A comparison of the kinetics of in vitro starch digestion in smooth and wrinkled peas by porcine pancreatic alpha-amylase.

This study describes the impact of crop genetics and processing in two pea lines (Pisum sativum L.) on starch digestion kinetics. Mutation at the rugosus (r) locus leads to wrinkled pea seeds, a reduction in starch content and a lower extent of in vitro starch digestibility. The Logarithm of Slope (LOS) kinetic model was used to analyse digestion curves obtained using porcine pancreatic α-amylase for a range of particle size fractions. Changes in starch structure induced by the r mutation led to clear differences in starch digestion kinetics for purified starches and pea flours. Larger particle size fractions showed slower starch digestion relative to the purified starch, but significant differences still existed between r and wild type pea lines. It is expected that this work will help inform the design of future studies where both starch structure and food structure are important determinants of digestion behaviour.

The effect of the protein corona on the interaction between nanoparticles and lipid bilayers.

HYPOTHESIS: It is known that nanoparticles (NPs) in a biological fluid are immediately coated by a protein corona (PC), composed of a hard (strongly bounded) and a soft (loosely associated) layers, which represents the real nano-interface interacting with the cellular membrane in vivo. In this regard, supported lipid bilayers (SLB) have extensively been used as relevant model systems for elucidating the interaction between biomembranes and NPs. Herein we show how the presence of a PC on the NP surface changes the interaction between NPs and lipid bilayers with particular care on the effects induced by the NPs on the bilayer structure. EXPERIMENTS: In the present work we combined Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) and Neutron Reflectometry (NR) experimental techniques to elucidate how the NP-membrane interaction is modulated by the presence of proteins in the environment and their effect on the lipid bilayer. FINDINGS: Our study showed that the NP-membrane interaction is significantly affected by the presence of proteins and in particular we observed an important role of the soft corona in this phenomenon.

Sodium alginate decreases the permeability of intestinal mucus.

In the small intestine the nature of the environment leads to a highly heterogeneous mucus layer primarily composed of the MUC2 mucin. We set out to investigate whether the soluble dietary fibre sodium alginate could alter the permeability of the mucus layer. The alginate was shown to freely diffuse into the mucus and to have minimal effect on the bulk rheology when added at concentrations below 0.1%. Despite this lack of interaction between the mucin and alginate, the addition of alginate had a marked effect on the diffusion of 500 nm probe particles, which decreased as a function of increasing alginate concentration. Finally, we passed a protein stabilised emulsion through a simulation of oral, gastric and small intestinal digestion. We subsequently showed that the addition of 0.1% alginate to porcine intestinal mucus decreased the diffusion of fluorescently labelled lipid present in the emulsion digesta. This reduction may be sufficient to reduce problems associated with high rates of lipid absorption such as hyperlipidaemia.

The adsorption-desorption behaviour and structure function relationships of bile salts.

The digestion of dietary components in the human gastrointestinal (GI) tract is a complex, dynamic, inherently heterogeneous process. A key aspect of the digestion of lipid in the GI tract is the combined action of bile salts, lipase and colipase in hydrolysing and solubilising dispersed lipid. The bile salts are a mixture of steroid acid conjugates with surfactant properties. In order to examine whether the different bile salts have different interfacial properties their dynamic interfacial behaviour was characterised. Differences in the adsorption behaviour to solid hydrophobic surfaces of bile salt species were studied using dual polarisation interferometry and atomic force microscopy (AFM) under physiological conditions. Specifically, the cholates adsorbed more slowly and a significant proportion were irreversibly adsorbed following buffer rinsing; whereas the deoxycholates and chenodeoxycholates adsorbed more rapidly and desorbed to a greater extent following buffer rinsing. The conjugating groups (taurine, glycine) did not influence the behaviour. AFM showed that the interfacial structures that remained following buffer rinsing were also different between these two groups. In addition, the adsorption-desorption behaviour affected the adsorption of colipase to a solid surface. This supports the idea that cooperative adsorption occurs between certain bile salts and colipase to facilitate the adsorption and activity of pancreatic lipase in order to restore lipolytic activity in the presence of bile salts. This study provides insights into how differences in bile salt structure could affect lipase activity and solubilisation of lipolysis products and other lipid-soluble bioactive molecules.

