Effect of extraction method on the calcium binding capacity of faba bean globulin fractions at various pH.

Faba bean ingredients are rich in proteins and good sources of calcium (Ca), although containing phytic acid (PA) molecules. PA, a polyphosphate compound, can affect the bioavailability of minerals/proteins through complex formation. This study evaluates the impact of two extraction processes, Alkaline Extraction-IsoElectric Precipitation (AE-IEP) and Sequential Extraction (SE), on the ability of faba bean globulin systems to bind added calcium ions. Increasing concentrations of CaCl2 were introduced into 2.5% (w/v) protein dispersions at pHs 4.5, 5.5, 6.5, and 7.5, and free Ca monitored. Near the isoelectric point of globulin (pH âˆ¼ 4-5), Ca binding capacity was found to be low. At higher pHs, significant Ca chelation occurred, initially attributed to free PA binding sites, resulting in the formation of insoluble complexes and subsequent protein precipitation. The AE-IEP globulin fraction exhibited a higher Ca binding capacity than the SE globulin, attributed to its higher PA and lower initial Ca concentrations.

In-situ speciation and estimation of iron(II) and iron(III) contents in anisotropic polysaccharide-based hydrogel by 1H low-field nuclear magnetic resonance.

This article aims to quantify and differentiate in-situ iron(II) and/or iron(III) in heterogeneous polygalacturonate hydrogels using the 1H-NMR relaxometry technique. This holds significant importance, for example, in addressing iron-deficiency anemia through the oral administration of iron(II) supplements. The NMR dispersion profiles of the gels exhibited markedly distinct relaxation behaviors corresponding to the different iron oxidation states. At 20 MHz, two primary relaxation mechanisms must be considered: relaxation arising from water molecules confined within the polygalacturonate fiber mesh and paramagnetic relaxation due to iron cations. When iron(III) serves as the cross-linking agent, paramagnetic interaction dominates the relaxation, while with iron(II) as the cross-linker, both mechanisms have to be considered. To distinguish labile from structuring iron, we monitored the evolution of iron concentrations within the gels during successive washes using NMR and atomic absorption spectroscopy. Eventually, a gel containing both iron(II) and iron(III) was analyzed, and successful differentiation between the two cations was achieved. NMR relaxometry demonstrates powerful capabilities in terms of in-situ experiments, rapid results, speciation (iron(II)/iron(III)), and quantification (labile/ bridging iron).

Advanced insight on the water dynamics of anisotropic hydrogels by field-cycling nuclear magnetic resonance: Application of 3-Tau model.

Fast field cycling (FFC) nuclear magnetic resonance (NMR) relaxometry is used to investigate an anisotropic polygalacturonate hydrogel formed by the diffusion of calcium ions from an external reservoir (external gelation). Such a hydrogel has a gradient of polymer density accompanied by a gradient of the mesh size of its 3D network. The NMR relaxation process is dominated by the interaction of proton spins between water molecules located at polymer interfaces and in nanoporous spaces. The FFC NMR experiment provides the spin-lattice relaxation rate R1ω as a function of Larmor frequency ω producing dispersion (NMRD) curves that are highly sensitive to the dynamics of the protons at the surfaces. The hydrogel is sliced into three parts and the NMR profile for each hydrogel slice is measured. The NMRD data for each slice is interpreted using the 3-Tau Model with the aid of user-friendly fitting software called 3TM. The key fit parameters include three nano-dynamical time constants and the average "mesh size" which collectively determine the bulk water and water surface layer contribution to the total relaxation rate. The results are consistent with independent studies where comparison is possible.

How Accurate Is the Egg-Box Model in Describing the Binding of Calcium to Polygalacturonate? A Molecular Dynamics Simulation Study.

