The concentration-modified physicochemical surface properties of sodium carbonate-soluble pectin from pears (Pyrus communis L.)

Food Hydrocolloids 113 (2021) 106524

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The concentration-modified physicochemical surface properties of sodium carbonate-soluble pectin from pears (Pyrus communis L.) ´ ska , Piotr Pieczywek , Justyna Cybulska , Artur Zdunek Jolanta Cie´sla *, Magdalena Koczan Institute of Agrophysics, Polish Academy of Sciences, Do´swiadczalna 4, 20-290, Lublin, Poland

A R T I C L E I N F O

A B S T R A C T

Keywords: Diluted alkali–soluble pectin Dissociation Gelation Ion binding Self–assembling Surface electrical charge

Cross–linking and gelation are important abilities affecting pectin functionality. Pectin concentration, pH and the composition of the dispersing medium play a crucial role in gel formation. The self-assembly of pectin in solution occurs simultaneously with changes in its physicochemical properties. However, this process has not been described in detail to date. Therefore, the effect of pectin concentration on such properties as its surface electrical charge (Q), hydrogen ions binding (βH+) and total counterions binding (βtotal) during intermolecular interactions in different media was studied. Low–methoxyl diluted alkali–soluble pectin (DASP) extracted from pear fruits by using sodium carbonate was examined. Investigations were performed on a wide range of its content (1.84⋅10− 4–1.84⋅100% w/v) in water and salt (NaCl and CaCl2) solutions with the ionic strength of 30 mM at 20 ◦ C. Back Dynamic Light Scattering, Laser Doppler Electrophoresis, potentiometry and Atomic Force Micro­ scopy methods were used. The DASP dissociation constant was calculated and used for determination of other physicochemical parameters. The relationship between changes in the physical and physicochemical properties of DASP during gelation was shown. An increase in the relative mean hydrodynamic diameter with DASP concentration was connected with the self–assembling (CaCl2) and cross–linking (water, NaCl) processes leading to gel formation. Simultaneously, the Q value decreased, βH+, βtotal and the contribution of βH+ to βtotal increased up to the obtained constant values at the gel state. Thus, the physicochemical properties of pectin may be in­ dicators of the particular stages of macromolecules interactions occurring during gelation.

1. Introduction Pectin is a structural polysaccharide of plant cell walls which is used in different branches of industry (e.g. the food, cosmetic and pharma­ ceutical industries) as a stabilizer, a gelling and thickening agent, emulsifier and as the carrier of bioactive substances in drug delivery systems (Gawkowska, Cybulska, & Zdunek, 2018a; Liu, Fishman, & Hicks, 2007; Sriamornsak, Prakongpan, Puttipipatkhachorn, & Ken­ nedy, 1997; Sundar, Rubila, Jayabalan, & Ranganathan, 2012). The macromolecules of pectin contain linear chains formed by differentially methyl-esterified (1–4)-α-D-galacturonosyl units with (1–2)-α-L-rhamnopyranosyl units serving as branch points which form the start of side chains composed of (1–5)-α-L-arabinofuranosyl or (1–4)β-D-galactopyranosyl residues (Schols & Voragen, 1994b; Schols, Vora­ gen, & Colquhoun, 1994a). The ability of pectin to form network and gel depends on such factors as the origin of this polysaccharide, the chem­ ical structure of its molecules (i.e. degree of methyl-esterification,

degree of amidation and the modification of hydroxyl groups), proper­ ties of pectin dispersion (i.e. the pectin concentration, the ionic strength and the composition of the dispersing medium, including pH and pre­ sence/valence of cations) and other conditions (such as temperature) (Axelos & Thibault, 1991; Liu, Fishman, Kost, & Hicks, 2003; Padival, Ranganna, & Manjrekar, 1979; Silva, Brito, de Paula, Feitosa, & Paula, 2003; Sriamornsak et al., 1997; Thom, Dea, Morris, & Powell, 1982; Tibbits, MacDougall, & Ring, 1998). In the case of low-methoxyl pectin, gelation occurs in the presence of divalent cations (e.g. Ca2+) due to intermolecular bridges formation (Axelos & Thibault, 1991; Basak & Bandyopadhyay, 2014; Capel, Nicolai, Duranda, Boulenguer, & Lan­ gendorff, 2006; Fang et al., 2008; Han et al., 2017; Ralet, Dronnet, Buchholt, & Thibault, 2001; Silva et al., 2003; Yuliarti & Mardyiah Binte Othman, 2018), in acidic conditions due to the formation of hydrogen bonds (Capel et al., 2006; Gawkowska, Cie´sla, Zdunek, & Cybulska, 2019a, b; Han et al., 2017; Yuliarti & Mardyiah Binte Othman, 2018) or in the presence of monovalent cations (e.g. Na+) at pH ≥ 7 (Fishman,

* Corresponding author. E-mail addresses: [email protected] (J. Cie´sla), [email protected] (M. Kocza´ nska), [email protected] (P. Pieczywek), j.cybulska@ipan. lublin.pl (J. Cybulska), [email protected] (A. Zdunek). https://doi.org/10.1016/j.foodhyd.2020.106524 Received 3 July 2020; Received in revised form 1 December 2020; Accepted 5 December 2020 Available online 9 December 2020 0268-005X/© 2020 Elsevier Ltd. All rights reserved.

