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Item Characterisation and protein complexation of an anthocyanin-bound pectin extracted from New Zealand blackcurrant (Ribes nigrum) : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2022) Salleh, NurhazwaniThe main objective of this thesis was to investigate the cause of physical instability in blackcurrant juice-milk system. Poor phase stability in fruit juice-milk beverages is a major challenge for the clean-label beverage industry as milk protein can interact with fruit components, like polysaccharides and polyphenols, generating unwanted characteristics such as coagulation of milk proteins and phase separation. Hence, the principal step to understand the causes of poor phase stability was to identify and study the key interactive components of the juice, which was extracted from the New Zealand blackcurrant (Ribes nigrum), and then investigate their interactions with milk proteins. The key components of the blackcurrant juice were first isolated using mild extraction procedures, via ethanol precipitation and dialysis, and were identified as a complex fraction particularly rich in pectin and anthocyanins (Chapter 4). Proximate analysis revealed that the fraction contained carbohydrate (78% w/w), uronic acid (21% w/w), protein (4.8% w/w), anthocyanin (3.9% w/w) and calcium (2.2% w/w). The pectin-rich fraction had a net negative surface charge of -23.1 mV (at pH 4.8), a pKₐ value of 1.7 and a relatively high degree of esterification (65.2%). Constituent sugar analysis showed that the fraction was mostly made of galacturonic acid, rhamnose, arabinose and galactose, and NMR spectroscopic analysis revealed that it was rich in rhamnogalacturonans with arabinogalactan side chains. This pectic fraction was unique as it was highly pigmented, with cyanidin 3-O-rutinoside as its major anthocyanin. Liquid chromatography revealed that the anthocyanins were tightly bound to the fraction as methanol used in the technique failed to separate them. Results from size-exclusion chromatography coupled with multi-angle laser light scattering showed that the blackcurrant juice contained two major pectic fractions—≈283 kDa present at 14.6% w/w and ≈97 kDa at 85.5% w/w—with the latter producing higher UV₂₈₀ ₙₘ signal, signifying that proteins and/or polyphenols were present mainly in the second fraction. Association of anthocyanins to biopolymers like pectin and protein can occur via multiple interactive forces (electrostatic, hydrophobic and hydrogen bonding forces), and pH is known to play a significant role as it can affect the associative mechanisms of anthocyanins by changing their molecular configuration and ability to electrostatically interact. An attempt to dissociate blackcurrant anthocyanins from the blackcurrant biopolymers was carried out by disrupting electrostatic interactions and changing the planarity of anthocyanins via pH adjustments and ultra-filtration (Chapter 5). Lowering the juice pH to 2 did not result in anthocyanins dissociation, likely because anthocyanins were bound to the biopolymers by other interactive forces apart from the electrostatic bonds. Increasing the juice pH to 4.5 might have dissociated some anthocyanins from the biopolymers, but this was not reflected in the analysis of anthocyanins, probably because the freed anthocyanins had degraded before the analysis was carried out. Overall, size segregation of the juice components via ultra-filtration was relatively effective. Regardless of the pH, majority of the anthocyanins were still tightly associated with the large molecular weight biopolymers, confirming the involvement of multiple interactive forces. In order to uncover the cause of phase instability in blackcurrant juice-milk system, a complexation study between the isolated pectin-rich fraction and whey proteins was conducted (Chapter 6). The impact of bound anthocyanins on pectin-protein interactions was studied by exploring the effects of pH (pH 3.5 and pH 4.5), heating (85 °C, 15 min) and heating sequence (mixed-heated or heated-mixed). The pH was found to influence the colour, turbidity, particle size and surface charge of the mixtures, but its impact was most drastic when heating was introduced. Heating increased the amount of blackcurrant pectin within the complexes—especially at pH 3.5, where 88% w/w of the initial pectin was found in the sedimented (insoluble) fraction. Based on physical stability measurements, the mixed-heated system at pH 4.5 displayed better stability than at pH 3.5. A noteworthy finding was that heating sequence was found to be effective in preventing the destabilisation of the systems. Mixing of components before heating produced a more stable system with small complexes (<300 nm) and relatively low polydispersity. However, heating whey proteins before mixing with blackcurrant pectin prompted