Origin of the extremely high elasticity of bulk emulsions, stabilized by Yucca Schidigera saponins
Tsibranska, Sonya ; Tcholakova, Slavka ; Golemanov, Konstantin ; Denkov, Nikolai ; Arnaudov, Luben ; Pelan, Eddie ; Stoyanov, Simeon D. - \ 2020
Food Chemistry 316 (2020). - ISSN 0308-8146
Drop aggregation - Drop-drop adhesion - Emulsion - Emulsion elasticity - Interfacial elasticity - Non-purified oil - Phytosterols - Saponin
We found experimentally that the elasticity of sunflower oil-in-water emulsions (SFO-in-W) stabilized by Yucca Schidigera Roezl saponin extract, is by >50 times higher as compared to the elasticity of common emulsions. We revealed that strong specific interactions between the phytosterols from the non-purified oil and the saponins from the Yucca extract lead to the formation of nanostructured adsorption layers which are responsible for the very high elasticity of the oil-water interface and of the respective bulk emulsions. Remarkably, this extra high emulsion elasticity inhibits the emulsion syneresis even at 65 vol% of the oil drops – these emulsions remain homogeneous and stable even after 30 days of shelf-storage. These results demonstrate that the combination of saponin and phytosterols is a powerful new approach to structure oil-in-water emulsions with potential applications for formulating healthier functional food.
Gravity-driven syneresis in model low-fat mayonnaise
Wu, Qimeng ; Punter, Melle T.J.J.M. ; Kodger, Thomas E. ; Arnaudov, Luben ; Mulder, Bela M. ; Stoyanov, Simeon ; Gucht, Jasper Van Der - \ 2019
Soft Matter 15 (2019)46. - ISSN 1744-683X - p. 9474 - 9481.
Low-fat food products often contain natural, edible polymers to retain the desired mouth feel and elasticity of their full-fat counterparts. This type of product, however, can suffer from syneresis: densification due to the expulsion of fluid. Gaining insight into the physical principles governing syneresis in such soft hybrid dispersions remains a challenge from a theoretical perspective, as experimental data are needed to establish a basis. We record non-accelerated syneresis in a model system for low-fat mayonnaise: a colloid polymer mixture, consisting of oil in water emulsion with starch in the aqueous phase. We find the flow rate of expelled fluid to be proportional to the difference in hydrostatic pressure over the system. The osmotic pressure of the added starch, while being higher than the hydrostatic pressure, does not prevent syneresis because the soluble starch is lost to the expelled fluid. From these findings, we conclude that forced syneresis in these systems can be described as a gravity-driven porous flow through the densely packed emulsion, explainable with a model based on Darcy's law.
Rheology of particle/water/oil three-phase dispersions : Electrostatic vs. capillary bridge forces
Georgiev, Mihail T. ; Danov, Krassimir D. ; Kralchevsky, Peter A. ; Gurkov, Theodor D. ; Krusteva, Denitsa P. ; Arnaudov, Luben N. ; Stoyanov, Simeon D. ; Pelan, Eddie G. - \ 2018
Journal of Colloid and Interface Science 513 (2018). - ISSN 0021-9797 - p. 515 - 526.
Capillary bridges - Capillary suspensions - Pendular state - Silica particles - Suspension rheology - Wet granular materials - Yield stress
Hypothesis Particle/water/oil three-phase capillary suspensions possess the remarkable property to solidify upon the addition of minimal amount of the second (dispersed) liquid. The hardening of these suspensions is due to capillary bridges, which interconnect the particles (pendular state). Electrostatic repulsion across the oily phase, where Debye screening by electrolyte is missing, could also influence the hardness of these suspensions. Experiments We present data for oil-continuous suspensions with aqueous capillary bridges between hydrophilic SiO2 particles at particle volume fractions 35–45%. The hardness is characterized by the yield stress Y for two different oils: mineral (hexadecane) and vegetable (soybean oil). Findings and modelling The comparison of data for the “mirror” systems of water- and oil-continuous capillary suspensions shows that Y is lower for the oil-continuous ones. The theoretical model of yield stress is upgraded by including a contribution from electrostatic repulsion, which partially counterbalances the capillary-bridge attraction and renders the suspensions softer. The particle charge density determined from data fits is close to that obtained in experiments with monolayers from charged colloid particles at oil/water interfaces. The results could contribute for better understanding, quantitative prediction and control of the mechanical properties of solid/liquid/liquid capillary suspensions.