Structural characterisation of parotid and whole mouth salivary pellicles adsorbed onto DPI and QCMD hydroxyapatite sensors.

In this study we investigated the differences in the properties of pellicles formed from stimulated parotid saliva (PS), which contains little or no mucin; and stimulated whole mouth saliva (WMS), which contains mainly two types of mucin: MUC5B and MUC7. By contacting WMS and PS with quartz-crystal microbalance with dissipation monitoring (QCM-D) and dual polarisation interferometer (DPI) hydroxyapatite (the main component of enamel) coated sensors, we observed the formation and structure of the respective salivary pellicles. As this was the first time that DPI hydroxyapatite sensors have been used to measure salivary pellicle adsorption; the techniques combined allowed us to measure the hydrated mass, dry mass, thickness and viscoelastic properties of the pellicle; but also to record the density of the PS and WMS formed pellicles. Subsequently, the PS pellicle was shown to form a denser layer than WMS pellicle; which would suggest that the proteins present in PS are also responsible for forming the dense basal layer of the acquired enamel pellicle. Whereas proteins present in the WMS are more likely to help form the softer outer layer of the pellicle. The data presented help to further define the mechanisms leading to the multi-layered structure of the salivary pellicle and demonstrate that salivary composition has an important effect on the structural properties of the adsorbed pellicle.

The effect of poly-L-lysine structure on the pH response of polygalacturonic acid-based multilayers.

The effect of poly-L-lysine (PLL) molecular weight and structure on pH stability of polygalacturonic acid (PGaLA)-based multilayer films is studied over a pH cycle 7.0-1.6-7.0. The multilayer assembled with the lowest molecular weight PLL (1 kDa) showed the largest pH response. Only 12% of the mass remained and a preferential loss of PLL was observed. Extensive structural reorganisation of the layer as the pH was increased was due to the PGaLA reionisation leading to extensive net loss of hydrated mass. The multilayers assembled with the higher molecular weight linear PLLs (10 kDa, 200 kDa) showed loss of about 50% of their initial polymer mass. The multilayer assembled with the dendrimer (22 kDa) showed a stronger response to pH compared to the linear higher molecular weight PLLs. Over the pH cycle a loss of about 60% polymer mass and a decrease in the film thickness was observed. Despite having a reduced density at pH 1.6, the density substantially recovered to 0.54 g mL(-1) on return to pH 7.0.

An electronic medication reminder, supported by a monitoring service, to improve medication compliance for elderly people living independently.

We conducted a pilot study of a personal medication reminder unit, supported by a monitoring service. A total of 31 elderly residents were offered the reminder service for a period of eight weeks (1736 person-days of service). A telephone call from the monitoring service was made after four weeks to check that each participant was happy with the service and keen to continue. No one opted out and users of the service found it easy to manage, helpful and acceptable. There was a significant improvement (P = 0.012) in the rate of self-assessed medication compliance, from pre (52%) to post (81%) service introduction. There was a significant improvement in people's perceived ability to look after themselves at home (self-care ability) (P = 0.001). The percentage of participants rating their ability to look after themselves at home as excellent increased from 42% to 68%. Health-related quality of life measures, such as physical and mental health, showed positive improvement, but the changes were not significant. The service has the potential to improve health and well-being outcomes for people on multiple medications living independently in the community.

Combined QCMD and AFM studies of lysozyme and poly-L-lysine-poly-galacturonic acid multilayers.