protein aggregation, producing large complexes (>400 nm) that worsened the destabilisation. The influence of bound anthocyanins on pectin-protein complexation was further studied by comparing two types of pectin-protein mixtures: (i) a mixture that is rich in anthocyanin (blackcurrant pectin-whey protein, BCP-WP) and (ii) a mixture that is free of anthocyanin (citrus pectin-whey protein, CP-WP) (Chapter 7). The mixtures were prepared at pH 4.5 with and without heat treatment at 85 °C. The study revealed that there was no direct relationship between anthocyanin presence and the destabilisation of mixtures. The Fourier-Transform Infrared (FTIR) spectrum of the heated and non-heated BCP-WP sedimented fractions showed the emergence of a peak at 800-1200 cm⁻¹, signifying the presence of anthocyanin-protein interactions. This peak, however, was absent in the spectrum of any of the anthocyanin-free CP-WP sedimented fractions, indicating that the bound anthocyanins of blackcurrant pectin provided the whey proteins with additional binding sites. The findings from FTIR analyses also indicated that non-electrostatic forces were most likely the governing forces of the heated BCP-WP mixture, via hydrophobic interactions and later reinforced by hydrogen bonds upon cooling. This thesis revealed that poor phase stability of the blackcurrant juice-milk system should not be attributed exclusively to the blackcurrant juice components, particularly the polyphenols. Environmental factors like pH and heat were likely the leading cause of phase instability as they could intensify the interactions that occurred in the mixed system, which eventually destabilised the mixture. This suggests that appropriate processing conditions can be applied to positively affect the blackcurrant juice-milk system.Item Process for recovery of smooth fibre ingredient from pomace : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Food Technology at Massey University, Manawatu, New Zealand(Massey University, 2020) Eblaghi, MarziehThis research aimed to develop a process to convert apple pomace into a food ingredient which can provide functional properties such as water-binding in baked goods, stability in aqueous suspension and smooth mouthfeel. This was achieved by modifying the apple pomace through three main steps: heating, shearing and enzymatic hydrolysis. Firstly, the effect of sample preparation (addition of water to fresh pomace, temperature and shearing apple pomace) on the solubility of pectin was investigated. Secondly, kinetics of main reactions involved in pomace while heating at temperatures between 90-140 °C (10 °C intervals) and incubation times between 0-360 min, were studied at bench scale. These reactions were: solubilisation and depolymerisation reactions of pectin, degradation of sugars and production of secondary products such as organic acids and 5-hydroxymethylfurfural (5-HMF). After that, the kinetics of these reactions were modelled for determining the rate constants and activation energies. The kinetic models of pectin solubilisation and 5-HMF formation were then used for scaling up the hydrothermal process in a more complex heat transfer situation, using a pilot scale retort. Finally, the effect of particle size distribution and molecular weight of solubilised components (mainly pectin) on physicochemical and sensorial properties of pomace material was investigated. Solubilisation of pectin at room temperature was independent of addition of water and shearing treatment of pomace. However, heating at temperatures > 100 °C, combined with increasing the amounts of water added to pomace (from 0 to 8 mL water/ g pomace) resulted in increasing the pectin solubility up to a pomace-water ratio of 1:2. The maximum amount of solubilised pectin (~ 605 μmol galacturonic acid/ g dry pomace) was determined when heating pomace at 130 and 140 °C for 15 and 7 min, respectively. Hydrothermal depolymerisation of pectin through acid hydrolysis and β-elimination reactions also showed temperature-dependent behaviour. Depolymerisation reactions resulted in degradation of pectin polymers into ethanol-soluble forms (galacturonic acid). Depolymerisation seemed more likely to happen from non-esterified sites of pectin polymers, as suggested by the high degree of esterification of the remaining insoluble pectin. Increasing amounts of glucose and fructose were observed in the serum phase of pomace to about five times their initial values when pomace was heated at temperatures >120 °C. This was accompanied by a reduction in sucrose content, suggesting hydrothermal hydrolysis of sucrose to its subunits. A complete conversion of sucrose was recorded at temperatures > 120 °C and times > ~30 min. Glucose, fructose and galacturonic acid underwent further transformation at temperatures > 100 °C forming secondary products of organic acids, (such as acetic