Novel mechanochemical approach for wheat starch-LPC complex formation
Rangelov, Antoan ; Stoyanov, S. ; Arnaudov, L. ; Spassov, Tony - \ 2017
Journal of Cereal Science 76 (2017). - ISSN 0733-5210 - p. 72 - 75.
Amylose-LPC - Ball-milling - DSC - Inclusion complex
Amylose-LPC inclusion complexes are acquired by a novel mechanochemical method and by hydrothermal method. The new synthetic method includes mechanical milling of suspension of LPC and starch under controlled conditions. Both methods applied resulted in similar microstructure of the complexes, studied by x-ray diffraction (XRD). Furthermore, Solid-state 13C NMR spectroscopy and differential scanning calorimetry (DSC) also confirm the efficient complexation by the new approach.
Cyclodextrin-Based Solid-Gas Clathrates
Pereva, Stiliana ; Himitliiska, Tsveta ; Spassov, Tony ; Stoyanov, S.D. ; Arnaudov, L.N. ; Dudev, Todor - \ 2015
Journal of Agricultural and Food Chemistry 63 (2015)29. - ISSN 0021-8561 - p. 6603 - 6613.
clathrates - crystallization - cyclodextrins - kinetics
"Cyclodextrin-gas" clathrates were obtained by crystallization from water solution of α-, β-, and γ-cyclodextrins (CDs) under pressure of the gas to be entrapped into the CD molecules. When the pressure is released, these clathrates are stable at ambient conditions and dissociate at elevated temperature, which makes them interesting for various applications as foam boosters in food and other industries. It was found that under these conditions α-CD forms clathrates with all of the gases used in this study (N2, N2O, CO2, Ar), whereas β- and γ-CDs can form clathrates only with N2. The concentration of the cyclodextrin and the temperature and pressure of the gas were varied for achieving higher clathrate yield and larger amount of embedded gas. Highest values of about 2 wt % were found for α-CD-N2O, as it releases in the temperature range of 40-60 °C.
Nanoemulsions obtained via bubble bursting at a compound interface
Feng, J. ; Roche, M. ; Vigolo, D. ; Arnaudov, L.N. ; Stoyanov, S.D. ; Gurkov, T.D. ; Tsutsumanova, G.G. ; Stone, H.A. - \ 2014
Nature physics 10 (2014). - ISSN 1745-2473 - p. 606 - 612.
surfactant solutions - gas-bubbles - sea spray - alkanes - aerosols - water
Bursting of bubbles at an air/liquid interface is a familiar occurrence relevant to foam stability, cell cultures in bioreactors and ocean–atmosphere mass transfer. In the latter case, bubble-bursting leads to the dispersal of sea-water aerosols in the surrounding air. Here we show that bubbles bursting at a compound air/oil/water-with-surfactant interface can disperse submicrometre oil droplets in water. Dispersal results from the detachment of an oil spray from the bottom of the bubble towards water during bubble collapse. We provide evidence that droplet size is selected by physicochemical interactions between oil molecules and the surfactants rather than by hydrodynamics. We demonstrate the unrecognized role that this dispersal mechanism may play in the fate of the sea surface microlayer and of pollutant spills by dispersing petroleum in the water column. Finally, our system provides an energy-efficient route, with potential upscalability, for applications in drug delivery, food production and materials science.
