Emulsification of caraway essential oil in water by lecithin and beta-lactoglobulin: emulsion stability and properties of the formed oil-aqueous interface

The stability and droplet size of protein and lipid stabilised emulsions of caraway essential oil as well as the amount of protein on the emulsion droplets have been investigated. The amount of added protein (beta-lactoglobulin) and lipid (phosphatidylcholine from soybean (sb-PC)) were varied and the results compared with those obtained with emulsions of a purified olive oil. In general, emulsions with triglyceride oil proved to be more stable compared with those made with caraway essential oil as the dispersed phase. However, the stability of the emulsions can be improved considerably by adding sb-PC. An increase in the protein concentration also promoted emulsion stability. We will also present how ellipsometry can be used to study the adsorption of the lipid from the oil and the protein from the aqueous phase at the oil-water interface. Independently of the used concentration, close to monolayer coverage of sb-PC was observed at the caraway oil-aqueous interface. On the other hand, at the olive oil-aqueous interface, the presence of only a small amount of sb-PC lead to an exponential increase of the layer thickness with time beyond monolayer coverage. The amounts of beta-lactoglobulin adsorbed at the caraway oil-aqueous interface and at the olive oil-aqueous interface were similar, corresponding roughly to a protein monolayer coverage.     

Wetting of β-casein layers adsorbed at the solid–aqueous interface

The wetting by water of the adsorbed layer of β-casein on hydrophobised silica and pure (hydrophilic) silica surface was investigated by dynamic contact angle measurements based on the Wilhelmy plate principle. The results are discussed in relation to adsorption data obtained for the protein on similar surfaces by in situ ellipsometry. β-casein adsorption on a hydrophobic surface leads to a significant decrease of the contact angle, in particular in terms of the receding contact angle, which decreased by about 70°. This indicates a strong shielding of the hydrophobic surface by the hydrophilic domain of β-casein. Adding a specific enzyme, endoproteinase Asp-N, which previously has been proposed to remove a large fraction of the hydrophilic segments, results in a significantly decreased wettability of the solid surface. The layer is now more hydrophobic and the hysterises is much smaller. The receding contact angle after the proteolysis is roughly 70°. The results are consistent with the hypothesis that β-casein adsorbs at the hydrophobic surface to form a monolayer with the hydrophobic part of the protein anchored at the surface, leaving the hydrophilic segments dangling into the solution. Less dramatic effects are observed in terms of changes of the wettability on the hydrophilic surface. The surface is still quite hydrophilic both after adsorbing β-casein and exposing the layer to endoproteinase Asp-N. These results confirm the differences in the structure of β-casein layers on the hydrophobic and hydrophilic surface.     

Effects of mucin addition on the stability of oil–water emulsions

In this work, bovine submaxillary gland mucin (BSM) was used as an emulsifier to stabilize oil–water emulsion systems. Prior to use, commercial BSM was purified by jacalin affinity chromatography. Emulsions consisting of 5% mineral oil in phosphate buffered saline (PBS) were prepared through the addition of different amounts of purified mucin followed by sonication using either of two methods: (1) low energy input for a long time (2 h), or (2) high energy input for a short time (20 s). The surfactancy property of mucin was investigated by surface tension measurements, which showed the BSM to greatly reduce the surface tension of PBS. Compared to several synthetic surfactants of the Pluronic® type, mucin showed comparable or better surface activity than F68, F88 and F108 products in dilute solutions. The formed emulsions had a mean droplet size that decreased monotonically with increasing concentration of mucin until a plateau was reached at concentrations around 0.1% by weight. The stability of these emulsions was evaluated by monitoring their average droplet size during a 33-day period. Emulsions with more than 0.25% mucin showed a constant mean size throughout the period. Specifically, an emulsion produced with 0.95% mucin showed a stable mean droplet size of about 300 nm. The stability of the mucin-emulsified systems was also evaluated by measuring turbidity changes with time, which allowed a comparison with similar emulsions stabilized by the Pluronic® surfactants in the same concentration. Thus, mucin showed its ability to establish more stable and more efficient oil–water emulsion systems. Since mucin is a glycoprotein, and hence biodegradable, our results suggest that mucin might serve as an ideal biological surfactant for the stabilization of emulsion systems intended for biomedical and pharmaceutical applications.     