acid, formic acid and lactic acid), furfural and 5-HMF. Modelling the kinetics of pectin solubilisation and 5-HMF production resulted in activation energies of 81 and 105 kJ/mol, respectively. The effects of hydrothermal treatment were modelled using COMSOL for heating a slab of pomace in a pilot scale retort with a maximum steam temperature of 125 °C. In this model, heat transfer through the pomace and chemical reactions of pectin solubilisation and 5-HMF production were predicted. The aim for this model was to identify conditions permitting solubilisation enough to double the amount of pectin from the amount initially present at room temperature while limiting the production of 5-HMF to the range permitted in food standards (bulk averaged). The validity of the model was confirmed in the pilot plant condition. Another objective of this research study was to investigate the effects of particle size distributions and molecular weight of pectin on sensory properties and physical stability of pomace samples. A controlled modification of particle size and molecular weight of solubilised pectin was achieved by fractionation of heat-treated pomace into insoluble solid and serum parts. Shearing insoluble particles for 5 min showed significant particle size reduction from 496 μm (initial shearing for 2 min) to 165 μm. Further shearing did not affect the average size of the particles. Microscopy revealed the effect of shearing in separating cell aggregations, resulting in individual cells. Shearing also fractured some individual cells, the oval shape of most cells was still visible. The pectin in the serum phase of pomace before and after heat treatment was analysed for molecular weight distribution. Results confirmed the effectiveness of heat treatment on solubilising high molecular weight pectins into the serum phase. Two ranges of high (between 4000-73 kDa) and low (< 73 kDa) pectin molecular weights were analysed in heat-treated pomace. These two ranges were separated from each other by ultrafiltration. High molecular weight of fraction (with the average size of 380 kDa) was enzymatically hydrolysed into two pectin components with average molecular weights of 30 and 150 kDa. Finally, six pomace ingredients were produced from the pomace fractions with two insoluble particle size distributions (with the average size ranges of 500 and 160 μm) and three ranges of molecular weights (380, 150 and 30 kDa) being blended. Particle size reduction had a significant effect on the physical stability of pomace suspensions. Samples containing particles >496 μm showed phase separation during storage (5 days at 4 °C), while samples with smaller particle size did not show any phase separation.Item Physical and chemical attachment of pectins to substrates : methods, characterisation and application : thesis presented by Abdenor Fellah for the degree of Doctor of Philosophy, Massey University, New Zealand & Fonterra, New Zealand(Massey University, 2012) Fellah, AbdenorThe plant cell wall is a complex biological matrix in which pectic polysaccharides play an instrumental role in regulating mechanical properties. Nanomechanical studies of single chains hold the promise of enabling the comprehension of fundamental aspects concerning the structural, mechanical and binding properties of pectin at an unprecedented level of molecular detail, using measured single polysaccharide force-extension behavior as a signature. However, before such promise can be fulfilled, a better understanding of the attachment of the polymer under study to the substrates between which it is stretched is required. Herein, chemoselective methodologies have been developed to covalently couple one end of a pectin chain onto a solid support. Prior to immobilization, pectin fine structure was investigated using accurate and non-invasive infrared spectroscopy. Comparison of experimental results with the predictions of quantum chemical calculations carried out using density functional theory confirmed this technique as an effective tool for the characterization of pectin fine structure. Subsequently, following appropriate functionalization of the support, pectin chains were anchored to polystyrene beads, specifically through their reducing end. These methods were shown to be efficient using IR spectroscopy, once more coupled with quantum chemical calculations, with the formation of specific newly introduced bonds being demonstrated. Finally, single-molecule force spectroscopy was used to stretch single pectin molecules covalently bonded to substrates using the previously described method applied to glass surfaces. Compared to physisorption, which was also extensively studied, tethering the pectin non-reducing end appeared to increase the average stretch length and improved significantly the probability of stretching a single chain to high forces.Item Pectin