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Chau, Kolpak, & Brady, 2001; Gawkowska et al., 2019a; Silva et al., ¨m, Schuster, & Goh, 2014; Wehr, Menzies, & Blamey, 2004). 2003; Stro The addition of monovalent cation to the pectin weak gel at a pH lower than 3.5 leads to the formation of a true gel, probably through the reduction of electrostatic repulsion between the chains (Silva et al., ¨m et al., 2014). 2003; Stro Pectin gelation in the presence of divalent cations is usually explained by a conventional ‘egg-box model’. This model was originally used to describe the interactions between the alginate molecules and Ca2+ ions. It assumes that over a dozen adjacent nonmethoxylated gal­ acturonic acid (GalA) residues should be present in two interacting macromolecules to chelate a divalent cation (Fang et al., 2007, 2008). The three steps of interaction are as follows: monocomplexation, dimerization and lateral association were proposed by Fang et al. (2007). However, further investigations, with the application of isothermal titration calorimetry and viscometry (Fang et al., 2008) showed the differences between alginate and pectin with respect to the course of gelation. Low-methoxyl pectin revealed a less demarked dimerization step starting below the stoichiometry of the ‘egg-box’ model and its lateral association was less significant than that of algi­ nate. Therefore, pectin-Ca gelation was described by the modified (shifted) ‘egg-box’ model. This model assumes that the mono­ complexation of Ca2+ and the free GalA units of the macromolecules occurred initially, and finally the ‘egg-box’ dimers were formed through the pairing of monocomplexes (Fang et al., 2008). The interaction processes of polysaccharide macromolecules in so­ lution have been intensively studied mainly using rheological methods (Fang et al., 2008; Gawkowska, Cybulska, & Zdunek, 2018b; Li, Liao, ¨m et al., 2014; Wei et al., 2009) or Thakur, Zhang, & Wei, 2018; Stro dynamic light scattering (Basak & Bandyopadhyay, 2014; Gawkowska et al., 2019a, b). Scanning electron microscopy (Basak & Bandyo­ padhyay, 2014) and atomic force microscopy (Fishman, Cooke, & Coffin, 2004; Gawkowska et al., 2019a, b; Jonassen, Treves, Kjøniksen, Smistad, & Hiorth, 2013; Pos´e, Kirby, Mercado, Morris, & Quesada, 2012; Wang, Wan, Wang, Li, & Zhu, 2018; Zdunek, Kozioł, Pieczywek, & Cybulska, 2014) are usually used to characterize the structural proper­ ties of pectin. Understanding and description of the processes of pectin interactions in solution is important both from the scientific and practical points of view, when optimal conditions of food production, waste reduction and the sustainable processing of biomass are required (European Commis­ sion, 2020). Pyrus communis L. belongs to the essential commercially cultivated fruit tree species in Europe. In 2017, about 12% of the world production of pear fruits was obtained from this region (Food and Agriculture Or­ ganization of the United Nations, 2019). Carbohydrates are the second component of pear fruit (about 14%) after water (about 85%) (Itai, 2007). Polysaccharides included in cell walls play a crucial role in maintaining the firm texture of fruit during postharvest ripening, this is important especially in the case of pears, which undergo dynamic quality changes during storage. The DASP (diluted alkali–soluble frac­ tion of pectin) fraction obtained through sequential extraction with so­ dium carbonate solution (Gawkowska et al., 2018b) represents polysaccharides which are covalently bound to the cell wall (Brummell, 2006). It was previously found, that molecules of DASP isolated from pear fruits had the ability to self–organize and form two–dimensional network on mica. The density of this network was cultivar–specific (Zdunek et al., 2014). Moreover, the content of GalA in DASP affected the stiffness of the primary cell walls of pear fruits (Zdunek, Kozioł, Cybulska, Lekka, & Pieczywek, 2016). To the best of our knowledge, the changes to the pectin surface electrical charge and the binding of counterions (H+, Na+ and Ca2+) during the cross–linking and gelation of sodium carbonate–soluble pectin have not been reported as yet. Some of the relationships between the structure and physicochemical properties of the DASP extracted from apples and dispersed in water have been marked in an earlier study

(Gawkowska et al., 2019a, b). However, the role of ionic strength adjusted by the salts of mono- and divalent cations was not considered there. It was hypothesized, that such properties of the DASP as the surface electrical charge, hydrogen ions binding and counterions binding are modified during the concentration–affected cross–linking process which occurs in the presence of cations with a different valence. Therefore, these parameters may be indicators of particular stages of interactions of polysaccharide macromolecules in solution and gel formation. The measurements of particles size (Dynamic Light Scattering), electrophoretic mobility (Laser Doppler Electrophoresis) and pH as well as a potentiometric titration were performed to verify the above as­ sumptions for DASP dispersions in pure water and salt solutions (NaCl or CaCl2 at 30 mM ionic strength). The analyses of liquid samples (1.84⋅10− 2 – 1.84⋅10− 1% w/v DASP) were supported by the AFM study of air–dried material. 2. Material and methods 2.1. Characterization of DASP fraction The parenchyma tissue without skin was obtained from pear fruits (Pyrus communis L.) cultivar ‘Conference’. The material was blended and then stored in a deeply frozen state at − 80 ◦ C. After de-freezing, the alcohol insoluble residue (AIR) and the DASP fraction were obtained using the previously described procedure of sequential extraction (Gawkowska et al., 2018b). The lyophilized DASP was dialysed against ultrapure water (MilliQ) using dialysis tubular membranes (Zellu­ Trans/Roth MWCO 3500) and lyophilized again. This material (here­ inafter referred to as DASP) was then used for further analyses. The water content in DASP was evaluated by drying the sample (~20 mg) to achieve a stable mass under a nitrogen atmosphere in DVS Intrinsic apparatus (Surface Measurement Systems Ltd., London, UK) which was calibrated using saturated LiCl, MgCl2 and NaCl slurries at 20 ◦ C. The GalA content in DASP, expressing the total content of uronic carboxylic groups, was determined colorimetrically at 530 nm (Continuous Flow Analyser Skalar, The Netherlands) in completely decomposed (96% sulphuric acid with di-sodium tetraborate) samples containing 3-phenyl phenol (Blumenkrantz & Asboe-Hansen, 1973). Mono-GalA solutions (20–100 μg/ml) were used as standards. The degree of methyl esterification (DM) of DASP was determined by analysing the FT-IR spectra (resolution of 4 cm− 1; area normalization was applied) at a range of 1800–1500 cm− 1 (Nicolet 6700 FT-IR spec­ trometer, Thermo Scientific, Waltham, MA, USA). DM was calculated as the ratio of peak intensity at 1730 cm− 1 to the sum of the peak intensities at 1730 cm− 1 and 1593 cm− 1. The theoretical frequencies of the FT-IR bands representing the stretching vibration of the carbonyl group in alkyl esters and the antisymmetric stretching of carboxylic anions are 1740 cm− 1 and 1630–1600 cm− 1, respectively, but the shift in the bands can be observed for natural samples due to e.g. the bridging of the polysaccharide chains by divalent cations (Kyomugasho, Christiaens, Shipingelman, Van Loey, & Hendrickx, 2015; Szymanska-Chargot & Zdunek, 2013). The content of potassium, sodium, calcium and magnesium in DASP was determined using Atomic Absorption Spectroscopy (ContrAA 300, Analytik Jena AG, Jena, Germany). DASP (about 200 mg per porcelain crucible) was ashed at 500 ◦ C overnight in a muffle furnace. The ash was dissolved in 5 ml of 20% HCl and then filtered into a 50-ml volumetric flask. The solution was diluted to a volume of 50 ml with ultrapure water (MiliQ) and mixed vigorously. All analyses were performed in triplicate. 2.2. Preparation of DASP dispersions DASP was dispersed in ultrapure water (MilliQ) and salt solutions (NaCl and CaCl2) with an ionic strength (I) of 30 mM to obtain a 2