Applicability of polymer theories to the thermodynamics of proteins at the air/water interface: example of β-lactoglobulin

Knowledge of the equation of state of adsorbed or deposited layers of proteins at the air/water interface is of fundamental interest in the understanding of the surface activity of these molecules. Using scaling laws of current polymer theories, it has been shown that the equation of state of the interfacial layer in the semi-dilute regime should relate the surface pressure to the surface concentration through a power law. The exponent of this power law should reflect the quality of the solvent and the conformation of the adsorbed polypeptide chain. In the case of β-lactoglobulin layers, in the range of surface concentrations that should correspond to the semi-dilute regime, the relationship between surface pressure and surface concentration is expressed as a power law. The exponent of this power law is strongly influenced by the nature of the aqueous substrate and by the net charge of the protein molecule. The use of scaling laws gives a coherent view of the expansion of the polypeptide chain in the interfacial layer and of the relationship between surface concentration and surface pressure in the semi-dilute regime. This result favours a strong similarity between β-lactoglobulin and a polymer chain in the interfacial layer. It is concluded that current theories of polymer adsorption could be applied to interfacial protein layers.     

Influence of permeate flux on fouling during the microfiltration of β-lactoglobulin solutions under cross-flow conditions

Experiments to investigate the microfiltration fouling behaviour of a β-lactoglobulin solution were performed on a constant-flux, computer-controlled, cross-flow membrane rig equipped with zirconium oxide membranes. Fouling was dependent upon the permeate flux, being light at low flux (50 l/m² h) and severe at high flux (200 l/m² h). The fouling increased in severity as the flux was increased from 50 to 200 l/m² h. At 50 l/m² h, protein transmissions of>90% were observed. At higher fluxes, the protein transmission decreased with increasing fouling resistance. The relationship between fouling resistance and protein transmission was similar for 50 and 100 nm membranes and was independent of the starting permeate flux. Standard poreplugging and pore-narrowing models did not describe the observed behaviour. Development of a model to predict protein transmission from the fouling resistance indicated that fouling occurred only in a small part of the membrane pore, most likely at the pore entrance. It is proposed that the microfiltration pore acts in a way similar to a pressure-relief valve where shear-induced protein denaturation has been observed. Shear forces on the protein perhaps lead to protein denaturation and aggregation, and narrowing of the pore in the immediate vicinity of the pore entrance.     

Interfacial characteristics of β-casein spread films at the air–water interface

Casein is well known to be a good protein emulsifier and β-casein is the major component of casein and commercial sodium caseinate. This work studies the behaviour of β-casein at the interface. The interfacial characteristics (structure and stability) of β-casein spread films have been examined at the air–water interface in a Langmuir-type film balance, as a function of temperature (5–40°C) and aqueous phase pH (pH 5 and 7). From surface pressure–area isotherms (π–A isotherms) as a function of temperature we can draw a phase diagram. β-Casein spread films present two structures and the collapse phase. That is, there is a critical surface pressure and a surface concentration at which the film properties change significantly. This transition depends on the temperature and the aqueous phase pH. The film structure was observed to be more condensed and β-casein interfacial density was higher at pH 5. β-Casein films were stable at surface pressures lower than equilibrium surface pressure. In fact, no hysteresis was observed in π–A isotherms after continuous compression-expansion cycles or over time. The relative area relaxation at constant surface pressure (10 or 20 mN m⁻¹) and the surface pressure relaxation at constant area near the monolayer collapse, can be fitted by two exponential equations. The characteristic relaxation times in β-casein films can be associated with conformation–organization changes, hydrophilic group hydration and/or surface rheology, as a function of pH.     