A quartz crystal microbalance with dissipation monitoring (QCMD) has been used to monitor the adsorption and structure of lysozyme monolayers and multilayers, and poly-L-lysine (PLL)-polygalacturonic acid (PGalA) multilayers at a solid-liquid interface using freshly-cleaved mica as a substrate. QCMD measurements were complemented with atomic force microscopy (AFM). AFM images revealed that lysozyme formed incomplete monolayers and provided a basis for calculation of the thickness of the protein film. Comparative studies of adsorption onto standard and mica-coated quartz crystals showed higher areal mass adsorption and a longer-time adsorption process for mica-coated quartz crystals. Simultaneous AFM images and QCMD data were obtained for lysozyme, linear PLL-PGalA and 7 nm PLL dendrimer-PGalA multilayers. The layer-by-layer deposited multilayer films exhibited viscoelastic properties and their growth followed a non-linear regime, associated with the PLL diffusion in and out of the film formation for linear PLL-PGalA films. For the PLL 7 nm dendrimer-PGalA films the AFM images revealed marked changes in surface roughness during layer by layer deposition: these changes influence the interpretation of the QCMD data and provide additional information on the growth and structure of the multilayers.

Environmental responsiveness of polygalacturonic acid-based multilayers to variation of pH.

The effect of pH on the stability of layer-by-layer deposited polygalacturonic acid (PGalA)-based multilayer films prepared with the polycations poly-L-lysine, chitosan, and lysozyme is studied. The response was characterized using a quartz crystal microbalance, dual polarization interferometry, and Fourier transform infrared spectroscopy which probe multilayer thickness, density, polymer mass (composition and speciation), and hydration. All multilayers showed irreversible changes in response to pH change becoming thinner due to the partial disassembly. Preferential loss of the polycation (50-80% w/w) and relative small losses of PGaLA (10-35% w/w) occurred. The charge density on the polycation has a strong influence on the response to the acid cycle. Most of the disassembly takes place at the pH lower that pK(a) of PGaLA, indicating that this factor was crucial in determining the stability of the films. The pH challenge also revealed a polycation-dependent shift to acid pH in the PGaLA pK(a).

Characterization of the rate of thermally-induced aggregation of ß-lactoglobulin and its trehalose mixtures in the glass state.

The thermally induced aggregation of bovine ß-lactoglobulin in the glass state was studied both as the pure protein and in trehalose mixtures. Calorimetric measurements and size exclusion chromatography were used to characterize the glass state and aggregation, respectively. The initial aggregation rate was characterized by the initial rate of tetramer formation (ß-lactoglobulin dimerizes under the elution conditions), which showed Arrhenius temperature dependence with an activation energy of 95 kJ mol(-1) in the temperature range 60-100 °C. The trehalose addition slowed the aggregation in a way that depended exponentially on volume fraction, exp(-φ/φ*). The volume fraction characterizing the retardation of the reaction, φ*, was 0.173 of the order required to fill the interstices between close-packed protein molecules. In this system, trehalose was acting as an inert glass-forming diluent. In a general way, this supports the water substitute hypothesis and the understanding of the mechanisms underlying anhydrobiosis and protein stabilization technologies.

Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices.

Biopharmaceutical proteins are often formulated and freeze dried in agents that protect them from deleterious reactions that can compromise activity and authenticity. Although such approaches are widely used, a detailed understanding of the molecular mechanisms of protein stabilization in low water content amorphous glasses is lacking. Further, whilst deterioration chemistries are well described in dilute solution, relatively little is known about the extent and mechanisms by which protein integrity is compromised in the glassy state. Here we have investigated the relationship between protein modification and rate thereof, with variation of pH, carbohydrate excipient, temperature and the glass transition temperature using a model protein, lysozyme. Mass spectrometry analysis and peptide mapping confirm that protein modifications do occur in the glassy state in a time-, temperature-, and carbohydrate excipient-dependent manner. There were clear trends between the buffer pH and the primary modification detected (glycation). Most importantly, there were differences in the apparent reactivities of the lysine residues in the glass compared with those previously determined in solution, and therefore, the well-characterized solution reactivity of this reaction cannot be used to predict likely sites of modification in the glassy state. These findings have implications for (i) the selection and combinations of formulation components, particularly with regard to glycation in the glassy state, and (ii) the design of procedures and methodologies for the improvement of protein stability in the glassy state.