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concentration range of about 2⋅10− 4 – 2⋅100% w/v (i.e.: 1.84⋅10− 4, 4.60⋅10− 4, 1.84⋅10− 3, 4.60⋅10− 3, 1.84⋅10− 2, 4.60⋅10− 2, 1.84⋅10− 1, 4.60⋅10− 1, 9.20⋅10− 1, 1.38⋅100 and 1.84⋅100% w/v). The samples were stirred in tightly closed tubes using a rotator for 24 h at 20 ◦ C and then their properties were studied.

participation of various functional groups in the formation of the total surface electrical charge (Jozefaciuk & Szatanik-Kloc, 2004; Nederlof, De Wit, Riemsdijk, & Koopal, 1993). The derivation of the formula for this function and the approximations used are precisely described in the literature (Jozefaciuk & Szatanik-Kloc, 2004; Nederlof et al., 1993). The following relationships were used for the f(pKapp,i) calculation (Jozefa­ ciuk & Szatanik-Kloc, 2004; Nederlof et al., 1993) ( ) ( ) Qv (pH) = Qv pKapp,i ​ and ​ f pKapp,i ( )( ( ) ) = 1/Qv ,total dQv pKapp ,i /dpKapp,i ,

2.3. Determination of particles size and electrophoretic mobility The particle size (Dynamic Light Scattering with a non-invasive back scatter (173◦ ) detection – DLS (International Standard ISO 22412, 2017)) and electrophoretic mobility (EM) (Laser Doppler Electropho­ resis – LDE (Mayinger, 1994)) of DASP dispersed in water and salt so­ lutions (NaCl and CaCl2, I = 30 mM) were determined using a Zetasizer Nano ZS (Malvern Ltd., Malvern, UK) apparatus equipped with a He-Ne laser (633 nm). Measurements were performed in triplicate at 20 ◦ C. The relative mean hydrodynamic diameter (Relative Zave) was defined as a given value of the mean hydrodynamic diameter divided by the lowest value among those obtained at a DASP concentration of 1.84⋅10− 4% w/v in water, NaCl and CaCl2 solutions. Knowing the mean hydrodynamic diameter (d) of the dispersed particles (DLS method assuming particle sphericity), their EM and the viscosity of the dispersing medium (η), the surface electrical charge (Q) was calculated as previously (Gawkowska et al., 2019a):

where Qv was the variable surface charge, Qv,total was the total surface electrical charge which corresponded to the total amount of titrant used, and dQv(pKapp,i)/dpKapp,i was the first derivative of Qv(pKapp,i) function. The general acid-base character of DASP was described by the apparent dissociation constant (pKapp), i.e. the pH at which the disso­ ciation degree was equal to 0.5, and the average apparent dissociation constant (pKapp,ave) which took into account the contribution of surface functional groups with a given pKapp,i (Jozefaciuk & Szatanik-Kloc, 2004; Nederlof et al., 1993) and was determined at pH range of 3–10: n ∑

pKapp,ave =

) ( pKapp,i f pKapp,i ,

i=1

where pKapp,i was the pKapp of a given functional group and f(pKapp,i) was a value of distribution function of apparent dissociation constants which was obtained for this group. The intrinsic dissociation constant (pK0) of DASP was calculated using the potentiometric data, following the procedure described by Ralet et al. (2001). The pH values of all of the DASP dispersions within the full range of the DASP concentration were determined using WP pH Spear Tester (Oakton Instruments, Vernon Hills, USA). The results of the pH mea­ surements and the pKapp,ave values were applied to the modified Henderson-Hasselbalch equation (Bodnarchuk et al., 2017) for the calculation of the degree of hydrogen ions binding (βH+): ( ) log[βH+ /(1 − ​ βH+ )] = pKapp,ave − pH /n

Q ​ = ​ (3⋅η⋅EM)/d In the electric field, the particle moves with the counterions which are most strongly bound to its surface. Therefore, the calculated Q corresponds to the surface electrical charge which is partially shielded. In these studies, it was assumed that counterions binding was the source of this shielding. Assuming that the highest absolute value of Q (i.e. Qmax), determined in a diluted DASP dispersion in water (pH = 7.58 ± 0.03), refers to almost fully ionized functional groups (generally, the dissociation of surface functional groups at pH < 7.00 is a source of 97–99% of the total variable surface electrical charge; see Fig. 3), the degree of counterions binding (βtotal) at a given Q was calculated: βtotal = (Qmax − Q)⋅(100 ​ %)/Qmax. 2.4. Potentiometric measurements

The values of n ranged from 1.5 to 3.6, depending on dispersing medium and the range of DASP concentration. Next, the contribution of hydrogen ions binding (βH+) to the total counterions binding ((βtotal) (i. e. 100%⋅ βH+/βtotal) was calculated.