Monitoring the structural alterations induced in β-lactoglobulin during ultrafiltration: learning from chemical and thermal denaturation phenomena

This work aims to identify of non-reversible structural changes induced in β-lactoglobulin by permeation through porous ultrafiltration membranes. The evaluation of these structural changes is performed using a fluorescence methodology, which combines the use of three different, complementary, fluorescence techniques: steady-state fluorescence, picosecond time-resolved fluorescence and steady-state fluorescence anisotropy. The identification of the nature of the structural changes induced upon permeation is possible through comparison of the fluorescence responses obtained for β-lactoglobulin solutions collected after permeation (permeates and retentates) with those induced by chemical (addition of Guanidine hydrochloride, GndHCl) and thermal denaturation of β-lactoglobulin.

The fluorescence approach used allowed to identify irreversible losses of structural integrity of β-lactoglobulin in the permeates, while β-lactoglobulin retentates seemed to be unaffected by the ultrafiltration process.

The mechanisms that regulate the structural alterations of β-lactoglobulin and the magnitude of these alterations depend on the protein to membrane pore size ratio, λ, being more substantial at higher λ (severe pore constriction). Under these conditions (permeation with a 10 kDa membrane) the structural changes induced in the proteins are dictated by the high shear stress at the membrane pore walls. The increase of the membrane cut-off (30 kDa membrane) induces a decrease in the magnitude of the shear stress and the effect of protein–membrane chemical interactions becomes noticeable.     

Adsorption kinetics of some carotenoids at the oil/water interface

The kinetic analysis of the adsorption of two carotenoids (i.e., ethyl ester of β-apo-8′-carotenoic acid and β-carotene, all trans-isomers) from n-hexane solutions at the oil/water interface is presented for several carotenoid concentrations in the oil phase. A new kinetic approach is developed and it addresses the diffusion adsorption associated with a reversible interfacial reaction, which describes the reorientation of surfactant molecules between two conformations. This approach leads to a general analytical expression that contains four physical parameters and describes with high accuracy the experimental dynamic interfacial tensions for the two carotenoids, which independently adsorb from n-hexane phase to the n-hexane/water interface. The calculations give the characteristic times for the carotenoid adsorption at the oil/water interface in terms of diffusion relaxation and kinetic relaxation times. The results explain the long time effects on the adsorption of these carotenoids at the oil/water interface. The data are in substantial agreement with the molecular structure of these carotenoids and with the earlier data recorded for cholesterol adsorption at the n-heptane/water interface. Based on these findings, we propose a molecular mechanism for the interfacial transformation of carotenoid molecules at a hydrophobic/hydrophilic interface.     

Evaluation of the interactions between lipids and γ-globulin protein at the air–liquid interface

The interactions between lipids (cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine and sphingomyelin) and the γ-globulin protein have been analyzed using the monolayer technique at the air–liquid interface. The analysis has been carried out using both state equations and an adequate thermodynamic formulation for the surface pressure (π)–molecular area (a) isotherms. Different parameters as the virial coefficients, have been estimated. For the uncharged lipid monolayers, the interactions between the molecules are of an attractive nature, at medium and long distance, and of a steric repulsive nature at short distance. At low surface pressures the lipid molecules form small domains. The net force between γ-Globulin molecules in the monolayers has been found to be attractive. Finally, it can be concluded that when the lipid monolayers are uncharged, there is practically no interaction between the protein and lipid molecules at the mentioned interface.     