degradation and metabolism in Monoglobus pectinilyticus 14T from human faeces : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand(Massey University, 2017) Kim, Caroline Chae-hyunPectin is a conspicuous plant polysaccharide, comprising one third of the dry weight of dietary fibre in common vegetables and fruit. Although pectin is almost completely digested by the human gut microbiota, few bacterial species are known to possess a comprehensive glycobiome to challenge the structurally complex pectin. The current understanding of the colonic degradation of pectin is incomplete, as the knowledge has almost exclusively derived from studying the sequestration system of Bacteroides spp. Here I report the isolation and characterization of Monoglobus pectinilyticus, and the sequencing of its genome which so far encodes the most pectin-specialized repertoire of carbohydrate active enzymes (CAZymes) found from the human gut. M. pectinilyticus also possesses an extracellular pectin degradation system consisting of novel protein constituents which did not find significant sequence homology and functional matches using the most up-to-date nucleotide and protein sequence databases. Proteome analysis of M. pectinilyticus using iTRAQ quantification revealed that pectin-degrading CAZymes and the potential constituents of the novel pectin degradation system were differentially up-regulated in response to the availability of pectin. Finally, using quantitative PCR, a positive correlation was observed between the prevalence of M. pectinilyticus and the consumption of fibre, vegetables, and pectin in individuals living in NZ. The discovery of M. pectinilyticus may add a new layer of complexity onto our interpretation of the colonic pectin degradation by presenting a system highly relevant to the pectin-rich diet of humans, and by suggesting a possibility outside the established paradigms of microbial polysaccharide degradation. The presence of M. pectinilyticus and the related uncultured bacteria in the gastrointestinal systems of humans and animals indicated that the organisms of this lineage are frequent terrestrial gut commensals, prompting an investigation into the genomic and molecular properties underlying their carbohydrate degradation potentials.Item Studies on the extraction and characterization of pectin and bitter principles from New Zealand grapefruit and Philippine calamansi : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University(Massey University, 1981) Nisperos, Myrna OrdoñaA study was conducted to determine the presence of bitter components in NZ grapefruit and Philippine calamansi; describe the effect of maturity on the bitter components and other chemical constituents of grapefruit; reduce the bitterness of grapefruit juice by adsorption on polyvinylpyrrolidone; and to extract and characterize pectin from grapefruit peel. Naringin (995 PPm), narirutin (187 ppm), and limonoids (7.9 ppm) were detected in NZ grapefruit juice concentrate (27° Brix). Naringin was not detected in the calamansi juice, and limonin was detected at the level of 10.5 ppm in juice containing 5% crushed seeds. Maturation of the grapefruit caused an increase in pH from 3.00 to 3.50, an increase in total soluble solids from 10.8 to 14.4 with a decline to 13.5° Brix later in the season, a steady fall in acidity from 2.50 to 1.31 g citric acid/100 mL, and a continuous rise in the Brix/acid ratio from 4.2 to 10.3. Juice yield fluctuated throughout the season. Ascorbic acid remained fairly steady in the early and mid-season fruit but decreased in the late-season fruit. Naringin content was highest at the beginning of the season and fluctuated throughout the season. Naringin content in the grapefruit peel remained constant as the fruit matured. Narirutin was detected in the early-season fruit but disappeared later in the season. Limonoid content in both unpasteurized and pasteurized juices decreased with ripening. The use of polyvinylpyrrolidone significantly reduced naringin in grapefruit juice by up to 78.1% and limonin by up to 17.5% depending on the amount and reaction time of the adsorbent. A loss of 23.1% in ascorbic acid occurred with 5% PVP with a reaction time of 1 h. Pectin extraction at 85°C and the use of acidified isopropyl alcohol yielded a product with the following characteristics: 8.9% yield; 1.3% moisture content; 1.9% ash; 759 equivalent weight; 9.2% methoxyl content; 82.2% anhydrogalacturonic acid; 63.2% degree of esterification; 4.2 intrinsic viscosity; 89,362 molecular weight and setting time of 0.55 minute.Item Structure and dynamics of biopolymer networks : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics at Massey University, Manawatu, New Zealand(Massey University, 2015) Mansel, Bradley WilliamThe aim of this work was to further understand the structural and dynamical properties of pectin-based biopolymer