A potentiometric titration (pH range of 3–10) of selected DASP sus­ pensions (1.84⋅10− 2, 4.60⋅10− 2, 1.84⋅10− 1% w/v) in water and salt so­ lutions (NaCl and CaCl2, I = 30 mM) was carried out using an automatic titrator (TitraLab® AT1000, Hach Lange Ltd., Wroclaw, Poland) at a temperature of 20 ± 2 ◦ C. Considering, that the dispersing medium composition and the DASP concentration may affect the spatial structure of DASP in liquid media and the dissociation of surface functional groups, the investigations were carried out for samples of various compositions. The samples were chosen on the basis of DLS results (Fig. 1a) covering the DASP concentration range where an increase in relative diameter started. The initial pH of the samples was established at 10, using 1 M NaOH in the case of the water and NaCl systems and 1 M Ca(OH)2 for the CaCl2 systems, 0.1 M HCl was used as a titrant. The experiment was performed in triplicate and the already used procedures ´ ska, Łukowska, Bieganowski, & Janc­ were applied (Cie´sla, Kopycin zarek, 2016; Jozefaciuk & Szatanik-Kloc, 2004). The obtained data were used for further calculations. In a case of biopolymer which is dispersed in a liquid, the surface functional groups with a different chemical structure as well as the groups with the same chemical structure (e.g. carboxyl groups) but different ability to dissociation (e.g. due to a different ‘local environ­ ment’) are exposed to water. Considering the ability of surface func­ tional groups to dissociate, the surface is heterogeneous. A given surface functional group (i) can be characterized by its apparent dissociation constant (expressed as pKapp,i). The so-called ‘distribution function of the apparent dissociation constants’ (f(pKapp,i) is used to show the

2.5. An AFM analysis of the DASP fraction The 60 μl of DASP suspensions (1.84⋅10− 2, 4.60⋅10− 2 and 1.84⋅10− 1% w/v in water, NaCl and CaCl2 solutions; the same disper­ sions as those used for potentiometric titration) were drop-deposited and uniformly distributed (spin coater, SPS-Europe B.V., Midden Eng­ weg 41, NL-3882 TS PUTTEN, The Netherlands) onto a freshly cleaved mica (EMS, Hatfield, PA, USA). The samples were air dried at room temperature and stored in a desiccator. The experiment was performed in ambient air (relative humidity around 28%) at room temperature (21 ± 1 ◦ C). A Multimode 8 with a Nanoscope V controller (Bruker, Billerica, MA, USA) was used for im­ aging in the automatic PeakForce Tapping mode (ScanAsyst). A silicon tip on a nitride cantilever (Bruker) with the nominal radius of the py­ ramidal tip at 2 nm and a nominal spring constant of 0.4 N/m was used. The scanning area was 4 μm2 (aspect ratio 1:1) at a resolution of 512 × 512 points and a scanning linear velocity of 0.9 Hz. Nine images were collected for each sample. The results were used to visualize the struc­ ture of the samples representing the DASP concentrations at which the self-assembly of the molecules was detected by DLS.

3

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Fig. 1. a) Effect of DASP concentration on the relative mean hydrodynamic diameter; bars mean standard deviation; different letters refer to significantly different results (two-way ANOVA, post-hoc Tukey HSD test, p < 0.05), b) The AFM images of selected samples.

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2.6. Statistical analyses

discussed above, also revealed differences in the DASP structure which were dependent on both the polysaccharide concentration and the dispersing medium used. A crosslinked structure formed by short mac­ romolecules was visible for water systems at DASP concentrations of 1.84⋅10− 2 and 4.60⋅10− 2% w/v. An increase in the pectin content to 1.84⋅10− 1% w/v led to the formation a fibrous network. In the case of NaCl solution as the dispersing medium, beginning from the DASP concentration of 1.84⋅10− 2% w/v, branched fibres could be observed and their number increased with increasing DASP content. At the DASP concentration of 1.84⋅10− 1% w/v a network similar to that determined for DASP dispersed in water was visible but its density was lower than that in a pure aqueous system. In the presence of CaCl2, the nano­ particles were visible after samples drying. An increase in DASP con­ centration resulted in an increase in their size which finally led to gel formation. A similar effect of the high concentration of calcium salt (i.e. the presence of rods and particles) was previously determined for algi­ nates (Wang et al., 2018). It was also pointed out by Basak and Ban­ dyopadhyay (2014), that obtaining a gel structure with pectin in the presence of Ca2+ was preceded by an increase in the size of their flocks in solution. Therefore, the AFM images obtained (Fig. 1b) confirmed that in the analysed range of DASP concentration, where the relative diam­ eter of the dispersed particles increased, the self-assembly of molecules occurred. It was also shown previously, based on the results of viscosity measurements and AFM analyses of a pure aqueous dispersion of DASP isolated from apples (Gawkowska, Cie´sla, Zdunek, & Cybulska, 2019b), that an increase in the relative hydrodynamic diameter with increasing pectin content is connected with the process of crosslinking which leads to the formation of gel-like structure (at about 2% w/v DASP). The starting point of a significant increase in Relative Zave was determined, in this case, at a DASP concentration range of 1⋅10− 1 – 5⋅10− 1% w/v (Gawkowska et al., 2019b). The electrophoretic mobility (EM) of DASP extracted from pears was negative and its value was strongly influenced by the dispersing medium used (Fig. 2a). The ionic strength of the NaCl and CaCl2 solutions was the same. Despite this, the absolute values of EM determined in the CaCl2 solution were close to zero, whereas in the NaCl solution they were in the range of 2.25–3.30 μmcmV− 1s− 1 being close to those obtained in H2O (2.25–4.2 μmcmV− 1s− 1). As it can be seen, there were no differ­ ences between the EM of the DASP dispersed in the H2O and NaCl so­ lution for two the lowest concentrations of DASP. An increase in DASP concentration from 4.60⋅10− 4 to 1.84⋅10− 3% w/v resulted in an increase of absolute value of the EM of the particles dispersed in water. For the concentration range of 1.84⋅10− 3 – 1.84⋅10− 1% w/v, the absolute value of EM (3.7–4.2 μmcmV− 1s− 1) was not affected by the DASP content. Next, an increase in the pectin concentration to 4.60⋅10− 1% w/v led to an increase in the absolute value of EM (3.2–3.5 μmcmV− 1s− 1) but raising the DASP concentration further did not have an effect on EM. In the case of the NaCl solution, the EM of the dispersed particles was not affected by the DASP content. For DASP in the CaCl2 solution at a con­ centration of 1.84⋅10− 4 – 1.84⋅10− 1% w/v, there was no effect of the amount of DASP on the absolute value of EM (0.30–0.45 μmcmV− 1s− 1). An increase in the DASP concentration from 1.84⋅10− 1 to 1.38⋅100% w/ v resulted in an increase in the absolute value of EM from about 0.4 to 1.2 μmcmV− 1s− 1. As the result of a further increase in DASP concen­ tration from 1.38⋅100 to 1.84⋅100% w/v a rapid decrease in the absolute value of EM to about 0.2 μmcmV− 1s− 1 occurred. Negative values of the EM of DASP dispersed in water and salt so­ lutions are consistent with the results previously obtained for low­ –methoxyl DASP extracted from apples (Gawkowska et al., 2019a) and high–methoxyl citrus pectin (Schmidt, Schütz, & Schuchmann, 2017) under different acid-base conditions. Most of the published data refers to the effect of pH (Harnsilawat, Pongsawatmanit, & McClements, 2006; Lutz, Aserin, Wicker, & Garti, 2009) and salt (Kuljanin et al., 2014) on the electrokinetic potential of the polysaccharide dispersions. This po­ tential characterizes the electrical double layer around the particle and is determined at the ‘slipping plane’ separating the layer of ions attached

