaka, T. Yoshimura, Synthesis and properties of superabsorbent hydrogels based on guar gum and succinic anhydride, J. Appl. Polym. Sci. 114 (2009) 612–616, https://doi.org/10.1002/app.30600. [28] Q. Xiao, S. Ye, Y. Zhang, J. Chen, F. Chen, H. Weng, A. Xiao, Gel properties transition from mono-succinylation to cross-linking of agar by attemperation with succinic anhydride, Food Chem. 381 (2022) 132164, https://doi.org/10.1016/j. foodchem.2022.132164. [29] E. De Giglio, S. Cometa, N. Cioffi, L. Torsi, L. Sabbatini, Analytical investigations of poly(acrylic acid) coatings electrodeposited on titanium-based implants: a versatile approach to biocompatibility enhancement, Anal. Bioanal. Chem. 389 (2007) 2055–2063, https://doi.org/10.1007/s00216-007-1299-7. [30] D.S. Seeli, M. Prabaharan, Guar gum succinate as a carrier for colon-specific drug delivery, Int. J. Biol. Macromol. 84 (2016) 10–15, https://doi.org/10.1016/j. ijbiomac.2015.12.002. [31] J. Li, M. Goto, X. Yang, S.L. Morris-Natschke, L. Huang, C.-H. Chen, K.-H. Lee, Fluorinated betulinic acid derivatives and evaluation of their anti-HIV activity, Bioorg. Med. Chem. Lett. 26 (2016) 68–71, https://doi.org/10.1016/j. bmcl.2015.11.029. [32] https://www.edana.org/, (n.d.). [33] R. Gnanasambandam, Determination of pectin degree of esterification by diffuse reflectance Fourier transform infrared spectroscopy, Food Chem. 68 (2000) 327–332, https://doi.org/10.1016/S0308-8146(99)00191-0. [34] High resolution XPS of organic polymers: the Scienta ESCA300 database (Beamson, G.; Briggs, D.),, J. Chem. Educ. 70 (1993) A25, https://doi.org/10.1021/ ed070pA25.5. [35] N. Harada, H. Uyama, Surface modification of cellulose with succinic anhydride in dimethyl sulfoxide using potassium carbonate as a catalyst, Carbohydr. Res. 528 (2023) 108812, https://doi.org/10.1016/j.carres.2023.108812. [36] R.M. Ribeiro-Viana, P.C.S. Faria-Tischer, C.A. Tischer, Preparation of succinylated cellulose membranes for functionalization purposes, Carbohydr. Polym. 148 (2016) 21–28, https://doi.org/10.1016/j.carbpol.2016.04.033. [37] M. Yang, Z. Liu, J. Zhang, X. Zhu, W. Xie, H. Lan, Y. Huang, X. Ye, J. Yang, Simultaneous quantification of cellulose and pectin in tobacco using a robust solidstate NMR method, Carbohydr. Res. 521 (2022) 108676, https://doi.org/10.1016/ j.carres.2022.108676. [38] V. Siracusa, S. Romani, M. Gigli, C. Mannozzi, J.P. Cecchini, U. Tylewicz, N. Lotti, Characterization of active edible films based on Citral essential oil, Alginate and Pectin, Materials 11 (2018) 1980, https://doi.org/10.3390/ma11101980. [39] E.E. Santos, R.C. Amaro, C.C.C. Bustamante, M.H.A. Guerra, L.C. Soares, R.E. S. Froes, Extraction of pectin from agroindustrial residue with an ecofriendly solvent: use of FTIR and chemometrics to differentiate pectins according to degree of methyl esterification, Food Hydrocoll. 107 (2020) 105921, https://doi.org/ 10.1016/j.foodhyd.2020.105921. [40] J.H. Bridson, D.J. Van De Pas, A. Fernyhough, Succinylation of three different lignins by reactive extrusion, J. Appl. Polym. Sci. 128 (2013) 4355–4360, https:// doi.org/10.1002/app.38664. [41] W. Zhang, J. Cao, W. Jiang, Effect of different cation in situ cross-linking on the properties of pectin-thymol active film, Food Hydrocoll. 128 (2022) 107594, https://doi.org/10.1016/j.foodhyd.2022.107594. [42] M.T. Tunç, H.˙ I. Odabas¸, Single-step recovery of pectin and essential oil from lemon waste by ohmic heating assisted extraction/hydrodistillation: a multi-response optimization study, Innovative Food Sci. Emerg. Technol. 74 (2021) 102850, https://doi.org/10.1016/j.ifset.2021.102850. [43] M. Falkeborg, P. Paitaid, A.N. Shu, B. P´ erez, Z. Guo, Dodecenyl succinylated alginate as a novel material for encapsulation and hyperactivation of lipases, Carbohydr. Polym. 133 (2015) 194–202, https://doi.org/10.1016/j. carbpol.2015.06.103. [44] R.-M.P. Karlsson, P.T. Larsson, P. Hansson, L. Wågberg, Thermodynamics of the water-retaining properties of cellulose-based networks, Biomacromolecules 20 (2019) 1603–1612, https://doi.org/10.1021/acs.biomac.8b01791. [45] G.T. Grant, E.R. Morris, D.A. Rees, P.J.C. Smith, D. Thom, Biological interactions between polysaccharides and divalent cations: the egg-box model, FEBS Lett. 32 (1973) 195–198, https://doi.org/10.1016/0014-5793(73)80770-7. [46] N. Wellner, M. Kaˇ cur´ akov´ a, A. Malovikova, R.H. Wilson, P.S. Belton, FT-IR study of pectate and pectinate gels formed by divalent cations, Carbohydr. Res. 308 (1998) 123–131, https://doi.org/10.1016/S0008-6215(98)00065-2. [47] E. ´ Alvarez-Castillo, M.J. Pelagio, C. Bengoechea, A. Guerrero, Plasma based superabsorbent materials modulated through chemical cross-linking, J. Environ. Chem. Eng. 9 (2021) 105017, https://doi.org/10.1016/j.jece.2020.105017. [48] E. ´ Alvarez-Castillo, I. Santana, J. G´ omez, C. Bengoechea, A. Guerrero, Effect of citric acid on porcine plasma protein bioplastics processed through injection moulding, React. Funct. Polym. 192 (2023), https://doi.org/10.1016/j. reactfunctpolym.2023.105709. [49] L. Cao, L. Zhong, Y. Li, J. Gao, G. Chen, Crosslinking dependence of direct current breakdown performance for XLPE-PS composites at different temperatures, Polymers 13 (2021) 219, https://doi.org/10.3390/polym13020219. [50] I. Kalathaki, K. Alba, H. Muhamedsalih, V. Kontogiorgos, Fabrication and characterisation of metal-doped pectin films, Food Hydrocoll. 92 (2019) 259–266, https://doi.org/10.1016/j.foodhyd.2019.01.065. [51] J. Kruk, J. Tkaczewska, M. Szuwarzy´ nski, T. Mazur, E. Jamr´ oz, Influence of storage conditions on functional properties of multilayer biopolymer films based on chitosan and furcellaran enriched with carp protein hydrolysate, Food Hydrocoll. 135 (2023) 108214, https://doi.org/10.1016/j.foodhyd.2022.108214. S. Liotino et al. Reactive and Functional Polymers 214 (2025) 106331 8