We performed molecular dynamics (MD) simulations of octameric galacturonate, GalA8, chains in the presence of Ca2+ in a ratio of R = [Ca2+]/[GalA] = 0.25 in order to determine to which extent the popular "egg-box model" (EBM) is able to describe the association between Ca2+ cations and polygalacturonate (polyGalA) chains. To this aim, we slightly revised the empirical parameters for the interaction between Ca2+ and the carboxylate oxygen atoms of GalA units so as to reproduce the experimental Ca2+-GalA association constant. We also defined an ad hoc order parameter, referred to as the egg-box score (EBS), that quantifies any deviation of the local coordination geometry of calcium cations with respect to an "ideal" EBM coordination geometry. The results reveal that the local coordination geometry of Ca2+ cations bound to polyGalA chains differs from that of the EBM. Moreover, polyGalA chains exhibit significant conformational disorder, and the cross-link angles formed between polyGalA chain axes are broadly distributed. Overall, the present study suggests that the EBM fails to describe accurately the association modes between calcium and polyGalA chains at a molar ratio R of 0.25.

In Situ Polymerization of Linseed Oil-Based Composite Film: Enhancement of Mechanical and Water Barrier Properties by the Incorporation of Cinnamaldehyde and Organoclay.

Linseed oil-based composite films were prepared with cinnamaldehyde (Cin) using a modified clay (organoclay) through in situ polymerization, which is the result of the interaction between Cin and organoclay. The incorporation of organoclay reduces the polymer chain's mobility and, therefore, increases the thermal stability of the composite films. In some experimental conditions, the clay is located both inside and on the surface of the film, thus, affecting the mechanical and thermal properties as well as the surface properties of the composite films. The incorporation of organoclay decreases the water contact angle of the composite film by more than 15%, whatever the amount of cinnamaldehyde. However, the incorporation of cinnamaldehyde has the opposite effect on film surface properties. Indeed, for the water vapor permeability (WVP), the effect of cinnamaldehyde on the film barrier properties is much higher in the presence of organoclay. The incorporation of hydrophobic compounds into the polymer films reduces the water content, which acts as a plasticizer and, therefore, decreases the WVP by more than 17%. Linseed oil has a natural antioxidant activity (~97%) due to the higher content of unsaturated fatty acids, and this activity increased with the amount of organoclay and cinnamaldehyde.

Slow water dynamics in polygalacturonate hydrogels revealed by NMR relaxometry and molecular dynamics simulation.

Diffusions in gels are of prime importance, but their measurements are mainly focused on the diffusion in the pores or through the mesh of the gels. In this study, we performed a deeper dynamic analysis of the water in close interaction with the fibers structuring two heterogeneous polygalacturonate (polyGalA) hydrogels formed by Ca and Zn ions (crosslinking agents). Nuclear magnetic resonance dispersion (NMRD) profiles recorded in-situ by fast-field cycling relaxometry allow to observe the very slow dynamics of water within the gels. Two distinct interpretations of the NMRD profiles are discussed, the first in regard of rotational and translational dynamics in the fibers and the second with respect to a Levy-walk on the fibers' surface. These discussions are confronted with molecular dynamics simulations on a model Ca-polyGalA fiber.

Core-shell polygalacturonate magnetic iron oxide nanoparticles: Synthesis, characterization, and functionalities.

This work aims to synthesize polygalacturonate-based magnetic iron oxide nanoparticles (INP-polyGalA). The synthesis consists of the diffusion of both Fe2+ and Fe3+ at a molar ratio of 1:2 through polyGalA solution followed by the addition of an alkaline solution. To form individual nanoparticle materials, the polyGalA concentration needs to be below its overlapping concentration (C*). The synthesized materials (INP-polyGalA) contain about 45% of organic compound (polyGalA), and they have an average particle size ranging from 10 to 50 nm as estimated by several techniques (DLS, TEM and AFM) and their surfaces are negatively charged in pH range 2 to 7. The synthesized NPs showed magnetic characteristics, thanks to the formation of magnetite (Fe3O4) as confirmed by X-ray diffractions (XRD). Moreover, AFM combined with Infra-red mapping allowed us to conclude that polyGalA is located in the core of the nanoparticles but also on their surfaces. More specially, both carboxylate (COO-) and carboxylic (COOH) groups of polyGalA are observed on the NPs surfaces. The presence of such functional groups allowed the synthesized material to (i) bind through the electrostatic interactions methylene blue (MB) which may have a great potential for r pollution control or (ii) to form hydrogel beads (ionotropic gelation) by using calcium as a crosslinking agent which can be used to encapsulate active molecules and target their release by using an external stimulus (magnetic field).

