Molecular dynamics simulations and X-ray scattering show the κ-carrageenan disorder-to-order transition to be the formation of double-helices.

Recent molecular dynamics simulations, verified experimentally by solution-state x-ray scattering experiments, have found that κ-carrageenan chains contain helical secondary structure, akin to that found in the solid-state, even in aqueous solution. Furthermore, upon the addition of ions to single chains the simulations found no evidence that any conformational transitions take place. These findings challenge the long-held assumption that the so-called disorder-to-order transition in carrageenan systems involves a uni-molecular 'coil-to-helix transition'. Herein, the results of further molecular dynamics simulations undertaken using pairs of κ-carrageenan chains in 0.1 M NaI solutions are reported, and are validated experimentally using state-of-the-art solution-state WAXS experiments. From initially separated chains double-helices are shown to form, leading the authors to propose 'two single helices-to-stabilized double-helix' as a description of the molecular events taking place during the disorder-to-order transition.

X-ray scattering and molecular dynamics simulations reveal the secondary structure of κ-carrageenan in the solution state.

The solution state structure of κ-carrageenan is typically described as a 'random coil', to indicate a lack of defined secondary structure elements. From this starting point the assignment of an optical-rotation-detected change that follows the introduction of particular ions to such solutions as a 'coil-to-helix transition' seems unambiguous, and thus the canonical description of this important biopolymer's gelling behaviour was born. However, the notion that κ-carrageenan exists in solution as a random coil, devoid of secondary structure, has been questioned a number of times previously in the literature, particularly by the molecular modelling and NMR communities. Regrettably, there has been little desire to-date to address these largely overlooked studies or consider their implications for the nature of the so-called 'coil-to-helix transition'. Despite evidence to the contrary, the random-coil-paradigm has prevailed. Here, new data from synchrotron-enabled solution-state x-ray scattering experiments, combined with state-of-the-art atomistic molecular dynamics simulations, are used to show that the solution-state structure of κ-carrageenan in fact retains many of the helical motifs present in the solid-state, as inferred from fibre diffraction data. Furthermore, no evidence is found to suggest that single chains undergo any uni-molecular conformational transition upon the addition of ions. These findings once again challenge the paradigm that κ-carrageenan exists as a 'random coil' in the solution state, and thereby question the long held assumption that a uni-molecular 'coil-to-helix transition' precedes the dimerization of helices.

Sodium Reduction in Bouillon: Targeting a Food Staple to Reduce Hypertension in Sub-saharan Africa.

Bouillon cubes are a staple ingredient used in Sub-saharan African countries providing flavor enhancement to savory foods. Bouillon has been identified as a vehicle for fortification to overcome micronutrient deficiencies in Sub-saharan Africa. However, bouillon has a high sodium content (and in addition with other foods) contributes to dietary sodium intake above recommended guidelines. High dietary sodium intake is a key risk factor for hypertension and cardiovascular disease (CVD). Africa has the highest rates of hypertension and CVD globally with nearly half the adult population above 25 years affected. This review presents current state of research on sodium reduction strategies in bouillon. The key challenge is to reduce sodium levels while maintaining optimal flavor at the lowest possible production cost to ensure bouillon continues to be affordable in Sub-saharan Africa. To produce lower sodium bouillon with acceptable flavor at low cost will likely involve multiple sodium reduction strategies; direct reduction in sodium, sodium replacement and saltiness boosting flavor technologies. Efforts to reduce the sodium content of bouillon in Sub-saharan Africa is a worthwhile strategy to: (i) lower the overall sodium consumption across the population, and (ii) deliver population-wide health benefits in a region with high rates of hypertension and CVD.

Structural Analysis of Polysaccharide Networks by Transmission Electron Microscopy: Comparison with Small-Angle X-ray Scattering.