Sonication–Microfluidics for Fabrication of Nanoparticle-Stabilized Microbubbles
Chen, H. ; Li, J. ; Zhou, W. ; Pelan, E.G. ; Stoyanov, S.D. ; Arnaudov, L.N. ; Stone, H.A. - \ 2014
Langmuir 30 (2014)15. - ISSN 0743-7463 - p. 4262 - 4266.
flow-focusing device - contrast agents - foams - emulsions - delivery - bubbles
An approach based upon sonication–microfluidics is presented to fabricate nanoparticle-coated microbubbles. The gas-in-liquid slug flow formed in a microchannel is subjected to ultrasound, leading to cavitation at the gas–liquid interface. Therefore, microbubbles are formed and then stabilized by the nanoparticles contained in the liquid. Compared to the conventional sonication method, this sonication–microfluidics continuous flow approach has unlimited gas nuclei for cavitation that yields continuous production of foam with shorter residence time. By controlling the flow rate ratios of the gas to the liquid, this method also achieves a higher production volume, smaller bubble size, and less waste of the nanoparticles needed to stabilize the microbubbles.
Colloids in Flatland: a perspective on 2D phase-separated systems, characterisation methods, and lineactant.
Bernardini, C. ; Stoyanov, S.D. ; Arnaudov, L.N. ; Cohen Stuart, M.A. - \ 2013
Chemical Society Reviews 42 (2013)5. - ISSN 0306-0012 - p. 2100 - 2129.
langmuir-blodgett-films - air-water-interface - perfluorinated carboxylic-acids - field optical microscopy - hydrogenated hybrid amphiphiles - fluorinated comb copolymers - brewster-angle microscopy - organized molecular films - atomic-force microscopy - mixed lan
In 1861 Thomas Graham gave birth to a new field of science, today known as colloid science. Nowadays, the notion “colloid” is often used referring to systems consisting of two immiscible phases, one of which is finely dispersed into the other. Research on colloids deals mostly with sols (solids dispersed in a liquid), emulsions (liquids dispersed in liquid), and foams (gas dispersed in a liquid). Because the dispersed particles are small, there is a lot of interface per unit mass. Not surprisingly, therefore, the properties of the interface have often a decisive effect on the behaviour of colloids. Water–air interfaces have a special relevance in this field: many water-insoluble molecules can be spread on water and, given the right spreading conditions and enough available surface area, their spreading proceeds until a monolayer (a one-molecule thick layer) eventually remains. Several 2D phases have been identified for such monolayers, like “gas”, “liquid expanded”, “liquid condensed”, and “solid”. The central question of this review is whether these 2D phases can also exist as colloidal systems, and what stabilizes the dispersed state in such systems. We shall present several systems capable of yielding 2D phase separation, from those based on either natural or fluorinated amphiphiles, to polymer-based ones. We shall seek for analogies in 3D and we shall try to clarify if the lines between these 2D objects play a similar role as the interfaces between 3D colloidal systems. In particular, we shall consider the special role of molecules that tend to accumulate at the phase boundaries, that is, at the contact lines, which will therefore be denoted “line-actants” (molecules that adsorb at a 1D interface, separating two 2D colloidal entities), by analogy to the term “surfactant” (which indicates a molecule that adsorbs at a 2D interface separating two 3D colloidal entities).
Hydrodynamic cavitation: a bottom-up approach to liquid aeration
Raut, J.S. ; Stoyanov, S.D. ; Duggal, C. ; Pelan, E.G. ; Arnaudov, L.N. ; Naik, V.M. - \ 2012
Soft Matter 8 (2012)17. - ISSN 1744-683X - p. 4562 - 4566.
converging-diverging nozzle - saccharomyces-cerevisiae - trichoderma-reesei - flows
We report the use of hydrodynamic cavitation as a novel, bottom-up method for continuous creation of foams comprising of air microbubbles in aqueous systems containing surface active ingredients, like proteins or particles. The hydrodynamic cavitation was created using a converging-diverging nozzle. The air bubble size obtained using this technique was found to be significantly smaller than that achieved using conventional mechanical entrapment of air via shearing or shaking routes, which are in essence top-down approaches. In addition, the technique provided the possibility of forming non-spherical bubbles due to the high elongational stresses experienced by the bubbles as they flow through the nozzle throat. We show that surface active agents with a high surface elasticity modulus can be used to stabilize the nascent air bubbles and keep their elongated shapes for prolonged periods of time. This combination of the cavitation process with appropriate surface active agents offers an opportunity for creating bubbles smaller than 10 microns, which can provide unique benefits in various applications.
