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Previous work has suggested the ability of 4-hydroxybenzoic acid (4HBA) to bind to β -casein following ultra high temperature-like processing (UHT) in model aqueous systems. The present work confirmed directly, using MALDI-TOF-MS, the presence of covalently bound 4HBA following UHT-like treatment. In subsequent molecular dynamics simulations, the 3D structure of the β -casein molecule was modified so that the meta-C of 4HBA ring and the side chain amino group of lys32 were linked covalently. Such simulations further indicated that the covalent addition of the phenolic compound had impacted the protein density and solvent accessibility. Hydrogen bond analysis between lys32 and the remainder of the protein structure revealed that the covalent complexation supported the formation of additional hydrogen bonds. These increased from potentially 9, in the single protein molecule, to 51 in the complex with 4HBA. However, the persistence of hydrogen bonds was reduced, leading overall to decreased stability and increased protein flexibility.

Interactions between the dimeric form of β-lactoglobulin and vanillic acid were investigated at pH 7.2, using a variety of spectroscopic techniques and molecular dynamics (MD) simulations. FTIR and CD studies showed alterations in the secondary structure of the protein upon its interaction with the ligand. Fluorescence measurements indicated that the dimeric complex with the phenolic acid produced a large dissociation constant (K

Changes in the structural properties of hydrogels made with gelatin and bovine serum albumin mixtures were recorded following exposure to high pressure at 300 MPa for 15 min at 10 and 80 °C. Dynamic oscillation, SEM, FTIR and blending law modelling were utilised to rationalise results. Pressurization at the low temperature end yielded continuous gelatin networks supporting discontinuous BSA inclusions, whereas an inverted dispersion was formed at the high temperature end with the continuous BSA network suspending the discontinuous gelatin inclusions. Lewis and Nielsen equations followed the mechanical properties of the composites thus arguing that solvent partition between the two phases was always in favour of the polymer forming the continuous network. As far as we are aware, this is the first attempt to elucidate the solvent partition in pressurised hydrogel composites using blending law theory. Outcomes were contrasted with earlier work where binary mixtures were subjected only to thermal treatment.

The present work investigated the physicochemical and structural properties of Tulsi, Alfalfa and two varieties of Manuka honey derived from medicinal plants. Chemical analysis yielded data on the content of reducing sugars (glucose and fructose) that dominate the honey matrix, and of the minor constituents of protein, phenols and flavonoids. Standard chemical assays were used to develop a database of water content, electrical conductivity, pH, ash content, visual appearance and colour intensity. Physicochemical characteristics were related to structural behaviour of the four honey types, as recorded by small-deformation dynamic oscillation in shear, micro- and modulated differential scanning calorimetry, wide angle X-ray diffraction and infrared spectroscopy. The preponderance of hydrogen bonds in intermolecular associations amongst monosaccharides in honey yields a semi-amorphous or semi-crystalline system. That allowed prediction of the calorimetric and mechanical glass transition temperatures that demarcate the passage from liquid-like to solid-like consistency at subzero temperatures.

Interactions between the dimeric form of β-lactoglobulin and vanillic acid were investigated at pH 7.2, using a variety of spectroscopic techniques and molecular dynamics (MD) simulations. FTIR and CD studies showed alterations in the secondary structure of the protein upon its interaction with the ligand. Fluorescence measurements indicated that the dimeric complex with the phenolic acid produced a large dissociation constant (KD) compared to the monomeric counterpart at acidic pH (part A of this series). Stoichiometry of 1:1 was identified for the β-lactoglobulin-vanillic acid complex by Job plot analysis at neutral pH suggesting two ligand molecules can participate in binding with the dimer. Molecular docking and MD simulations suggested that the top-ranked binding sites of the ligand were located at the entrance of each β-barrel structure of the dimer. These simulations also allowed identification of the contribution of water molecules, in the form of protein-water-ligand bridging interactions, to the complexes.

This work examines the effect of including hydrated NaCl and CaCl2 (up to 6% w/w) on the physicochemical properties of condensed tapioca starch. Samples were prepared by hot pressing at 120°C to produce condensed systems that covered a range of moisture contents from 7.34% w/w (23% relative humidity) to 19.52% w/w (75% relative humidity). Tensile storage modulus and heat flow measurements were taken using DMA and MDSC, which were accompanied by FTIR, WAXD and ESEM. Increasing the salt level enhances the mechanical strength of starch in the glassy state and shifts the glass transition temperature to a higher value. Antiplasticising effects of NaCl and CaCl2 on the non-phosphorylated tapioca starch are indistinguishable from each other. Observations are complemented by intensification of absorbance peaks in FTIR spectra and a systematic change in shape and intensity of diffraction patterns with increasing addition of salt consistent with interactions between added ions and macromolecule.