Polysaccharide gels assembled from the anionic biopolymers pectin and carrageenan have been studied using transmission electron microscopy (TEM). Gels were formed in several different ways: for pectin, hydrogen bonding was used to form junction zones between strands, whereas for carrageenan systems, several different ion types were used to form ionotropic networks. Using this approach, several distinct network architectures were realized. In addition to preparing gelled samples for electron microscopy, a set of samples was taken without performing the additional treatment necessitated by the TEM measurements, and these were studied directly by small-angle X-ray scattering (SAXS). Taking careful consideration of the relative merits of different image sizes and available processing techniques, the real-space images acquired by TEM were used via radial integration of the Fourier transform to produce simulated scattering patterns. These intensity-versus-wavevector plots were compared with the results of SAXS experiments carried out on the unadulterated gels using synchrotron radiation. Although information regarding chain thicknesses and flexibilities was found to be modified by labeling and changes in the dielectric constant and mechanical properties of the surroundings in the TEM, the studies carried out here show that careful protocols can produce data sets where information acquired above ∼20 nm is broadly consistent with that obtained by SAXS studies carried out on unadulterated samples. The fact that at larger length scale the structure of these water-rich networks seems largely preserved in the TEM samples suggests that three-dimensional (3D) TEM tomography experiments carried out with careful sample preparation will be valuable tools for measuring network architecture and connectivity; information that is lost in SAXS owing to the intrinsic averaging nature of the technique.

Effects of Agar Gel Strength and Fat on Oral Breakdown, Volatile Release, and Sensory Perception Using in Vivo and in Vitro Systems.

The density and composition of a food matrix affect the rates of oral breakdown and in-mouth flavor release as well as the overall sensory experience. Agar gels of increasing concentration (1.0, 1.7, 2.9, and 5% agarose) with and without added fat (0, 2, 5, and 10%) were spiked with seven aroma volatiles. Differences in oral processing and sensory perception were systematically measured by a trained panel using a discrete interval time intensity method. Volatile release was measured in vivo and in vitro by proton transfer reaction mass spectrometry. Greater oral processing was required as agar gel strength increased, and the intensity of flavor-related sensory attributes decreased. Volatile release was inversely related to gel strength, showing that physicochemical phenomena were the main mechanisms underlying the perceived sensory changes. Fat addition reduced the amount of oral processing and had differential effects on release, depending on the fat solubility or lipophilicity of the volatiles.

Zooming in: Structural Investigations of Rheologically Characterized Hydrogen-Bonded Low-Methoxyl Pectin Networks.

Self-assembled hydrogen-bonded networks of the polysaccharide pectin, a mechanically functional component of plant cell walls, have been of recent interest as biomimetic exemplars of physical gels, and the microrheological and strain-stiffening behaviors have been previously investigated. Despite this detailed rheological characterization of preformed gels, little is known about the fundamental arrangement of the polymers into cross-linking junction zones, the size of these bonded regions, and the resultant network architecture in these hydrogen-bonded materials, especially in contrast to the plethora of such information available for their well-known calcium-assembled counterparts. In this work, in concert with pertinent rheological measurements, an in-depth structural study of the hydrogen-bond-mediated gelation of pectins is provided. Gels were realized by using glucona-delta-lactone to decrease the pH of solutions of pectic polymers that had a (blockwise) low degree of methylesterification. Small-angle X-ray scattering and transmission electron microscopy were utilized to access structural information on length scales on the order of nanometers to hundreds of nanometers, while complementary mechanical properties were measured predominantly using small amplitude oscillatory shear rheology.

Challenges in computational modelling of food breakdown and flavour release.

A dynamic, three dimensional (3D) computational model that predicts the breakdown of food and the release of tastants and aromas could enhance the understanding of how food is perceived during consumption. This model could also shorten the development process of new foods because many virtual foods could be assessed, and discarded if unsuitable, before any physical prototyping is required. The construction and testing of a complete 3D model of mastication presents many challenges including an accurate representation of: the anatomical movements of the oral cavity (including the teeth, tongue, cheeks and palates), the breakdown behaviour of the food, the interactions between comminuted food and saliva as the bolus is formed, the release and transport of taste and aromas and how these physical and chemical processes are perceived by a person. These challenges are discussed in reference to previous experimental and simulation work and using results of new applications of a coupled biomechanical-smoothed particle hydrodynamics (B-SPH) model. The B-SPH model is demonstrated to simulate several complicated aspects of mastication including: (1) the sensitivity of particle size to changes in the movements of the jaw and tongue; (2) large strain behaviour of food due to softening by heating; (3) interactions between solid and liquid food components; (3) the release of tastants into the saliva; and (4) the transport of tastants to the taste buds. These applications show the possibilities of a model to viably simulate mastication, but highlight the many modelling and experimental challenges that remain.