Interfacial layers from the protein HFBII hydrophobin: Dynamic surface tension, dilatational elasticity and relaxation times
Alexandrov, N.A. ; Marinova, K.G. ; Gurkov, T.D. ; Danov, K.D. ; Kralchevsky, P.A. ; Stoyanov, S.D. ; Blijdenstein, T.B.J. ; Arnaudov, L.N. ; Pelan, E.G. ; Lips, A. - \ 2012
Journal of Colloid and Interface Science 376 (2012). - ISSN 0021-9797 - p. 296 - 306.
class-ii hydrophobins - air-water-interface - trichoderma-reesei - structural-analysis - crystal-structures - curved interfaces - latex-particles - beta-casein - adsorption - rheology
The pendant-drop method (with drop-shape analysis) and Langmuir trough are applied to investigate the characteristic relaxation times and elasticity of interfacial layers from the protein HFBII hydrophobin. Such layers undergo a transition from fluid to elastic solid films. The transition is detected as an increase in the error of the fit of the pendant-drop profile by means of the Laplace equation of capillarity. The relaxation of surface tension after interfacial expansion follows an exponential-decay law, which indicates adsorption kinetics under barrier control. The experimental data for the relaxation time suggest that the adsorption rate is determined by the balance of two opposing factors: (i) the barrier to detachment of protein molecules from bulk aggregates and (ii) the attraction of the detached molecules by the adsorption layer due to the hydrophobic surface force. The hydrophobic attraction can explain why a greater surface coverage leads to a faster adsorption. The relaxation of surface tension after interfacial compression follows a different, square-root law. Such behavior can be attributed to surface diffusion of adsorbed protein molecules that are condensing at the periphery of interfacial protein aggregates. The surface dilatational elasticity, E, is determined in experiments on quick expansion or compression of the interfacial protein layers. At lower surface pressures (
Polymer compatibility in two dimensions. Modeling of phase behavior of mixed polymethacrylate Langmuir films
Bernardini, C. ; Cohen Stuart, M.A. ; Stoyanov, S.D. ; Arnaudov, L.N. ; Leermakers, F.A.M. - \ 2012
Langmuir 28 (2012)13. - ISSN 0743-7463 - p. 5614 - 5621.
field optical microscopy - interacting chain molecules - block-copolymer - statistical-theory - hybrid lipids - separation - monolayer - surface - adsorption - size
We analyze the possibility of polymer blends undergoing phase separation in two dimensions. To this end, we investigate a model system consisting of water-supported Langmuir monolayers, obtained from binary polyalkyl-methacrylate mixtures (PXMA, where X stands for any of the type of ester side groups used: M, methyl-; E, ethyl-; B, butyl-; H, hexyl-; O, octyl-; L, lauryl-methacrylate), by means of self consistent field (SCF) calculations. In particular, we address the conditions which determine demixing and phase separation in the two-dimensional system, showing that a sufficient chain length mismatch in the ester side group moieties is able to drive the polymer demixing. When the difference in length of the alkyl chain of the ester moieties on the two types of polymers is progressively reduced, from 11 carbon atoms (PMMA/PLMA) to 4 carbons only (POMA/PLMA), the demixing tendency is also reduced. The polymer/subphase interactions affect more the distribution of the polymer coils in the POMA/PLMA blend monolayer. Mixing of the two polymers is observed, but also a partial layering along the vertical direction. We also add, to a PMMA/PLMA blended monolayer, a third component, namely, a symmetrical diblock copolymer of the type PLMA-b-PMMA. We observe adsorption of the diblock copolymer exclusively at the contact line between the two homopolymer domains, and a concomitant lowering of the line tension. The line tension varies with the chemical potential of the diblock copolymer according to Gibbs’ law, which demonstrates that PLMA-b-PMMA can act as a “lineactant” (the equivalent of a surfactant in two-dimensional systems) in the binary demixed PMMA/PLMA Langmuir monolayer.