Importance of binary and ternary complex formation on the functional and nutritional properties of legume proteins in presence of phytic acid and calcium.

Nowadays, legumes are considered as a good source of plant-based proteins to replace animal ones. They are more favorable regarding environmental aspects and health benefits, therefore many people consider moving toward a greener diet. Interestingly, recent consumer trends are promoting pea and faba bean as alternatives to soybean. Both are rich in protein and a good source of essential nutrients and minerals (calcium). However, these advantages can be partially impaired due to their high phytic acid content. This natural polyphosphate is a major antinutrient in plant-based foods, as it can bind minerals (particularly calcium) and proteins, thereby reducing their digestibility and subsequent bioavailability. Indeed, complexes formed are insoluble and limiting the absorption of nutrients, thus lowering the nutritional value of pulses. To understand and overcome these issues, the present review will refine specific mechanisms involved in assemblies between these three essential compounds in legumes as soluble/insoluble binary or ternary complexes. Molecular interactions are influenced by the environmental medium including pH, ionic strength and molar concentrations modulating the stability of these complexes during protein extraction. Protein/phytic acid/calcium complexes stability is of high relevance for food processing affecting not only structure but also functional and nutritional properties of proteins in legume-based foods.

Impact of Glucose on the Nanostructure and Mechanical Properties of Calcium-Alginate Hydrogels.

Alginate is a polysaccharide obtained from brown seaweed that is widely used in food, pharmaceutical, and biotechnological applications due to its versatility as a viscosifier and gelling agent. Here, we investigated the influence of the addition of glucose on the structure and mechanical properties of alginate solutions and calcium-alginate hydrogels produced by internal gelation through crosslinking with Ca2+. Using 1H low-field nuclear magnetic resonance (NMR) and small angle neutron scattering (SANS), we showed that alginate solutions at 1 wt % present structural heterogeneities at local scale whose size increases with glucose concentration (15-45 wt %). Remarkably, the molecular conformation of alginate in the gels obtained from internal gelation by Ca2+ crosslinking is similar to that found in solution. The mechanical properties of the gels evidence an increase in gel strength and elasticity upon the addition of glucose. The fitting of mechanical properties to a poroelastic model shows that structural changes within solutions prior to gelation and the increase in solvent viscosity contribute to the gel strength. The nanostructure of the gels (at local scale, i.e., up to few hundreds of Å) remains unaltered by the presence of glucose up to 30 wt %. At 45 wt %, the permeability obtained by the poroelastic model decreases, and the Young's modulus increases. We suggest that macro (rather than micro) structural changes lead to this behavior due to the creation of a network of denser zones of chains at 45 wt % glucose. Our study paves the way for the design of calcium-alginate hydrogels with controlled structure for food and pharmaceutical applications in which interactions with glucose are of relevance.

Silica-coated calcium pectinate formulations for controlling carbendazim release: water and soil release studies.

This study aims to encapsulate the fungicide carbendazim using a biodegradable polymer (pectin). First, we have obtained calcium pectinate beads (CPG-Carb) by ionotropic gelation using calcium ions as a crosslinking agent. These beads were then coated with silica starting from tetraethoxysilane (TEOS), by a sol-gel process to form hybrid beads (CPG-Carb-SG). The morphology, composition and structure of both beads were characterized and the controlled release assays of the fungicide were studied in both water and soil columns. The encapsulation efficiency for CPG-Carb was slightly higher (75%) compared to CPG-Carb-SG (67%) due to carbendazim loss during the impregnation and condensation steps. The release rate in water and soil columns was about 4 times lower for CPG-Carb-SG than CPG-Carb demonstrating the efficiency of the silica coating to delay the release of carbendazim. Moreover, the release of CPG-Carb-SG is due to the erosion of the silica layer during the first two weeks. After this period, the silica layer was degraded, and the release is then controlled by the swelling of the organic part of the bead as observed for CPG-Carb. Finally, the biodegradability of the pectin, and the release profile make such systems promising candidates for sustained and economical pesticide delivery systems.