Influence of heat and shear induced protein aggregation on the in vitro digestion rate of whey proteins.

Protein intake is essential for growth and repair of body cells, the normal functioning of muscles, and health related immune functions. Most food proteins are consumed after undergoing various degrees of processing. Changes in protein structure and assembly as a result of processing impact the digestibility of proteins. Research in understanding to what extent the protein structure impacts the rate of proteolysis under human physiological conditions has gained considerable interest. In this work, four whey protein gels were prepared using heat processing at two different pH values, 6.8 and 4.6, with and without applied shear. The gels showed different protein network microstructures due to heat induced unfolding (at pH 6.8) or lack of unfolding, thus resulting in fine stranded protein networks. When shear was applied during heating, particulate protein networks were formed. The differences in the gel microstructures resulted in considerable differences in their rheological properties. An in vitro gastric and intestinal model was used to investigate the resulting effects of these different gel structures on whey protein digestion. In addition, the rate of digestion was monitored by taking samples at various time points throughout the in vitro digestion process. The peptides in the digesta were profiled using SDS-polyacrylamide gel electrophoresis, reversed-phase-HPLC and LC-MS. Under simulated gastric conditions, whey proteins in structured gels were hydrolysed faster than native proteins in solution. The rate of peptides released during in vitro digestion differed depending on the structure of the gels and extent of protein aggregation. The outcomes of this work highlighted that changes in the network structure of the protein can influence the rate and pattern of its proteolysis under gastrointestinal conditions. Such knowledge could assist the food industry in designing novel food formulations to control the digestion kinetics and the release of biologically active peptides for desired health outcome.

Thermomechanical effects of co-solute on the structure formation of bovine serum albumin.

The effect of glucose syrup on the structural properties of bovine serum albumin has been addressed in preparations from low to high solids. Fifteen percent protein was mixed with the co-solute at concentrations up to 65% and subjected to thermal treatment to examine the changes in phase and state transitions. Thermomechanics were the working protocol being carried out with micro differential scanning calorimetry and small deformation dynamic oscillation. Results argue that protein molecules have been extensively stabilised by the addition of a co-solute, recorded via a delayed thermal denaturation. Further, increasing the glucose syrup enhanced polymer-polymer interactions leading to stronger networks following thermal denaturation of the globular protein. Condensed BSA/glucose syrup mixtures, i.e. at 80% solids, were cooled at subzero temperatures to exhibit a considerable state of vitrification. Molecular relaxation phenomena were successfully followed using theoretical concepts from synthetic polymer research to yield the mechanical glass transition temperature.

Fundamental studies on the structural functionality of whey protein isolate in the presence of small polyhydroxyl compounds as co-solute.

The present work deals with the changing network morphology of whey protein isolate (15%, w/w) in the presence of glucose syrup (co-solute) with concentrations ranging from 0% to 65% (w/w) in 10 mM CaCl2 solution, thus producing formulations with a total level of solids of up to 80% (w/w). Denaturation behaviour and aggregation of whey protein systems were investigated using small deformation dynamic oscillation on shear, micro and modulated differential scanning calorimetry, and confocal laser scanning microscopy. A progression in the mechanical strength of protein aggregates was observed resulting from enhanced protein-protein interactions in the presence of glucose syrup. Addition of the co-solute resulted in better thermal stability of protein molecules by shifting the process of denaturation to higher temperature, as observed by calorimetry. Observations are supported by micrographs showing coherent networks with reduced size of whey protein aggregates in the presence of high levels of glucose syrup, as opposed to thick and random clusters for systems of whey protein by itself. Glass transition phenomenon was observed for condensed protein/co-solute systems, which were treated with theoretical concepts adapted from synthetic polymer research to pinpoint the mechanical glass transition temperature.