PMMA highlights the layering transition of PDMS in Langmuir films
Bernardini, C. ; Stoyanov, S.D. ; Cohen Stuart, M.A. ; Arnaudov, L.N. ; Leermakers, F.A.M. - \ 2011
Langmuir 27 (2011)6. - ISSN 0743-7463 - p. 2501 - 2508.
air-water-interface - molecular layers - polymer-films - surface - poly(dimethylsiloxane) - methacrylate - ellipsometry - behavior - spread
We report a system consisting of a mixed Langmuir monolayer, made of water-insoluble, spreadable, fluid-like polymers polydimethylsiloxane (PDMS) and polymethylmethacrylate (PMMA) with a minority P(DMS-b-MMA) copolymer. We have performed both Langmuir trough pressure/area isotherm measurements and Brewster angle microscopy (BAM) observations and complement the experiments with molecularly detailed self-consistent field (SCF) calculations. PDMS undergoes a layering transition that is difficult to detect by BAM. Addition of PMMA gives contrast in BAM, now showing a two-phase system: if this would consist of separate two-dimensional (2D) PMMA and PDMS phases, a PDMS-PMMA diblock should accumulate at the phase boundary. However, the diblock copolymer of PDMS-PMMA failed to show the expected “lineactant” behavior, i.e., failed to accumulate at the phase boundary. The calculations point to a nontrivial arrangement of the polymer chains at the interface: in mixtures of the two homopolymers, in a rather wide composition ratio, we find a vertical (with respect to the air/water interfacial plane) configuration, with PMMA sitting preferably at the PDMS/water interface of the thicker PDMS film, during the PDMS layering phase transition. This also explains why the diblock copolymer is not a lineactant. Both PMMA and P(DMS-b-MMA) are depleted from the thin-thick PDMS film interface, and the line tension between the phases is, consequently, increased, in the binary mixtures as well as in the ternary ones
Polymers at the water/air interface, surface pressure isotherms, and molecularly detailed modeling
Bernardini, C. ; Stoyanov, S.D. ; Cohen Stuart, M.A. ; Arnaudov, L.N. ; Leermakers, F.A.M. - \ 2010
Langmuir 26 (2010)14. - ISSN 0743-7463 - p. 11850 - 11861.
interacting chain molecules - air/water interface - light-scattering - langmuir monolayers - methyl-methacrylate - monomolecular films - statistical-theory - air - spread - poly(dimethylsiloxane)
Surface pressure isotherms at the air/water interface are reproduced for four different polymers, poly-l-lactic acid (PLLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and poly(isobutylene) (PiB). The polymers have the common property that they do not dissolve in water. The four isotherms differ strongly. To unravel the underlying details that are causing these differences, we have performed molecularly detailed self-consistent field (SCF) modeling. We describe the polymers on a united atom level, taking the side groups on the monomer level into account. In line with experiments, we find that PiB spreads in a monolayer which smoothly thickens already at a very low surface pressure. PMMA has an autophobic behavior: a PMMA liquid does not spread on top of the monolayer of PMMA at the air/water interface. A thicker PMMA layer only forms after the collapse of the film at a relatively high pressure. The isotherm of PDMS has regions with extreme compressibility which are linked to a layering transition. PLLA wets the water surface and spreads homogeneously at larger areas per monomer. The classical SCF approach features only short-range nearest-neighbor interactions. For the correct positioning of the layering and for the thickening of the polymer films, we account for a power-law van der Waals contribution in the model. Two-gradient SCF computations are performed to model the interface between two coexistent PDMS films at the layering transition, and an estimation of the length of their interfacial contact is obtained, together with the associated line tension value.