Layer-by-layer assembly of viral nanoparticles and polyelectrolytes: the film architecture is different for spheres versus rods.

The development of tuneable thin film assemblies that contain (bio)nanoparticles is an emerging field in nanobiosciences/nanotechnology. Our research focuses on the utilisation of viral nanoparticles (VNPs) as tools and building blocks for materials science. In previous reports we studied multilayered arrays of chemically modified cowpea mosaic virus (CPMV) particles and linker molecules. To extend these studies and to gain more insights into the architecture of the arrays, we report here on the construction of multilayered assemblies of native plant viral particles and polyelectrolytes. We specifically addressed the question of whether the shape of the VNPs influences the overall structures of the arrays. To study this, we have chosen two particles with similar surface properties but different shapes: CPMV was used as a sphere-like VNP, and tobacco mosaic virus (TMV) served as a rod-shaped VNP. The multilayers were self-assembled on solid supports through electrostatic interactions. Multilayer build-up was followed by quartz crystal microbalance with dissipation monitoring and UV/Vis spectroscopy. Scanning electron microscopy was used to characterize the topologies of the thin films. Our studies show that shape indeed matters. Incorporation of CPMV in alternating arrays of VNPs and polyelectrolytes is demonstrated; in stark contrast, TMV particles were found to be excluded from the arrays, and floated atop the architecture in an ordered structure.

Deposition of pectin/poly-L-lysine multilayers with pectins of varying degrees of esterification.

The effect of pectin esterification on the assembly of multilayers consisting of poly-L-lysine (PLL) and pectin was studied using surface plasmon resonance (SPR), Fourier transform infrared-attenuated total reflection spectroscopy (FTIR-ATR), and a quartz crystal microbalance with dissipation monitoring (QCMD). With each layer deposited, there was a progressive increase in mass. The net charge of the multilayers was positive and increased with increasing degree of esterification of the pectin. Multilayer fabrication involved a limited fractionation of the pectin preparations, with the more highly esterified pectins having a weaker affinity for PLL. The multilayers were relatively hydrated structures with estimates of solids content in the range 10-32% w/w. The more highly esterified pectins had a tendency to form more hydrated structures, which showed a strong deswelling when PLL was added to a freshly deposited pectin layer.

The effect of thermal history on the maillard reaction in a glassy matrix.

To test whether the extent of physical aging affected the reaction rate, Maillard reaction kinetics were studied in glassy model preservation systems subjected to two different thermal histories. The glass transition temperature and physical aging of the matrix were determined using differential scanning calorimetry, and the normalized heat capacities were modeled using the Tool-Narayanaswamy-Moynihan approach. Samples prepared using the different thermal histories initially had different degrees of aging, but these were practically indistinguishable after 10 h under the reaction conditions (65 degrees C); the samples underwent rapid structural relaxation at that temperature. The reaction of glucose and lysine in an amorphous trehalose/sucrose matrix was followed using spectrophotometric and chromatographic analysis. A difference in reaction rate could only be distinguished in the rate of consumption of glucose, which was approximately 20% faster in the minimally aged matrix; no significant differences were seen in any other indicator of reaction.

Physical aging of starch, maltodextrin, and maltose.

The physical aging of low water content, amorphous starch/water, maltodextrin/water, and maltose/water mixtures in the glassy state was examined using mechanical testing and calorimetry. Stress relaxation measurements showed that upon storage of the glassy materials there was a time-dependent increase in both flexural modulus and mechanical relaxation time. The mechanical relaxation time increased with depth of quench below the calorimetric glass transition temperature and with aging time at the quench temperature. Calorimetry of the aged materials showed an overshoot in heat capacity in the vicinity of the glass transition. The logarithm of the mechanical relaxation time showed a simple linear relationship with the size of the overshoot expressed as an enthalpy change. The calorimetric behavior could be modeled using the Tool-Narayanaswamy-Moynihan method.