Using SAXS to reveal the degree of bundling in the polysaccharide junction zones of microrheologically distinct pectin gels.

The results of microrheological studies carried out on ionotropic pectin gels, particularly the manifest power law behavior observed at high frequencies, indicate that by using different assembly conditions gels can be formed in which the elementary network strands have different stiffnesses. It has been hypothesized that these differences reflect different network architectures, the extreme cases of which might be described as (i) dimeric calcium-chelating junction-zones of limited extent, linked by considerably longer, flexible, single-chain sections, or (ii) semiflexible bundles consisting of extensively aggregated dimeric junction zones that latterly become entangled and cross-linked. To test this hypothesis directly, microrheologically distinct pectin gels have been generated using different assembly modalities, in particular by using different concentrations of polymer and cross-linking ions and by contrasting the controlled-release of ions or ion-binding groups, and the resulting systems have been studied by small-angle X-ray scattering. The results straightforwardly reveal that gels that are clearly more semiflexible from a microrheological point-of-view contain larger scattering entities than those with a more flexible character. Furthermore, a more detailed interpretation of the scattering data with the aid of molecular modeling suggests that for the gels formed here those with a semiflexible microrheological signature consist predominantly of network filaments consisting of four or more chains, whereas those with a more flexible signature are predominantly single-chain sections linked by dimeric associations with no more that a few percent of the chains bundled to any higher extent. The ability to generate differing network architectures from the same polymer that fulfill different functional requirements, either in vivo in the plant cell wall, where pectin plays a crucial structural and mechanical role, or in vitro in a myriad of applications, makes these biomimetic biopolymer networks of considerable interest.

Dynamic rheological properties of plant cell-wall particle dispersions.

The rheological behaviour of plant cell-wall particle dispersions was investigated using dynamic oscillatory measurements. Two starting plant materials, broccoli stem and carrot were used and two types of particles were obtained by mechanically shearing blanched (80°C, 10 min) or cooked (100°C, 15 min) plant tissues. Blanching resulted in cell-wall particles made up of a collection of clusters of cells with an average particles size of ∼200 µm, while cooking generated nearly all single-cell particles with an average particle size of ∼80 µm. The rheological measurements showed that in the range of weight concentrations considered (∼0.5% to ∼8%) the dispersions behaved as elastic materials with the elastic modulus G' higher than G″ within the frequency range (0.01-10 Hz). This study shows that the behaviour of the complex modulus G* as a function of the effective volume fraction ϕ can be modelled using different theoretical equations. To do so, it is assumed that below a critical volume fraction ϕc a network of plant cell-wall particles was formed and G* as a function of ϕ obeys a power-law relationship. However above ϕc, where the particles were highly packed, G* could be modelled using theoretical equations developed for concentrated emulsions and elastic particle dispersions.

Chemical modification of gelatin by a natural phenolic cross-linker, tannic acid.

Chemical modification of gelatin by a natural phenolic compound tannic acid (TA) at pH 8 was studied, and the properties of the modified gelatin materials were examined. The cross-linking effect was predominant when the TA content was lower, resulting in the formation of a partially insoluble cross-link network. The cross-linking structure was stable even under boiling, and the protein matrix became rigid, whereas the mechanical properties were enhanced. An effective cross-linking effect on gelatin matrix was achieved when the amount of TA was around 3 wt %. Further increase in the TA content enhanced the grafting and branching reactions between gelatin and TA in conjunction with the hydrogen bonding between gelatin and TA molecules. These effects produced an increase in molecular mobility of gelatin matrix, and the materials displayed a behavior similar to that of plasticized protein materials.

Chemical cross-linking gelatin with natural phenolic compounds as studied by high-resolution NMR spectroscopy.