networks. This is pertinent to furthering our understanding of the plant cell wall and has further implications for the food and pharmaceutical industries where biopolymer networks play a fundamental role in thickening and stabilizing food products and controlling the rate of drug release. Firstly, microrheological studies on an acid-induced pectin network revealed previously unseen slow motions of the network at times longer than one second. This "slow mode" is reminiscent of so-called alpha processes that are predicted with mode coupling theory in colloidal glasses. Such slow motions present in the networks are a signature of an outof- equilibrium system and lead to further work on studying slow relaxation processes in pectin networks. Secondly, structural and rheological measurements were performed on the acid-formed pectin networks. It was found using small-angle x-ray scattering that the network was composed of flexible cylindrical entities with a radius of 7 Å. At larger length scales these entities were arranged in a clustered confirmation that upon heating increased in density, indicating the importance of kinetic trapping for the initial network formation. Finally, multi-speckle dynamic light scattering experiments were performed on three different ionotropic pectin gels formed with calcium to study the dependence of the slow dynamics on the junction length (and binding energy) between pectin chains. It was found that increasing the junction length slows the dynamics until a point where the internal stress becomes so large that the dynamics increase again. Spatially resolved photon correlation spectroscopy measurements revealed previously unmeasured millimetre sized heterogeneity in the networks. Angle-resolved multi-speckle photon correlation spectroscopy showed conclusively that the dynamics are driven by internal stresses and further more allowed the temporal heterogeneity to be measured.Item Development and applications of a low-field portable NMR system : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics at Massey University, Manawatu, New Zealand(Massey University, 2011) Ward, Robert LNuclear magnetic resonance (NMR) is a phenomenon similar to MRI in which radio frequency signals are used to excite and manipulate atomic nuclei within a static magnetic field. Following excitation, the nuclei return to equilibrium, all the while offering valuable molecular level information pertaining to the sample. Within the last decade, the development of small and inexpensive NMR spectrometers and permanent magnet NMR sensors has been a significant focus within the NMR community. More recently, application scientists have sought practical applications for the new technologies. In this thesis, a prototype NMR apparatus consisting of a spectrometer and 3.2MHz permanent magnet sensor was extended to enable scientifc measurements. This involved developing radio frequency electronic circuitry for the spectrometer front-end, and electromagnetic noise shielding and temperature regulation for the magnetic sensor. Experimental results confirmed that repeatable measurements using the modified apparatus were indeed possible. The NMR apparatus was thereafter successfully used to study flow, diffusion and kiwifruit using several different experimental techniques. A significantly larger effort was then expended upon the study of T2 relaxation in pectin model systems using pH as the adjustable parameter. The fascinating experimental results were successfully interpreted and modeled across three pH zones in terms of a proton chemical exchange model and molecular conformational changes. In addition, it was found that pectin carboxyl de-protonation was significantly less than expected. Further experiments performed upon galacturonic acid monomers, dimers and trimers appeared to further illuminate the pectin results. Future experiments are planned. Also while studying pectin solutions, an unexpected pH-dependent water transverse relaxation behavior was observed at both 3.2MHz and 400MHz. The only references found in the literature were from a small publication almost 50 years ago, and a 2011 publication. Altogether, this thesis contributed to original knowledge in several ways: it showed how a low- eld apparatus and single-sided sensor could be improved and utilized for a variety of scientific measurements; it showed both experimentally and theoretically how T2 for pectin solutions change with pH; it revealed an unexpected de-protonation limit for pectin molecules; it revealed a T2 pH dependence for water.Item Computational approaches to the calculation of spectroscopic, structural and mechanical properties of polysaccharide chains : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University(Massey University, 2010) Anjukandi, PadmeshIn this thesis atomistic, statistical mechanical and coarse grained simulation