The influence of both the dispersing medium and the DASP con­ centration on the values of the studied physicochemical parameters were analysed using two-factors ANOVA and a post-hoc Tukey HSD test at the 0.05 significance level (Statistica 13.1 software, StatSoft, Cracow, Poland). 3. Results and discussion DASP (stored in a vacuum chamber) contained 8.04 % wt. of water, which was determined by drying under a nitrogen atmosphere. The results of chemical analyses showed that 1 g of dry sample contained 2.76 ± 0.15 mmol of GalA, 0.04 ± 0.01 mmol of potassium, 3.08 ± 0.01 mmol of sodium, 0.02 ± 0.01 mmol of magnesium and 0.09 ± 0.01 mmol of calcium. DM was estimated on the basis of FT-IR spectra to be about 3.3 ± 2.6%. A high content of GalA in DASP corresponded to the other results obtained for this fraction of pectin isolated from pears (Zdunek et al., 2014, 2016) and apples fruit (Gawkowska et al., 2019a). The amount of GalA in the DASP extracted from the pear (Pyrus com­ munis L.) cultivar ‘Conference’ is very sensitive to the time of the post­ harvest storage of the fruits and decreases with the extension of the storage period (Zdunek et al., 2016). The process of DASP extraction (the use of sodium carbonate and alkaline conditions) could lead to de-methyl-esterification (Pos´ e et al., 2012) and result in a high sodium content in the obtained fraction. The processes of the self-assembly of DASP in H2O and salt (NaCl and CaCl2) solutions at an ionic strength of 30 mM was monitored by the measurement of the mean hydrodynamic diameter of the dispersed particles at different concentrations of DASP. Even for the lowest con­ centration of DASP (1.84⋅10− 4% w/v) the particles with diameters of a few-hundred-nanometres (162 ± 13 nm in H2O, 203 ± 126 nm in NaCl solution and 1350 ± 323 nm in CaCl2 solution) were detected using the DLS method. Values of about 270 nm were obtained by Gawkowska et al. (2019a) for the DASP extracted from apples and dispersed in water (5⋅10− 3% w/v) under acidic conditions. It should be noted that the hydrodynamic diameter determined using the DLS method refers to a spherical particle with its hydration layer (International Standard ISO 22412, 2017). In the case of a polyelectrolyte, the diffusion of dispersed particles, which is the basis of the diameter calculation, is affected by their concentration and interaction with counter-ions as well as the ionic ¨rster & Schmidt, strength and composition of the dispersing medium (Fo 1995; Muthukumar, 2016). It was found previously (Gawkowska et al., 2019a, b), that the relative mean hydrodynamic diameter can be a good indicator of the morphological changes occurring in the dispersion of DASP extracted from apples. In the present investigations, in order to compare the three systems studied, the hydrodynamic diameter of the dispersed particles was divided by the lowest diameter among those determined for the most diluted DASP suspension in H2O, NaCl and CaCl2 solutions. The obtained results are shown in Fig. 1a. In the full range of DASP concentration, the size of the particles dispersed in the CaCl2 solution was significantly higher than in the H2O and NaCl solu­ tion, which may have been the result of the bridging action of the divalent cation leading to flocks formation (Fang et al., 2008; Yuliarti & Mardyiah Binte Othman, 2018). By analysing the shape of the depen­ dence between the relative mean diameter and the content of DASP in the samples, two distinct areas can be distinguished. The first one was specific for a low-concentration dispersion, where the relative diameter of the particles was not influenced by the DASP content. In the second region, an increase in the DASP concentration resulted in an increase in the relative diameter. The range of the DASP concentration, at which the transition between these two areas occurred, was dependent on the dispersing medium. For DASP dispersed in CaCl2, H2O and NaCl this was: 1.84⋅10− 2 – 4.60⋅10− 2% w/v, 4.60⋅10− 2 – 1.84⋅10− 1% w/v and 1.84⋅10− 1 – 4.60⋅10− 1% w/v, respectively. The AFM images (Fig. 1b), which were obtained for the DASP concentration covering the ranges 5

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Fig. 2. The influence of DASP concentration on a) electrophoretic mobility and b) the surface electrical charge of pectin dispersed in different media; bars mean standard deviation; different letters refer to significantly different results (two-way ANOVA, post-hoc Tukey HSD test, p < 0.05).