This study examined the release of vitamin B6 from a hydrogel made of whey protein isolate (WPI). Work was carried out at ambient temperature without preheating the whey protein. Native-state macromolecules were crosslinked with a nontoxic compound, genipin. Experimentation included a ninhydrin assay with UV–vis absorbance, FTIR, 13C NMR, compression testing, SEM imaging, WPI matrix swelling and vitamin release protocols. It was confirmed that geninin crosslinked effectively the protein chains whose network strength was reinforced with increasing crosslinker concentrations. The modified Flory-Rehner theory predicted the molecular weight between crosslinks, network mesh size and crosslinking density in the swollen WPI gels as a function of added crosslinker. Transport patterns of vitamin B6 through the polymeric matrix were monitored over prolonged periods of observation. These were examined with the generalised Fick’s equation and the Peppas-Sahlin equation to unveil the interplay between diffusion and relaxation dynamics in the anomalous transport of the bioactive compound.

The effect of calcium chloride on the structural properties of condensed potato starch undergoing a thermally induced glass transition has been studied using dynamic mechanical analysis and modulated differential scanning calorimetry. Extensive starch gelatinisation was obtained by hot pressing at 120°C for 7 min producing materials that covered a range of moisture contents from 3.7% w/w (11% relative humidity) to 18.8% w/w (75% relative humidity). FTIR, ESEM and WAXD were also performed in order to elucidate the manner by which salt addition affects the molecular interactions and morphology of condensed starch. Experimental protocol ensured the development of amorphous matrices that exhibited thermally reversible glassy consistency. Both moisture content and addition of calcium chloride affected the mechanical strength and glass transition temperature of polymeric systems. Highly reactive calcium ions form a direct interaction with starch to alter considerably its structural properties via an anti-plasticizing effect, as compared to the polymer-water matrix.

We examined the morphology of a network made with native BSA molecules being crosslinked with genipin at ambient temperature. Ninhydrin assay, FTIR, WAXD, SEM and mechanical tests documented successful crosslinking that enhanced the structural properties of the three dimensional structure. Its hydrophilic nature allows swelling with water absorption, which can be monitored with the modified Flory-Rehner theory to predict the molecular weight between adjacent crosslinks, network mesh size and crosslinking density as a function of crosslinker addition. Characterisation studies were carried out with a view to developing a delivery vehicle for the controlled release of vitamin B6 over a prolonged period of observation. Moving boundaries associated with swelling of the protein matrix resulted in vitamin transport that could be described with the interplay of diffusional and relaxational kinetics via the Peppas-Sahlin equation. Combination of diffusion and swelling equilibrium theories unveils a measurable effect of network characteristics on vitamin B6 release.

We examine the morphology of hydrogels made of bovine serum albumin and gelatin following high pressure processing at 300 MPa for 15 min at 10 and 80 °C. Emphasis is on the distribution of added calcium counterions between the polymeric phases seen in changes in the structural properties of the composite gel. Protocol includes thermal and HPP treatments, dynamic oscillation rheology, ESEM, and modeling from the "synthetic polymer approach" to rationalize results. Pressurization at 10 °C produced continuous gelatin networks with dispersed BSA inclusions whereas pressurization at 80 °C yielded an inverse dispersion of BSA as the continuous phase supporting liquid gelatin inclusions. Lewis and Nielsen equations were adapted to predict the counterion distribution between the polymeric phases that profoundly affected the structural properties of the pressurized gels. The concept of counterion partition (p

The present investigation deals with the structural properties of condensed potato starch-sodium chloride systems undergoing a thermally induced glass transition. Sample preparation included hot pressing at 120°C for 7 min to produce extensive starch gelatinisation. Materials covered a range of moisture contents from 3.6% to 18.8%, which corresponded to relative humidity values of 11% and 75%. Salt addition was up to 6.0% in formulations. Instrumental work was carried out with dynamic mechanical analysis in tension, modulated differential scanning calorimetry, Fourier transform infrared spectroscopy, scanning electron microscopy and wide angle X-ray diffraction. Experimental conditions ensured the development of amorphous matrices that exhibited thermally reversible glassy consistency. Both moisture content and addition of sodium chloride affected the mechanical strength and glass transition temperature of polymeric systems. Sodium ions interact with chemical moieties of the polysaccharide chain to alter considerably structural properties, as compared to the starch-water matrix.