Measuring the three-phase contact angle of nanoparticles at fluid interfaces
Arnaudov, L.N. ; Cayre, O.J. ; Cohen Stuart, M.A. ; Stoyanov, S.D. ; Paunov, V.N. - \ 2010
Physical Chemistry Chemical Physics 12 (2010)2. - ISSN 1463-9076 - p. 328 - 331.
gel trapping technique - water-air interfaces - langmuir trough - particles - energy - silica - size - flow
We report a generic technique to image and study the wettability of spherical nanoparticles adsorbed at liquid surfaces and demonstrate that nanoparticle monolayers can be imprinted at air–water and oil–water interfaces and their three-phase contact angle at the original liquid interface can be determined by an atomic force microscopy scan on a replica of the interface; the technique is tested using four different types of nanoparticles, the smallest one having a radius of 37 nm
Colloid fabrication by co-extrusion
Arnaudov, L.N. ; Stoyanov, S.D. ; Cohen Stuart, M.A. - \ 2008
Colloids and Surfaces. A: Physicochemical and Engineering Aspects 323 (2008)1-3. - ISSN 0927-7757 - p. 94 - 98.
We propose a novel technique that allows assembling composite particles by manipulating three (initially fluid) phases, e.g. gas bubbles or liquid droplets (phase 1) in one liquid (phase 3), coated by another one (phase 2). In this way, we can control (i) the type of disperse phase fluid and its flow rate, (ii) the type of the coating material, its composition, and its flow rate, and (iii) the type of the continuous phase and its composition. All this gives us numerous opportunities to prepare new disperse systems with interesting applications. We describe two sets of experiments. In the first one we produce gas bubbles coated with oil. In the second one we produce stable foam that is stabilized by a surfactant formed in situ on the surface of each separate bubble. The surfactant is formed by a chemical reaction between a fatty acid solution spread on the bubble surface and an aqueous solution of NaOH as a continuous phase. The foam grows linearly with time during the supply of the fatty acid solution. When we stop the supply of the fatty acid the foam growth stops. The simple examples show that with carefully chosen phases and precise control of the experimental conditions we can produce a whole range of different colloids: composite capsules, shell particles or fluid dispersions, etc.
Theoretical modeling of the kinetics of fibrilar aggregation of bovine beta-lactoglobulin at pH 2
Arnaudov, L.N. ; Vries, R.J. de - \ 2007
Journal of Chemical Physics 126 (2007)14. - ISSN 0021-9606 - 7 p.
heat-induced denaturation - scattering - protein
The authors propose a kinetic model for the heat-induced fibrilar aggregation of bovine ß-lactoglobulin at pH 2.0. The model involves a nucleation step and a simple addition reaction for the growth of the fibrils, as well as a side reaction leading to the irreversible denaturation and inactivation of a part of the protein molecules. For the early stages of the aggregation reaction, the authors obtain an analytical solution of the model. In agreement with their experimental results, the model predicts a critical protein concentration below where almost no fibrils are formed. The model agrees well with their experimental data from in situ light scattering. By fitting the experimental data with the model, the authors obtain the ionic strength dependent kinetic rate constants for ß-lactoglobulin fibrilar aggregation and the size of the critical nucleus.
Time-resolved small angle neutron scattering during heat-induced fibril formation from bovine beta-lactoglobulin
Arnaudov, L.N. ; Vries, R.J. de; Cohen Stuart, M.A. - \ 2006
Journal of Chemical Physics 124 (2006). - ISSN 0021-9606 - p. 084701 - 084701.