Intestinal release of biofilm-like microcolonies encased in calcium-pectinate beads increases probiotic properties of Lacticaseibacillus paracasei.

In this study, we show that calcium pectinate beads (CPB) allow the formation of 20 µm spherical microcolonies of the probiotic bacteria Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 with a high cell density, reaching more than 10 log (CFU/g). The bacteria within these microcolonies are well structured and adhere to a three-dimensional network made of calcium-pectinate through the synthesis of extracellular polymeric substances (EPS) and thus display a biofilm-like phenotype, an attractive property for their use as probiotics. During bacterial development in the CPB, a coalescence phenomenon arises between neighboring microcolonies accompanied by their peripheral spatialization within the bead. Moreover, the cells of L. paracasei ATCC334 encased in these pectinate beads exhibit increased resistance to acidic stress (pH 1.5), osmotic stress (4.5 M NaCl), the freeze-drying process and combined stresses, simulating the harsh conditions encountered in the gastrointestinal (GI) tract. In vivo, the oral administration of CPB-formulated L. paracasei ATCC334 in mice demonstrated that biofilm-like microcolonies are successfully released from the CPB matrix in the colonic environment. In addition, these CPB-formulated probiotic bacteria display the ability to reduce the severity of a DSS-induced colitis mouse model, with a decrease in colonic mucosal injuries, less inflammation, and reduced weight loss compared to DSS control mice. To conclude, this work paves the way for a new form of probiotic administration in the form of biofilm-like microcolonies with enhanced functionalities.

Resveratrol Favors Adhesion and Biofilm Formation of Lacticaseibacillus paracasei subsp. paracasei Strain ATCC334.

Bacterial strains of the Lactobacillaceae family are widely used as probiotics for their multifaceted potential beneficial properties. However, no official recommendations for their clinical use exist since, in many cases, oral administrations of these bacteria displayed limited beneficial effects in human. Additional research is thus needed to improve the efficiency of existing strains with strong potential. In this context, we assess in vitro the effects of nine polyphenols to stimulate biofilm formation by lactobacilli, a feature enhancing their functionalities. Among these polyphenols, we identify trans-Resveratrol (referred to hereafter as Resveratrol) as a potent inducer of biofilm formation by Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 strain. This effect is strain-dependent and relies on the enhancement of L. paracasei adhesion to abiotic and biotic surfaces, including intestinal epithelial cells. Mechanistically, Resveratrol modify physico-chemical properties of the bacterial surface and thereby enhances L. paracasei aggregation, subsequently facilitating adhesion and biofilm development. Together, our in vitro data demonstrate that Resveratrol might be used to modulate the behavior of Lactobacilli with probiotic properties. Combination of probiotics and polyphenols could be considered to enhance the probiotic functionalities in further in vivo studies.

Controlled Loading and Release of Beta-Lactoglobulin in Calcium-Polygalacturonate Hydrogels.

We show here how the structure of polygalacturonate (polyGalA) hydrogels cross-linked by Ca2+ cations via external gelation controls the loading and release rate of beta-lactoglobulin (BLG), a globular protein. Hydrogels prepared from a polyGalA/BLG solution are found to be similar to those obtained from a polyGalA solution in our previous study (Maire du Poset et al. Biomacromolecules 2019, 20 (7), 2864-2872): they exhibit similar transparencies and gradients of mechanical properties and polyGalA concentrations. The nominal BLG/polyGalA ratio of the mixtures is almost recovered within the whole mixed hydrogel despite such strong concentration gradients, except in the part of the hydrogels with the largest mesh size, where more BLG proteins are present. This gradient enables one to tune the amount of protein loaded within the hydrogel. At a local scale, the proteins are distributed evenly within the hydrogel network, as shown by small-angle neutron scattering (SANS). The release of proteins from hydrogels is driven by Fickian diffusion, and the release rate increases with the mesh size of the network, with a characteristic time of a few hours. The specific structure of these polysaccharide-based hydrogels allows for control of both the dosage and the release rate of the loaded protein and makes them good candidates for use as oral controlled-delivery systems.