High pressure processing (HPP) can induce structure development in macromolecules which are distinct from those of conventional thermal treatments. Gelation properties of whey protein (5-20% w/w) upon 15min HPP at 600MPa and 5 or 30°C (initial sample temperatures) were examined in the presence and absence of 5% w/w gelatin. The values of storage modulus (G') in pressure treated mixed gels were below those of their counterparts thermally treated at 80°C. Mixed systems subjected to HPP in the solution state possessed higher G' than the mixed systems subjected to HPP in the form of gels. The cooling profile of G' in pressurised mixed solutions was similar to that of the gelatin solution, which indicates that HPP resulted in a high degree of gelatin continuity. Confocal images confirmed that gelatin was the continuous phase whilst whey protein aggregated in discontinuous inclusions within the pressurised mixed systems.

This work investigates the effect of whey protein agglomeration on the survivability of Saccharomyces boulardii within spray dried microcapsules. It attempts to go beyond phenomenological observations by establishing a relationship between physicochemical characteristics of the polymeric matrix and its effect on probiotic endurance upon spray drying. It is well known that this type of thermal shock has lethal consequences on the yeast cells. To avoid such undesirable outcome, we take advantage of the early agglomeration phenomenon observed for whey protein by adjusting the pH value of preparations close to isoelectric point (pH 4-5). During the subsequent process of spray drying, development of whey protein agglomerates induces formation of an early crust, and the protein in this molten globular state creates a cohesive network encapsulating the yeast cells. It appears that the early crust formation at a given sample pH and temperature regime during spray drying benefits the survivability of S. boulardii within microcapsules.

There is an increasing demand to tailor the functional properties of mixed biopolymer systems that find application in dairy food products. The effect of static high pressure processing (HPP), up to 600 MPa for 15 min at room temperature, on milk–gelatin mixtures with different solid concentrations (5%, 10%, 15% and 20% w/w milk solid and 0.6% w/w gelatin) was investigated. The viscosity remarkably increased in mixtures prepared with high milk solid concentration (15% and 20% w/w) following HPP at 300 MPa, whereas HPP at 600 MPa caused a decline in viscosity. This was due to ruptured aggregates and phase separation as confirmed by confocal laser scanning microscopy. Molecular bonding of the milk–gelatin mixtures due to HPP was shown by Fourier-transform infrared spectra, particularly within the regions of 1610–1690 and 1480–1575 cm−1, which reflect the vibrational bands of amide I and amide II, respectively.

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.

The effect of varying concentrations of co-solute (polydextrose) on thermomechanical and physicochemical properties of deacylated gellan matrices is presented. Modulated differential scanning calorimetry, micro differential scanning calorimetry, small deformation dynamic oscillation in shear, Fourier transform infrared spectroscopy, wide angle X-ray diffraction and environmental scanning electron microscopy have been used to investigate the structural transformations in aqueous, low-solid and condensed systems. There was a rise in values of storage modulus as the level of co-solute was increased, followed by a significant decline at intermediate concentrations, with high modulus values being regained as more of the co-solute was incorporated. These results confirm the hypothesis of a structural transformation from a highly enthalpic aggregated assembly in the aqueous/low-solid environment to a lightly cross linked polysaccharide network in the high solids regime. Time-temperature superposition (TTS) phenomena observed for amorphous synthetic polymers have been utilised to generate master curves of viscoelasticity, which afforded rationalisation of results on the basis of the free volume theory.