to the surface from the bulk solution. However, in the case of soft par­ ticles, the ‘slipping plane’ is not well defined. The electrokinetic po­ tential is calculated using the EM value applied to different models and its sign (negative/positive) is the same as the sign for EM (Delgado, ´lez-Caballero, Hunter, Koopal, & Lyklema, 2007). Gonza The negative values of the DASP EM were a consequence of the negative surface electrical charge of macromolecules, which results mainly from the dissociation of the carboxyl groups of the uronic acid units (Kohn, 1971; Thakur, Singh, Handa, & Rao, 1997). The visible (Fig. 2a) extensive difference between the EM of DASP dispersed in NaCl and CaCl2 solutions resulted from the different action of the mono- and divalent cations. The presence of calcium cations led to the neutraliza­ tion of the negative charge of the surface –COO- groups, as in the case of the sodium cations, and the formation of intermolecular bridges and molecule complexes – unlike in NaCl solution (Fig. 1b). The movement of large self-assemblies in the electrical field was slower than that of single macromolecules or their small groups (Fig. 1a). Considering the changes in the relative hydrodynamic diameter in all of the systems studied, a decrease in the absolute value of EM with increasing con­ centration of DASP would be expected. However, the EM value depends not only on the size of the particle but also on its surface electrical charge (Delgado et al., 2007). Therefore, the effect of DASP concentra­ tion on the electrical charge revealed by the particle surface area unit (Q) was determined. It should be stressed, that this Q value, which was calculated using EM and the hydrodynamic diameter with the assump­ tion of the particle sphericity, is an approximated value of the real electrical charge of the particle surface (Lyklema, 2011). The results obtained are shown in Fig. 2b. The lines illustrate the shapes of the functions (f(x) = a/(b + c⋅exp(d⋅x))) which were fitted to the mea­ surement data using a non-linear regression with the determination coefficients equaled to 0.9966, 0.9890 and 0.9093 for DASP dispersed in water, NaCl and CaCl2 solutions, respectively. The highest values of Q were obtained for DASP dispersed in water and NaCl solution at the pectin concentration range of 1.84⋅10− 4 – 1.84⋅10− 2% w/v and 1.84⋅10− 4 – 4.60⋅10− 2% w/v, respectively. In these ranges of DASP concentration there were no differences between the two systems analysed above (pectin in H2O and NaCl). A further increase in DASP concentration to 9.20⋅10− 1% w/v led to a decrease in the Q value. Simultaneously, the Q value at a given concentration of DASP in NaCl solution was higher than in H2O. This could be the result of differences in DASP structure formed in these two dispersing media (Fig. 1b). In the case of DASP present in NaCl solution, more surface functional groups could be exposed to contact with the bulk of the liquid. Some of the hydrogen cations were removed from the particle surface to the bulk solution due to the competition between them and the sodium ions.

However, the sodium cations, which were not strongly connected to the surface –COO- groups, under the electrical field action were also moved far from the particle surface, that led to an increase in the negative value of Q. In the case of the DASP content ranged from 9.20⋅10− 1 – 1.84⋅100% w/v the difference between the H2O and NaCl systems ceased. The Q value of the particles was close to zero and its value was not affected by the DASP concentration. For the DASP dispersed in CaCl2 solution, the Q values were very low. As the DASP concentration ranged from 1.84⋅10− 4 to 1.84⋅10− 2% w/v the values of Q were constant and corresponded to those of particles dispersed in the H2O and NaCl sys­ tems at the highest DASP content. An increase in DASP concentration from 1.84⋅10− 2 to 4.60⋅10− 2% w/v resulted in a further decrease in the value of Q. Starting from a DASP concentration of 4.60⋅10− 2% w/v, the Q value was independent of the DASP content. Therefore, based on the Q analysis for all of the dispersing media used, three ranges of DASP concentration could be distinguished; the first one – for the low content of DASP, where Q was high and not-affected by DASP concentration, the second one – where Q decreased with increasing DASP concentration (in the process of crosslinking in H2O and NaCl solution and large selfassembly formation in CaCl2 solution), and the last one – where Q was almost totally neutralized and independent of the DASP content (the pectin network/gel formed). To the best of our knowledge, there have not been any works published to date showing the connection between the processes of concentration-affected DASP gelation in different media, and the changes in EM and Q values for this polysaccharide. In order to characterize the electrical and acid-base properties of DASP extracted from pears, a potentiometric titration was carried out. An experiment was performed for the DASP concentration of 1.84⋅10− 2, 4.60⋅10− 2 and 1.84⋅10− 1% w/v, for which a decrease in the Q values with increasing DASP content was observed (Fig. 2b). The distribution functions of the apparent dissociation constants (f(pKapp,i)) of the sur­ face functional groups of DASP dispersed in different media are shown in Fig. 3. In all cases, the shape of the functions was similar, indicating that the highest influence on the surface electrical charge was from the groups dissociating at low pH. The functional groups with pKapp,i > 7.0 generated only 1.1–3.2% of the total variable surface electrical charge. These results correspond to those obtained earlier using EM data for DASP extracted from apples (Gawkowska et al., 2019a). It was found using potentiometric titration, that the carboxyl (pKapp 3–5) and hy­ droxyl (pKapp > 9) groups played a significant role in the generation of a surface electrical charge for pectic acid in citrus peels (Schiewer & Iqbal, 2010) and commercially available low–methoxyl citrus pectin (Balaria & Schiewer, 2008). However, considering the pH of plant cells and the pH conditions applied in food processing, the acidic functional groups 6

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Food Hydrocolloids 113 (2021) 106524

Table 1 The intrinsic (pK0), apparent (pKapp) and the average apparent (pKapp,ave) dissociation constants calculated for different concentrations of DASP in water and salt solutions. pK pK0

Dispersing medium

pKapp, ave

1.84⋅10−

2

4.60⋅10−

1.84⋅10−

2.85 ± 0.02ab 2.92 ± 0.06ab

2.90 ± 0.00ab

3.02 ± 0.11a

4.51 ± 0.23a

4.33 ± 0.03ab

NaCl CaCl2

4.26 ± 0.14ab 3.48 ± 0.19c 3.42 ± 0.17c

3.92 ± 0.09b 3.49 ± 0.17c

4.05 ± 0.07b 3.52 ± 0.13bc

H2O

4.72 ± 0.71a

4.58 ± 0.53ab

NaCl

3.85 ± 0.56bc

4.18 ± 0.33abc 3.87 ± 0.21bc

4.40 ± 0.18abc 4.17 ± 0.36abc 3.77 ± 0.26bc

CaCl2

c

3.74 ± 0.37

b

1

2.71 ± 0.12 2.95 ± 0.07ab

H2O

b

2

2.70 ± 0.13 2.79 ± 0.11ab 3.02 ± 0.11a

H2O NaCl CaCl2

pKapp

DASP concentration (%w/v)

*different letters mean statistically significantly different results obtained from the two-way ANOVA and post-hoc Tukey HSD test (p < 0.05).