Cross-linking gelatin with natural phenolic compound caffeic acid (CA) or tannic acid (TA) above pH 9 resulted in formation of insoluble hydrogels. The cross-linking reactivity was controlled by variation of pH, the concentration of the gelatin solution, or the amount of CA or TA used in the reaction. The cross-linking chemistry was studied by high-resolution NMR technique in both solution and solid state via investigation on small molecular model systems or using (13)C enriched caffeic acid (LCA) in the reaction with gelatin. Direct evidence was obtained to confirm the chemical reactions occurring between the phenolic reactive sites of the phenolic compounds and the amino groups in gelatin to form C-N covalent bonds as cross-linking linkages in gelatin matrix. The cross-linked network was homogeneous on a scale of 2-3 nm. The cross-linking resulted in a significant decrease in the molecular mobility of the hydrogels, while the modulus of the films remained at high values at high temperatures.

Influence of boron on carrot cell wall structure and its resistance to fracture.

Plant cell wall structure integrity and associated tissue mechanical properties is one of key determinants for the perceived texture of plant-based foods. Carrots (Daucus carota) were used to investigate the effect of mineral supply of boron (B) and/or calcium (Ca), during plant growth, on the plant cell wall structure and mechanical properties of matured root tissues. Five commercial cultivars of carrots, Kuroda (orange), Dragon Purple, Kuttiger White, Yellow, and Nutri-Red, were cultivated under controlled glasshouse conditions over two seasons. Significant increases in the accumulation of B and Ca were found for all cultivars of carrots when additional B and Ca were included in the nutrient feeding solutions throughout the plant growth period. Elevated levels of B in carrot root tissue reduced the uptake of Ca and other mineral nutrients and enhanced plant cell wall structural integrity, its resistance to fracture, and the weight and size (both diameter and length) of carrots. Although higher amounts of Ca were accumulated in the plant materials, the additional supply of Ca did not have a significant effect on the mechanical properties of mature plant tissues or on the uptake of B by the plant. The results suggest that B cross-linking of pectin (rhamnogalacturonan II) has a greater influence on mature tissue mechanical properties than Ca cross-linking of pectin (homogalacturonan) when supplied during plant growth.

Influence of pectin fine structure on the mechanical properties of calcium-pectin and acid-pectin gels.

The in vitro and in vivo functionality of the anionic plant polysaccharide pectin depends not only on the amount of ion-binding groups attached to the polymer but also on the distribution of such groups along the backbone. It has been proposed recently that information regarding this intramolecular distribution can be quantified by defining a degree of blockiness (DB or DB(abs)), and the usefulness of such measures in discriminating qualitatively between pectins originating from different sources has been demonstrated. Despite this, the value of these parameters in predicting the pseudoequilibrium elastic modulus of gels remains untested. This study seeks to address this problem through the sourcing and in-house modification of a variety of pectins in order to produce a library of distinct representative fine structures. These were subsequently characterized in terms of their relevant properties, including the determination of the proposed DB and DB(abs), and the formation of gels of these samples was monitored using small deformation mechanical spectroscopy. In addition to ionotropic calcium gels the effect of the fine structure on acid gelation was also studied.

Associative and segregative phase separations of gelatin/kappa-carrageenan aqueous mixtures.

The effects of ionic strength, temperature, and pH on the phase separation behavior of type B pigskin gelatin/sodium-type kappa-carrageenan aqueous mixtures were investigated. Depending on the different combinations of temperature and sodium chloride (NaCl) concentration, the mixtures showed compatible, associative, and segregative phase separation behaviors. Additionally, a coexistence of associative and segregative (associative-co-segregative) phase separations was expected at low temperature and low NaCl concentration. These different phase separation events were observed using confocal scanning laser microscopy. Moreover, it was found that the segregative phase separation when alone is induced by the ordering of kappa-carrageenan chains, while that in the coexistence region is induced by the ordering of gelatin chains. pH had a significant effect on the associative phase separation, resulting in morphologies changing from compatible solution to liquid coacervate and further to solid precipitate with decreasing pH. These were attributed to the dramatic changes of the charge density of amphoteric gelatin during the pH decrease.