The dynamic interfacial tension (DIFT) at oil-water interface, diffusion coefficients, surface hydrophobicity, zeta potential and emulsifying properties, including emulsion activity index (EAI), emulsion stability index (ESI) and droplet size of lentil protein isolate (LPI), were measured at different pH and LPI concentration, in order to elucidate its emulsifying behaviour. Sodium caseinate (NaCas), whey protein isolate (WPI), bovine serum albumin (BSA) and lysozyme (Lys) were used as benchmark proteins and their emulsifying property was compared with that of LPI. The speed of diffusion-controlled migration of these proteins to the oil/water interface, was in the following order: NaCas>LPI>WPI>BSA>Lys, while their surface hydrophobicity was in the following order: BSA>LPI>NaCas>WPI>Lys. The EAI of emulsions stabilised by the above proteins ranged from 90.3 to 123.3 m(2)/g and it was 93.3 ± 0.2 m(2)/g in LPI-stabilised emulsion. However, the stability of LPI-stabilised emulsions was slightly lower compared to that of WPI and NaCas-stabilised emulsions at the same protein concentration at pH 7.0. The ESI of LPI emulsions improved substantially with decrease in droplet size when protein concentration was increased (20-30 mg/ml). Reduction of disulphide bonds enhanced both the EAI and ESI compared to untreated samples. Heat treatment of LPI dispersions resulted in poor emulsion stability due to molecular aggregation. The stability of LPI-stabilised emulsions was found to decrease in the presence of NaCl. This study showed that LPI can be as effective emulsifiers of oil-in-water emulsions as are WPI and NaCas at ≥20 mg/ml concentrations both at low and neutral pH. The emulsifying property of LPI can be improved by reducing the intra and inter-disulphide bond by using appropriate reducing agents.

In an effort to draw attention to the subject of structure/function relationships in high-solid biopolymer mixtures, this investigation produces binary composites of agarose with gelatin and systematically increases the amount of glucose syrup, which is the co-solute in this system. Experimental work was carried out using small-deformation dynamic oscillation on shear and modulated differential scanning calorimetry. Agarose/gelatin mixtures in an aqueous low-solid environment form non-interactive bicontinuous networks. Addition of glucose syrup to the polymeric blend prevents the formation of stable double helices in the environment. Gelatin, on the other hand, better withstands the co-solute-induced change in solvent quality. At subzero temperatures, materials go through the rubber-to-glass transition whose DSC glass transition temperature (Tg) is governed by the total level of solids in the system. Estimation of the mechanical Tg acquires physical significance by utilising the theory of free volume, as modelled by the Williams, Landel and Ferry (WLF) equation. The single value of Tg estimated by this approach argues in accordance with experimental observations for the predominance of the gelatin network in the high-solid mixture.

The structural properties of medium molecular weight chitosan (CHT), whey protein isolate (WPI) and native wheat starch (WS) from low- to intermediate-solid single systems and composite matrices were investigated. Analysis involved monitoring the thermal behavior of these biopolymers during controlled heating from 25 up to 95 °C and subsequent cooling to 5 °C under small deformation dynamic oscillation in-shear and micro differential scanning calorimetry experiments. Further information regarding the molecular interactions of components and overall network morphology of the systems was revealed through subjecting thermally developed gels to large deformation compression testing, scanning electron microscopy and infrared spectroscopy. Our study found a significant change in the structure of WPI networks upon incorporation of CHT in preparations due to electrostatic forces developing between the two polymeric constituents. In the tertiary system, the presence of low levels of starch contributed to a reduction in the firmness of the gel matrix. However, at higher additions of the polysaccharide, a recovery in the stored energy of composite materials was apparent, as recorded in the thermomechanical protocol.

The present study shows that application of high hydrostatic pressure of 600 MPa for 15 min at ambient temperature on condensed bovine serum albumin systems (BSA) with up to 80% w/w solids content has a limited effect on the conformational structure of the protein, as compared to thermal treatment. This was demonstrated throughout the experimental concentration range using small-deformation dynamic oscillation, differential scanning calorimetry and infrared spectroscopy. BSA possesses seventeen disulfide linkages per molecule, which constitutes a stable arrangement with high energy requirements for substantial structure alteration. Upon cooling, pressurised materials undergo vitrification and networks exhibit comparative mechanical strength to that of thermally treated counterparts. The mechanical manifestation of the glass transition region and glassy state for atmospheric and pressurised samples was examined by the method of reduced variables and the combined framework of WLF/free volume theory producing disparate predictions of the glass transition temperature for the two types of polymeric network.