The nonequilibrium phase and glass transition behavior of beta-lactoglobulin.

Concentrated solutions of bovine beta-lactoglobulin were studied using osmotic stress and rheological techniques. At pH 6.0 and 8.0, the osmotic pressure was largely independent of NaCl concentration and could be described by a hard sphere equation of state. At pH 5.1, close to the isoelectric point, the osmotic pressure was lower at the lower NaCl concentrations (0 mM, 100 mM) and was fitted by an adhesive hard sphere model. Liquid-liquid phase separation was observed at pH 5.1 at ionic strengths of 13 mM and below. Comparison of the liquid-liquid and literature solid-liquid coexistence curves showed these solutions to be supersaturated and the phase separation to be nonequilibrium in nature. In steady shear, the zero shear viscosity of concentrated solutions at pH 5.1 was observed at shear rates above 50 s(-1). With increasing concentration, the solution viscosity showed a progressive increase, a behavior interpreted as the approach to a colloidlike glass transition at approximately 60% w/w. In oscillatory shear experiments, the storage modulus crossed the loss modulus at concentrations of 54% w/w, an indication of the approaching glass transition. Comparison of the viscous behavior with predictions from the Krieger-Dougherty equation indicates the hydrodynamic size of the protein decreases with increasing concentration, resulting in a slower approach to the glass transition than a hard sphere system.

Oxygen solubility and permeability of carbohydrates.

The saturated oxygen concentration in a series of aqueous solutions of sorbitol (up to 35% w/w) and maltitol (up to 50% w/w) was measured using colorimetric reagent vials based on Rhodazine D. The results indicate that the solubility of oxygen in low-water carbohydrates is considerably lower than its solubility in pure water. It was concluded that the low-oxygen solubility is a major factor contributing to the barrier properties of low-water content carbohydrates used in the encapsulation of flavours, lipids, peptides and other oxidisable species.

Starch crystal solubility and starch granule gelatinisation.

The solubility and dissolution behaviour of A- and B-type crystals of short chain amylose were measured both directly and using differential scanning calorimetry in the temperature range 30-110 degrees C. Dissolution in the calorimeter was affected by super-heating to the extent of 24-28 degrees C. Following trends previously found by calorimetry the B-type crystal polymorph was more soluble than the A-type. Analysis of the chain composition of the dissolved material revealed a preferential solubilisation of the short chains at the lower temperatures. The solubility of both crystal polymorphs and the magnitude of the preferential solubilisation effect was reduced in the presence of 30% w/w sucrose. A comparison of calorimetric measurements of crystal dissolution and the gelatinisation of native granular waxy maize and potato starches found some broad similarities, such as transition temperatures and their composition dependence, and some differences, such as the relatively narrow temperature range of granular gelatinisation, which reflects its cooperative nature.

The glass transition behavior of the globular protein bovine serum albumin.

The glass-like transition behavior of concentrated aqueous solutions of bovine serum albumin was examined using rheological techniques. At mass fractions >0.4, there was a marked concentration dependence of viscosity with a glass-like kinetic arrest observed at mass fractions in the region of 0.55. At mass fractions >0.6 the material behaved as a solid with a Young's modulus rising from approximately 20 MPa at a mass fraction of 0.62-1.1 GPa at 0.86. The solid was viscoelastic and exhibited stress relaxation with relaxation times increasing from 33 to 610 s over the same concentration range. The concentration dependence of the osmotic pressure was measured, at intermediate concentrations, using an osmotic stress technique and could be described using a hard sphere model, indicating that the intermolecular interactions were predominantly repulsive. In summary, a major structural relaxation results from the collective motion of the globules at the supra-globule length scale and, at 20 degrees C, this is arrested at water contents of 40% w/w. This appears to be analogous to the glass transition in colloidal hard spheres.