some functional groups. To avoid the polyelectrolyte-effect, the pK0 which describes the dissociation of the monomer unit of polysaccharide, is usually determined at a high ionic strength. However, such obtained values are not an appropriate way to describe systems at a low ionic strength (Cooper et al., 2006). The values of pK0 published for pectin ranged from 2.75 to 3.05, they were independent of the degree of esterification (Ralet et al., 2001). The apparent dissociation constant (pKapp) is sensitive to the ionic strength of the dispersing medium and the type of counterions in solu­ tion (Vleugels, Ricois, Voets, & Tuinier, 2018). In the case of the systems studied (Table 1), the pKapp values increased in the following order ac­ cording to the dispersing media used: CaCl2 < NaCl < H2O, showing that in the presence of calcium cations the strongest acidic groups are deprotonated (as can be seen in Fig. 3). A similar effect was observed for pectic acid, when pKapp was equal to 4.41 ± 0.02 with KOH used as a titrant and 3.81 ± 0.01 with Ca(OH)2, whereas the pKapp of GalA was 3.48 ± 0.01 which was independent of the base solution applied (Kohn, 1973). The values of pKapp determined by potentiometric titration for pectin dispersed in water and 10 mM KCl solution were 4.65 ± 0.07 and 4.52 ± 0.19, respectively (Vleugels et al., 2018). The values of pKapp (Table 1) were slightly affected by the DASP content (a tendency to increase with increasing concentration of DASP). The average apparent dissociation constant (pKapp,ave) characterizes the general acid-base character of the surface at a given range of pH (Jozefaciuk & Szatanik-Kloc, 2004; Nederlof et al., 1993). Its values were slightly higher than pKapp because they included the effect of surface functional groups dissociating at high pH values. The influence of the dispersing medium on pKapp,ave was the same as that observed for pKapp. The results obtained for DASP dispersed in water (4.40–4.72) were comparable with a pKapp,ave of 4.64 ± 0.41 determined using EM for this fraction extracted from apples (Gawkowska et al., 2019a). The content of DASP in solution had an influence on the pH value (Fig. 4a). For an aqueous dispersion over the full range of DASP con­ centration, the pH decreased (from 7.58 ± 0.03 to 4.56 ± 0.01) with increasing DASP content. The pH of the DASP dispersions in salt solu­ tions was lower than in the water as a consequence of the exchange of hydrogen ions of surface groups and the sodium and calcium cations. In the case of DASP dispersed in NaCl solution, for a pectin concentration range of 1.84⋅10− 4 – 4.60⋅10− 3% w/v the pH decreased from 6.43 ± 0.06 to 5.75 ± 0.01. Next, an increase in the DASP content to 1.84⋅10− 1% resulted in a pH decrease to 4.57 ± 0.01. The change in DASP content from 1.84⋅10− 1 to 1.84⋅100% w/v led to pH of 4.29 ± 0.01 being obtained. For the samples where CaCl2 solution was used as the

Fig. 3. The distribution functions of apparent dissociation constants (f(pKapp,i)) of surface functional groups, which were determined for a) 1.84⋅10− 2% w/v, b) 4.60⋅10− 2% w/v and c) 1.84⋅10− 1% w/v DASP in different media.

are essential. Pectin is an important component of the fruits as well as the other organs of plants. The presence of pectin in the cell walls of wheat, rye, clover and lupine roots is the source of about half of their surface electrical charge (Szatanik-Kloc, Szerement, Cybulska, & Joze­ faciuk, 2017). Dissociation constants were used to describe the acid-base character of DASP extracted from pears. The data are summarized in Table 1. The intrinsic dissociation constant (pK0) was not affected by the DASP concentration. Also, the dispersing medium did not have an in­ fluence on the pK0 value with the exception of 1.84⋅ 10− 2% w/v DASP dispersion for which the results obtained in H2O were slightly lower than in NaCl solution and significantly lower than those produced by the CaCl2 solution. The presence of salt should facilitate the process of DASP deprotonation but at a low content of DASP and with a relatively high amount of salt the macromolecules could stick to each other blocking 7

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Food Hydrocolloids 113 (2021) 106524

Fig. 4. Effect of DASP concentration in different media on a) pH, b) degree of hydrogen ion binding, c) degree of ion binding and d) the contribution of hydrogen ion binding to total counterions binding; the bars indicate standard deviation; different letters refer to significantly different results (two-way ANOVA, post-hoc Tukey HSD test, p < 0.05).

dispersing medium, at a DASP concentration range of 1.84⋅10− 4 – 4.60⋅10− 3% w/v the pH changed from 6.34 ± 0.01 to 6.17 ± 0.02. An increase in the DASP concentration to 1.84⋅10− 1% w/v led to a decrease in pH to 3.69 ± 0.01, however, an increase in concentration to 4.60⋅10− 1% w/v did not have an effect on pH. However, further increase in DASP content to 1.84⋅100% w/v resulted in an increase of pH to 4.14 ± 0.01. A decrease in pH from about 5.80 to 4.20 with the DASP con­ centration increasing from 1⋅10− 3 to 2⋅100% w/v was determined by Gawkowska et al. (2019b) for DASP extracted from apples and dispersed in pure water. Such changes in pH with increasing DASP content in solution confirm the acidic character of this polysaccharide. The values of pH and pKapp,ave were applied to determine the degree of hydrogen ions binding (βH+) by DASP (Fig. 4b). Up to a DASP con­ centration of 1.84 ± 10− 2% w/v in salt solutions and 4.60⋅10− 2% w/v in water, the mean value of βH+ was lower than 10% and independent of DASP concentration. For pure aqueous dispersions of pectin, the βH+ increased with increasing DASP content until a value of 41 ± 8% was obtained for the most concentrated system. A similar effect was previ­ ously observed for this pectin fraction extracted from apples, however, βH+ was higher due to the higher content of GalA in samples (Gaw­ kowska et al., 2019b). For DASP, a concentration increase from 1.84⋅10− 2 to 4.60⋅10− 2% w/v in NaCl solution produced a βH+ increase from 8 ± 5% to 24 ± 11%. Further increase in DASP content resulted in a slight increase of βH+ to 44 ± 17% in the most concentrated dispersion. In the case of the DASP dispersion in CaCl2 solution, a significant