Network formation of whey protein isolate (WPI) with increasing concentrations of native wheat starch (WS) has been examined. Small deformation dynamic oscillation in shear and modulated temperature differential scanning calorimetry enabled analysis of binary mixtures at the macro- and micromolecular level. Following heat induced gelation, textural hardness was measured by undertaking compression tests. Environmental scanning electron microscopy provided tangible information on network morphology of polymeric constituents. Experiments involving in vitro starch digestion also allowed for indirect assessment of phase topology in the binary mixture. The biochemical component of this work constitutes an attempt to utilise whey protein as a retardant to the enzymatic hydrolysis of starch in a model system with α-amylase enzyme. During heating, rheological profiles of binary mixtures exhibited dramatic increases in G' at temperatures more closely related to those observed for single whey protein rather than pure starch. Results from this multidisciplinary approach of analysis, utilising rheology, calorimetry and microscopy, argue for the occurrence of phase separation phenomena in the gelled systems. There is also evidence of whey protein forming the continuous phase with wheat starch being the discontinuous filler, an outcome that is explored in the in vitro study of the enzymatic hydrolysis of starch.

The isothermal phase behaviour at 5 °C of mixtures of sodium caseinate and oat β-glucan isolates varying in molecular weight (MW) was investigated by means of phase diagram construction, rheometry, fluorescence microscopy and electrophoresis. Phase diagrams indicated that the compatibility of the β-glucan/sodium caseinate system increases as β-glucan MW decreases. Images of mixtures taken at various biopolymer concentrations revealed phase separated domains. Results also revealed that at the state of thermodynamic equilibrium, lower MW samples yielded considerable viscosity in the mixture. At equivalent hydrodynamic volume of β-glucan in the mixtures, samples varying in molecular weight exhibited similar flow behaviour. A deviation dependent on the protein concentration was observed for the high MW sample in the concentrated regime due to the size of β-glucan aggregates formed. Results demonstrate that by controlling the structural features of β-glucan in mixtures with sodium caseinate, informed manipulation of rheological properties in these systems can be achieved.

Co-solute requirements for high methoxy pectin gelation were observed by the addition of glucose syrup and polydextrose at concentrations varying from 50% to 78% (w/w). Pectin content was fixed at 2% (w/w) in formulations. Studies from small deformation dynamic oscillation in shear, modulated differential scanning calorimetry and environmental scanning electron microscopy are reported. Structural properties of pectin preparations were recorded in relation to the molecular weight and concentration of added co-solute in an acidic environment (pH ∼3.0). High levels of co-solute induce formation of weak pectin gels at elevated temperatures (even at 95 °C), which upon subsequent cooling exhibit increasing strength and convert to a clear glass at subzero temperatures. Fourier Transform Infrared Spectroscopy and wide angle X-ray diffraction were practised to examine the nature of interactions between polymer and co-solute and the extent of amorphicity of preparations. Glucose syrup is an efficient plasticiser leading to a reduction in the glass transition temperature (Tg) of the pectin network, whereas polydextrose assists in the formation of stronger pectin gels in the rubbery state.

Investigation on the phase behaviour of a biopolymer mixture has been performed using 7.5% (w/w) gelatin and 1.5% (w/w) agarose in the presence of variable amounts of polydextrose as the co-solute from low to high levels of total solids. Mechanical observation of the system was performed using small deformation dynamic oscillation in shear along with thermal studies using modulated differential scanning calorimetry. Micrographs provided images of the changing morphology of the network with the addition of co-solute. Agarose and gelatin form non-interactive bicontinuous phases in the aqueous environment. Systematic increase in the concentration of polydextrose prevents the formation of a stable agarose network, with the polysaccharide chains dispersing in the high solids environment. Gelatin, on the other hand, retains its conformational stability even at a saturating co-solute environment through enhanced protein structuring. Vitrification studies on the high solids system at subzero temperatures provides information on the structural and molecular relaxation identified as a glass transition phenomenon. Fourier transform infrared spectroscopy was used to analyse potential direct interaction between polymers and co-solute. The extent of amorphicity in the system was confirmed using wide angle X-ray diffraction.

This investigation focuses on understanding the phase behaviour of gelatin when mixed with polydextrose (co-solute) primarily at high solid concentrations. The experimental work was carried out using small deformation dynamic oscillation in shear, modulated differential scanning calorimetry, Fourier transform infrared spectroscopy, wide angle X-ray diffraction and environmental scanning electron microscopy. A progression in the mechanical strength and thermal stability of the gelatin network was observed with the addition of polydextrose to the system. Combined thermomechanical and microscopy evidence argues for the development of phase separation phenomenon between protein and co-solute in high-solid preparations, where gelatin maintains helical conformation to provide network integrity as well as glassy consistency at subzero temperature. At the high solids regime, glassy consistency was treated with theoretical frameworks from the synthetic polymer research to pinpoint the glass transition temperature of the system.