aggregation - light
We study in situ the kinetics of heat-induced fibrilar aggregation of bovine -lactoglobulin at pH 2.0 and 80 °C for the first time by time-resolved small-angle neutron scattering. A simple model for the scattering from a mixture of monodisperse charged spheres (monomeric -lactoglobulin) interacting via a screened electrostatic repulsion and noninteracting long cylinders (protein fibrils) is used to describe the data. The experimental data are fitted to the model and the concentration of the monomeric protein and the protein incorporated in fibrils are obtained as adjustable parameters. Thus, a simple physical model allows the determination of realistic kinetic parameters during fibrilar protein aggregation. This result constitutes an important step in understanding the process of irreversible fibrilar aggregation of proteins
Strong impact of ionic strength on the kinetics of fibrilar aggregation of bovine beta-lactoglobulin
Arnaudov, L.N. ; Vries, R.J. de - \ 2006
Biomacromolecules 7 (2006)12. - ISSN 1525-7797 - p. 3490 - 3498.
heat-induced gelation - atomic-force microscopy - globular-proteins - induced denaturation - light-scattering - gels - ph - temperature - growth - model
We investigate the effect of ionic strength on the kinetics of heat-induced fibrilar aggregation of bovine -lactoglobulin at pH 2.0. Using in situ light scattering we find an apparent critical protein concentration below which there is no significant fibril formation for all ionic strengths studied. This is an independent confirmation of our previous observation of an apparent critical concentration for 13 mM ionic strength by proton NMR spectroscopy. It is also the first report of such a critical concentration for the higher ionic strengths. The critical concentration decreases with increasing ionic strength. Below the critical concentration mainly "dead-end" species that cannot aggregate anymore are formed. We prove that for the lowest ionic strength this species consists of irreversibly denatured protein. Atomic force microscopy studies of the morphology of the fibrils formed at different ionic strengths show shorter and curvier fibrils at higher ionic strength. The fibril length distribution changes non-monotonically with increasing ionic strength. At all ionic strengths studied, the fibrils had similar thicknesses of about 3.5 nm and a periodic structure with a period of about 25 nm.
Thermally induced fibrilar aggregation of hen egg white lysozyme
Arnaudov, L.N. ; Vries, R.J. de - \ 2005
Biophysical Journal 88 (2005)1. - ISSN 0006-3495 - p. 515 - 526.
atomic-force microscopy - angle x-ray - amyloid fibrils - beta-lactoglobulin - peptide fragment - ethanol solution - protein - scattering - gels - ph
We study the effect of pH and temperature on fibril formation from hen egg white lysozyme (HEWL). Fibril formation is promoted by low pH and temperatures close to the midpoint emperature for protein unfolding (detected using far-UV circular dichroism (CD)). At the optimal conditions for fibril formation (pH 2.0, T = 57°C), on-line static light scattering shows the ormation of fibrils after a concentration independent lag time of around 48 h. Nucleation resumably involves a change in the conformation of individual lysozyme molecules. Indeed, long term CD measurements at pH 2.0, T = 57°C show a marked change of the secondary structure of lysozyme molecules after about 48 h of heating. From atomic force microscopy we find that most of the fibrils have a thickness of about 4 nm. These fibrils have a coiled structure with a periodicity of about 30 nm and show characteristic defects after every 4 or 5 turns.
Kinetics of fibrilar aggregation of food proteins
Arnaudov, L.N. - \ 2005
Wageningen University. Promotor(en): Martien Cohen Stuart; Erik van der Linden, co-promotor(en): Renko de Vries. - [S.l.] : S.n. - ISBN 9789085042020 - 128
bèta-lactoglobuline - lysozym - uitvlokking - colloïdale eigenschappen - eiwitten - voedsel - beta-lactoglobulin - lysozyme - flocculation - colloidal properties - proteins - food
In this thesis we study the kinetics of fibrilar aggregation of two model proteins widely used in the food industry -b-lactoglobulin (b-lg) and hen egg white lysozyme (HEWL). The kinetics of protein aggregation is studied mostly experimentally and, when possible, theoretically. The process of fibrilar (or linear) protein aggregation is the process of formation of elongated structures from otherwise compact (globular) proteins. Studying the kinetics of this process for different proteins can lead to a better understanding of the mechanism of the process and to a possible generalisation of this mechanism. The investigation of the morphology of the formed aggregates at different stages of the process of aggregation could also lead to a more complete picture of the detailed mechanism of the process. Last, but not least, is the influence of the protein stability on the type of the formed aggregates and the kinetics of fibrilar aggregation.