Evidence for an egg-box-like structure in iron(ii)-polygalacturonate hydrogels: a combined EXAFS and molecular dynamics simulation study.

The local structure of Fe2+ in Fe2+-polygalacturonic acid (polyGalA) hydrogels has been studied by coupling Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy and molecular dynamics (MD) simulation. The EXAFS fitting results reveal an octahedral coordination geometry of Fe2+ both in aqueous solution and in the hydrogel, with similar Fe-O distances (2.09 ± 0.01 Å in the hydrogel and 2.11 ± 0.01 Å in aqueous solution). The MD simulations evidence that standard empirical force fields are unable to accurately reproduce the EXAFS spectra of Fe2+ in both aqueous solution and hydrogel. Based on the EXAFS distance determinations, we then performed restrained MD simulations of hypothetical octahedral coordination modes of Fe2+ with polyGalA chains. The best agreement between experimental and simulated EXAFS spectra was found when Fe2+ is monodentately coordinated to two carboxylate and two hydroxyl oxygens from a pair of polyGalA chains as well as to two water oxygens in an octahedral coordination geometry compatible with the "egg-box model".

Tuning the Structure of Galacturonate Hydrogels: External Gelation by Ca, Zn, or Fe Cationic Cross-Linkers.

We show here how the nature of various divalent cations M2+ (Ca2+, Zn2+, or Fe2+) influences the structure and mechanical properties of ionotropic polygalacturonate (polyGal) hydrogels designed by the diffusion of cations along one direction (external gelation). All hydrogels exhibit strong gradients of polyGal and cation concentrations, which are similar for all studied cations with a constant ratio R = [M2+]/[Gal] equal to 0.25, showing that every M2+ cation interacts with four galacturonate (Gal) units all along the gels. The regions of the hydrogels formed in the early stages of the gelation process are also similar for all cations and are homogeneous, with the same characteristic mesh size (75 ± 5 Å, as measured by small angle neutron scattering (SANS)) and the same storage modulus G' (∼5 × 104 Pa). Conversely, in the regions of the gels formed in later stages of the process there exist differences in mechanical properties, turbidity, and local structure from one cation to another. Zn(II)-polyGal and Fe(II)-polyGal hydrogels display mesoscopic heterogeneities, more marked in case of Fe than for Zn, that are not present in Ca(II)-polyGal hydrogels. This comes from the mode and the strength of association between the cation and the Gal unit (bidentate for Ca2+ and monodentate "egg-box" for Zn2+ and Fe2+). Cross-links formed by Zn2+ and Fe2+ have a higher stability (lower ability to untie and reform) that induces the formation of local heterogeneities in the early stages of the gelation process whose size progressively increases during the gel growth, a mechanism that does not occur for cross-links made by Ca2+ that are less stable and enable possible reorganizations between polyGal chains.

Insights into gelation kinetics and gel front migration in cation-induced polysaccharide hydrogels by viscoelastic and turbidity measurements: Effect of the nature of divalent cations.

Polysaccharide-based hydrogels were prepared by the diffusion of various divalent cations (X2+) into the polygalacturonate (polyGal) solution through a dialysis membrane. The diffusion of various divalent cations (Mg2+, Ca2+, Zn2+ and Ba2+) was investigated. The polyGal gel growth was studied as a function of the initial cation concentration by both viscoelastic and turbidity measurements. We have demonstrated for the first time that the determination of the spatiotemporal variation of turbidity during the gelation process allowed to study the gel front migration. For Ca-polyGal, Zn-polyGal and Ba-polyGal, the gel front migration was characterized by the presence of a peak at the sol/gel interface. This peak was not observed in the case of Mg-polyGal where the gel was not formed. The apparent diffusion coefficient of the gel front (Dapp) which was calculated from the evolution of this peak increased when the initial cation concentration was increased. Moreover, we have suggested a gelation mechanism based on the presence of a threshold molar ratio R* (=[X2+]/[Galacturonic unit]) in which some point-like crosslinks are precursors of the formation of dimers and multimers inducing the contraction of the gel and thus the formation of the gel front.

Design of polygalacturonate hydrogels using iron(II) as cross-linkers: A promising route to protect bioavailable iron against oxidation.