techniques are used to study the properties of biopolymers and in particular the plant polysaccharide pectin. Spectroscopic aspects, structural and conformational behavior, and mechanical properties of the molecule in di erent physical states are addressed. After an introduction to the area and the theoretical techniques utilised herein (chapter 1), chapter 2 deals with the spectroscopic characterisation of pectin. Spectra were obtained theoretically by undertaking complete energy minimisation and Hessien calculations using DFT techniques implemented in Gamess (PC & US) packages. The calculated IR absorptions of di erent pectinic species and oligomers coupled on di erent surfaces were compared with experimental results. Herein, it is con rmed that experimental FTIR studies coupled with DFT calculations can be used as an e ective tool for the characterisation of pectin, and studying chemical coupling of the biopolymer to surfaces. In chapter 3, the properties of single chain polymer systems in controlled solvent conditions were studied using Brownian dynamics simulations, motivated by the formation of secondary structure architectures in biopolymer systems. We focus on the conformational properties of the chain in the presence of an additional torsional potential. New, interesting, and biologically relevant structures were found at the single molecule scale when a torsional potential was considered in the calculations. In chapter 4, results from DFT calculations carried out on single pectin sugar molecules (lengths and the free energies) are incorporated into a statistical mechanical model of polymer stretching, in order to obtain the force-extension behaviour of a single molecule pectin. This captures a good deal of the phenomenology of the single molecule stretching behavior of pectin. Chapter 5 summarises the conclusions of the work and nally chapter 6 suggests direction for further work.Item Rheo-NMR studies of macromolecules : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Physics at Massey University, Palmerston North, New Zealand(Massey University, 2008) Kakubayashi, MotokoIn this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.Item Microrheological investigations of biopolymer networks : PhD thesis, research conducted at the Institute of Fundamental Sciences, Massey University of Palmerston North, New Zealand(Massey University, 2008) Vincent, Romaric Remy Raoulis a major polysaccharide of the plant cell wall which is known to play a role in many mechanical functionalities, especially when a gel is formed in the presence of calcium. Understanding the gelling abilities of pectin is of great interest to the food industry also, since pectin is a widely used as a gelling agent and thickener. The aim of this study was to apply two complementary microrheological techniques to these systems, multiple particle tracking (MPT) and a light scattering technique called diffusing wave spectroscopy (DWS). While the first one provides fundamental information about the homogeneity of the studied gel, the second gives access to the high frequency behaviour, related to the nature of the basic strands of the network. Firstly, after verifying the validity of the experimental apparati and analysis approaches in a series of careful control experiments on archetypal systems, a regime where pectin gels exhibit the signatures of semi-flexible networks was identified in experiments carried out on gels made of pectin chains pre-engineered by enzymatic deesterification and subsequently assembled with the release of Ca2+. These results were the first showing that polysaccharides networks could be accommodated within the framework of semi-flexible networks, which have become a paradigm for biological gels, such as the well-known F-actin solutions present in the cell cytoskeleton. However, in the plant cell wall, where calcium is already present, the assembly mechanism could be controlled in a different manner, and a more biologically relevant system was studied where the action of the plant enzyme pectinmethylesterase was used to liberate ion-binding groups in the presence of Ca2+. Gels formed according to this alternative methodology were found to behave as punctually cross-linked flexible networks, strikingly different from the first results. This would be explained by the presence of short blocks of charged residues. Finally, experiments on pectins carried out with controlled blocky structures showed that a pectin made of short blocks can exhibit both sorts of network, depending on the polymer and Ca2+ concentrations. This lead naturally to the construction of a state diagram for the regimes of assembly, with proposed control parameters being the polymer concentration and the ratio of the amount of Ca2+ to the quantity of pectic residues which can effectively bind the calcium into cross-links, christened Reff.