increase of βH+ (from 3 ± 2% to 60 ± 10%) was visible when the DASP concentration increased from 1.84⋅10− 2 to 1.84⋅10− 1% w/v. Next an increase in DASP content to 9.20⋅10− 1% w/v led to a slight lowering of βH+ to 41 ± 12%. Finally, βH+ was equal to 31 ± 11%. An increase in βH+ with the increasing concentration of acidic DASP was connected with the intermolecular interactions leading to hydrogen bonds formation (Capel et al., 2006; Gawkowska et al., 2019b; Han et al., 2017). A decrease in βH+ determined at the highest concentrations of DASP in CaCl2 solution could be a consequence of syneresis, i.e. the process of sol and gel separation. The ion selective electrode was sensitive to free hydrogen ions in sol, the concentration of which was lower than that determined in homogeneous systems, which gave higher values of pH and resulted in a lower βH+ being calculated. The DASP samples at concentrations ranging from 9.20⋅10− 1 – 1.84⋅100% w/v in CaCl2 had a gel form. The counterions binding (βtotal) calculated on the basis of surface electrical charge of the dispersed DASP was close to 100% for all of the range of the DASP concentration in CaCl2 solution (Fig. 4c). In the case of DASP dispersed in NaCl solution, for the concentration range of 1.84⋅10− 4 to 4.60⋅10− 2% w/v the mean values of βtotal were at a level of 33–46% and did not differ significantly. An increase in DASP content resulted in an increase in βtotal up to 97% at the highest concentration of DASP. For the low concentrations of DASP in water (1.84⋅10− 4 to 1.84⋅10− 2% w/v) the mean values of βtotal varied from 17 to 27% and were not affected by the DASP content. A further increase in DASP 8

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Food Hydrocolloids 113 (2021) 106524

concentration led to an increase in βtotal of up to 97% in the most concentrated system, similar to the result for DASP dispersed in the NaCl solution. In the case of the dependence between DASP concentration and the contribution of hydrogen ions binding to the total counterions binding, the shape of the curve obtained for DASP dispersions in the NaCl solu­ tion was similar to that of the dispersions in the CaCl2 solution (Fig. 4d). For the DASP concentration range of 1.84⋅10− 4 – 4.60⋅10− 3% w/v in the NaCl solution the βH+ contribution was lower than 10%. An increase in DASP concentration to 4.60⋅10− 2% w/v resulted in an increase of the βH+ contribution to 62 ± 12%. A further increase in the DASP content led to a slight reduction in this contribution to 45 ± 17%. In the CaCl2 solution at a DASP concentration of 1.84⋅10− 4 – 1.84⋅10− 2% w/v, the βH+ contribution was the lowest (below 6%) and it was not affected by the DASP content. In this range of DASP concentration at pH > 4.5, an interaction between Ca2+ and polysaccharide is very effective what leads to self-assembling and even precipitation (Capel et al., 2006). An increase in DASP concentration to 1.84⋅10− 1% w/v led to an increase in the contribution of βH+ to 60 ± 10% but a further rise in the concen­ tration resulted in a slight decrease in this contribution to a value of 31 ± 11%. For a pure aqueous dispersion of DASP at a concentration range of 1.84⋅10− 4 – 4.60⋅10− 2% w/v the contribution of βH+ to βtotal was lower than 10% (as it was for DASP dispersed in salts solutions) and there was no significant effect of DASP content on its value. An increase in the DASP concentration to 1.84⋅100% w/v resulted in an increase of this contribution to 42 ± 8%. It should be noted that other cations present in the extracted fraction, especially Na+ at the Na+: GalA mole ratio of 3.08:2.76, participated in the neutralization of the negative electrical surface charge of DASP. The results presented in Fig. 4b and d showed, that independent of the dispersing medium used, an increase in the DASP content over some threshold level was connected with an increase in both βH+ and the contribution of βH+ to βtotal. The final contribution of βH+ to βtotal which was determined for gel-like samples ranged from 30 to 45% and was obtained or even exceeded at a lower DASP concentration in salt solu­ tions than in water. This confirmed that H+ ions were involved in hydrogen bonds formation between DASP macromolecules in selfassemblies (CaCl2 solution), branched fibres (NaCl solution), well cross-linked structures (NaCl solution and water) and, finally, gels.

of DASP in NaCl and water and the increase in size of DASP selfassemblies in CaCl2 confirmed by AFM studies; 3) 9.20⋅10− 1 – 1.84⋅100% w/v – the insignificant effect of DASP con­ centration on the physicochemical parameters in all dispersing media; the Q values close to zero, whereas βtotal values close to 100%; βH+ and its contribution to βtotal at the level of 30–40%. The studied physicochemical properties of DASP dispersed in different media could be indicators of the particular stages of poly­ saccharide interactions leading to gel formation. CRediT authorship contribution statement Jolanta Cie´sla: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Visualization, Writing - original draft, ´ ska: Investigation, Writing - review & editing. Magdalena Koczan Validation, Writing - original draft, Writing - review & editing. Piotr Pieczywek: Investigation, Validation, Data curation, Visualization, Writing - original draft, Writing - review & editing. Justyna Cybulska: Resources, Writing - review & editing. Artur Zdunek: Supervision, Funding acquisition, Writing - review & editing. Declaration of competing interest No conflicts of interest are declared for any of the authors. Acknowledgements We would like to express our gratitude to Prof. Monika Szymanska – Chargot from the Department of Microstructure and Mechanics of Bio­ materials at the Institute of Agrophysics Polish Academy of Sciences for the valuable instructions and the possibility of the FTIR spectrometer use, to Dr. Diana Gawkowska from the same department, for her help during the sequential extraction and GalA determination and to Dr. Patrycja Boguta from the Department of Physical Chemistry of Porous Materials for performing the Na, K, Ca and Mg analysis using AAS method. This work was supported by the National Science Centre, Poland [grant number DEC-2015/17/B/NZ9/03589].

4. Conclusions

References

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1) 1.84⋅10− 4 – 4.60⋅10− 2% w/v – no effect of DASP content; very low (<10%) values of both the βH+ and its contribution to βtotal; the βtotal values lower than 60% and the high Q values resulting in electro­ static repulsion between the dispersed particles (except for the CaCl2 system, where the Q values were close to zero and the βtotal values close to 100%; DASP self-assemblies); 2) 4.60⋅10− 2 – 9.20⋅10− 1% w/v – decrease in the Q values and an in­ crease in βH+, βtotal (except for CaCl2) and the contribution of βH+ to βtotal with increasing DASP concentration; the cross-linking process 9

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