We designed stable and highly reproducible hydrogels by external unidirectional diffusion of Fe2+ ions into aqueous solutions of polygalacturonate (polyGal) chains. The Fe2+ ions act as cross-linkers between the Gal units in such a way that both the molar ratio R ([Fe2+]/[Gal units] = 0.25) and the mesh size of the polyGal network at the local scale (ξ = 75 ±â€¯5 Å) have constant values within the whole gel, as respectively determined by titration and Small Angle Neutron Scattering. From macroscopic point of view, there is a progressive decrease of polyGal concentration from the part of the gel formed in the early stages of the gelation process, which is homogeneous, transparent and whose Young modulus has a high value of ∼105 Pa, up to the part of the gel formed in the late stages, which is heterogeneous, highly turbid and has a much lower Young modulus of ∼103 Pa. Since the local organization of the polyGal chains remains identical all along the hydrogels, this macroscopic concentration gradient originates from the formation of heterogeneities at a mesoscopic length scale during the gelation process. In addition, X-ray Absorption Spectroscopy measurements remarkably reveal that Fe2+ ions keep their +II oxidation state in the whole gels once they have cross-linked the Gal units. These polyGal hydrogels thus protect iron against oxidation and could be used for iron fortification.

Specific binding of trivalent metal ions to λ-carrageenan.

Carrageenans are a family of sulphated cell wall polysaccharides extracted from seaweeds and are widely used in different industrial sectors. Relative to κ-carrageenan (κ-car) and ι-carrageenan (ι-car), the ionic binding behavior of λ-carrageenan (λ-car) is far less studied. In this work, the interaction and binding behavior between λ-car and metal ions of different valency (Na+, K+, Mg2+, Ca2+, Fe2+, Fe3+, Al3+, Cr3+) have been investigated. In contrast to the non-specific interaction of the monovalent and divalent cations, specific binding has been identified between λ-car and Fe3+/Al3+. The specific binding could lead to either precipitation or gelation of λ-car, depending on the way of introducing Fe3+/Al3+ ions. Fe3+ and Al3+ exhibit the same binding stoichiometry of [M3+]/[repeating unit] = 1.0, with the former having a relatively larger binding constant. Cr3+, though having very similar physical properties with Fe3+/Al3+, is incapable of binding specifically to Cr3+. The phenomena could not be interpreted in terms of counterion condensation, and are rather attributable to a mechanism in which hexa-coordination of Fe3+/Al3+ and entropy-driven cation dehydration play crucial roles in driving the binding of the trivalent metal ions to λ-car.

Binding of Divalent Cations to Polygalacturonate: A Mechanism Driven by the Hydration Water.

We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* < 0.1) than for zinc and barium ions (R* > 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*).

Structural behaviour differences in low methoxy pectin solutions in the presence of divalent cations (Ca(2+) and Zn(2+)): a process driven by the binding mechanism of the cation with the galacturonate unit.

In this paper, we compare the interactions between low methoxy pectin (LMP) and either Ca(2+) or Zn(2+) in semi-dilute solutions. Intrinsic viscosity and turbidity measurements reveal that pectin-calcium solutions are more viscous, but yet less turbid, than pectin-zinc ones. To get a molecular understanding of the origin of this rather unexpected behavior, we further performed isothermal titration calorimetry, small angle neutron scattering experiments, as well as molecular dynamics simulations. Our results suggest that calcium cations induce the formation of a more homogeneous network of pectin than zinc cations do. The molecular dynamics simulations indicate that this difference could originate from the way the two cations bind to the galacturonate unit (Gal), the main component of LMP: zinc interacts with both carboxylate and hydroxyl groups of Gal, in a similar way to that described in the so-called egg-box model, whereas calcium only interacts with carboxylate groups. This different binding behavior seems to arise from the stronger interaction of water molecules with zinc than with calcium. Accordingly, galacturonate chains are more loosely associated with each other in the presence of Ca(2+) than with Zn(2+). This may improve their ability to form a gel, not only by dimerization, but also by the formation of point-like cross-links. Overall, our results show that zinc binds less easily to pectin than calcium does.