The specific aims of this thesis are the following: 1) To investigate the kinetics of heat-induced fibrilar aggregation of two model proteins, bovineb-lg and HEWL, in as much detail as possible; 2) To study the morphology of the fibrils formed from both proteins; 3) To study the influence of the environment such as temperature, pH, and ionic strength on the kinetics of fibrilar aggregation and the morphology of the formed fibrils.
The heat-induced fibrilar aggregation ofb-lg is investigated at pH 2.0, 80 °C, and at various ionic strengths. Fibril formation is followed in situ using static (SLS) and dynamic light scattering (DLS), small angle neutron scattering (SANS), and proton NMR techniques. The fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. Even after prolonged heating, an appreciable fraction of the protein molecules is incorporated into fibrils, only when theb-lg concentration is above some critical concentration that is ionic strength dependent.The linear aggregation ofb-lg upon prolonged heating at pH 2.0 at80 °Cappears to be a multistep process. Competing reactions lead to two products: long linear aggregates and low molecular weight "dead end" species. The "dead end" species comprises monomeric non-native protein molecules and cannot form fibrils. Fibril formation involves at least two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon subsequent slow cooling.
We have also shown that time-resolved SANS can be used with success in studying protein aggregation and that with enough additional information for the aggregation process one can in practice obtain complete information about the aggregation kinetics of the process.
Tapping mode atomic force microscopy results indicate that the fibrils formed at pH 2.0 upon heating at 80 °Chave a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. The main difference between the fibrils observed at different ionic strengths is their length and curvature. Fibrils obtained at higher ionic strength are shorter and more curved as opposed to longer and straighter fibrils obtained at lower ionic strengths. In case of higher ionic strength the fibril formation is faster, more fibrils are formed and as a result the mean length of the fibrils is shorter. Fibrils obtained at all ionic strengths exhibit similar type of periodic morphology, which suggests that the detailed mechanism of fibril formation might be independent of the ionic strength, but specific forb-lg.
In the case of HEWL we study the effect of pH and temperature on the fibril formation. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism (CD)). The stability of HEWL toward heat treatment is greatly influenced by the pH. The lower the pH, the lower the stability of the protein is. The conditions at pH 2.0 are unique in promoting the fibrilar aggregation of HEWL since heating of solutions at pH 3.0 and 4.0 to temperatures just above the midpoint of the unfolding transition of the molecule does not lead to the appearance of fibrilar aggregates.
HEWL fibrils are formed after a lag time that is practically concentration independent. This means that the governing process for the fibril formation is the change in the structure of single protein molecules caused by a prolonged exposure to a temperature close to the midpoint of the unfolding transition. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long term CD measurements at pH 2.0, T = 57°C show a marked change of the secondary structure of lysozyme molecules after about 48 h of heating.
The fibril morphology is complex. The fibrils formed at pH 2.0 are long and straight with a length of the order of 5mm and predominant thickness of about 4 nm and consist of stiff rod-like subunits with length either 124 or 157 nm. On smaller scale the fibrils consist of a coiled structure with a period of ca. 30 nm that gives the appearance of the rod-like subunits probably because of defects occurring every 4 or 5 turns.
The fibrils consist mostly of full-length HEWL, although, some fragments due to hydrolysis at pH 2.0 and 57°C are probably incorporated into the fibrils. At any rate the hydrolysis of the protein is not the cause of the aggregation since at pH 3.0 no hydrolysis is detected but fibrils do form.
In conclusion we can say that for a full and general description of the processes of fibrilar aggregation of globular proteins the type of specific interaction responsible for the aggregation must be identified. The interacting parts of the protein must also be identified. The last and most difficult task is to characterise the conformation of the protein in solution at conditions suitable for aggregation.