The structure and dynamic properties of mixed adsorption and penetration layers of α-dipalmitoylphosphatidylcholine/β-lactoglobulin at water/fluid interfaces

The interfacial tensions of mixed α-dipalmitoylphosphatidylcholine (DPPC)/β-lactoglobulin layers at the chloroform/water interface have been measured by the pendent drop and drop volume techniques. In certain intervals, the adsorption kinetics of these mixed layers was strongly influenced by the concentrations of both protein and DPPC. However, at low protein concentration, C_{β-lactoglobulin}=0.1 mg l⁻¹, the adsorption rate of mixed interfacial layers was mainly controlled by the variation of the DPPC concentration. As C_{β-lactoglobulin} was increased to 0.8 mg l⁻¹, the interfacial activity was abruptly increased, and within the concentration range of C_DPPC=10⁻⁴–10⁻⁵ mol l⁻¹, the DPPC has very little effect on the whole adsorption process. In this case, the adsorption rate of mixed layers was mainly dominated by the protein adsorption. This phenomenon also happened as the protein concentration was further increased to 3.6 mg l⁻¹. When C_DPPC>3 · 10^–5 mol l⁻¹, the adsorption behaviour was very similar to that of the pure DPPC although the protein concentration was changed. The equilibrium interfacial tensions of the mixed layers are dramatically effected by the lipid as compared to the pure protein adsorption at the same concentration. It reveals the estimation of which composition of lipid and protein decreases the interfacial tension. The combination of Brewster angle microscopy (BAM) with a conventional LB trough was applied to investigate the morphology of the mixed DPPC/β-lactoglobulin layers at the air/water interface. The mixed insoluble monolayers were produced by spreading the lipid at the water surface and the protein adsorbed from the aqueous buffer subphase. The BAM images allow to visualise the protein penetration and distribution into the DPPC monolayer on compression of the complex film. It is shown that a homogeneous distribution of β-lactoglobulin in lipid layers preferentially happens in the liquid fluid state of the monolayer while the protein can be squeezed out at higher surface pressures.     

Interfacial dilational behaviour of adsorbed β-lactoglobulin layers at the different fluid interfaces

β-Lactoglobulin adsorption layers at the interfaces solution/air, /tetradecan and /sunflower oil were characterised by dynamic interfacial tension measurements and harmonic drop oscillation experiments in a time scale of some seconds. Axialsymmetric drop shape analysis (ADSA) was used to calculate drop volume, area and interfacial tension. Within a definite range of drop volume amplitude, the oscillation of the surface tension is harmonic and interfacial dilation parameters can be determined. Dependence of the dilational parameters on the amplitude and frequency of drop volume oscillation were determined and methodical demands are given for this special kind of ADSA application. The concentration of interfacial saturation is minimal at the interface with sunflower oil. Interfacial dilational elasticities, and viscosities are maximal at the saturation concentration of all systems investigated. The dilational viscosities are maximal in the frequency range 0.007–0.011 Hz and characterise molecular rearrangement processes in the adsorption layer. Interfacial dilational elasticity and viscosity are the largest at the interface with air. They are the smallest at the interface with sunflower oil. Similarities and differences of the systems investigated are discussed by taking into account the adsorption behaviour and the solvatation of different apolar and polar parts of the protein molecules in the neighbouring phase.     

Simultaneous monitoring of protein adsorption at the solid–liquid interface from sessile solution droplets by ellipsometry and axisymmetric drop shape analysis by profile

In this paper two in situ techniques are combined to simultaneously examine protein adsorption at the solid–liquid interface from sessile solution droplets. With axisymmetric drop shape analysis by profile (ADSA-P) the change in solid–liquid interfacial tension is determined, while ellipsometry is employed to measure the amount of protein adsorbed from the same solution droplet at the solid–liquid interface. Three proteins (human serum albumin (HSA), immunoglobulin G (IgG) and fibrinogen (Fb)) were dissolved to a concentration of 0.05 mg ml⁻¹ in PBS (pH 7) and sessile droplets were placed for 2 h on a 47.8 nm thick gold coating on glass. The gold coated glass was positioned onto a quartz prism with immersion oil. The prism was aligned in a rotating analyser ellipsometer and the optical beam was thus allowed to be reflected at the hydrophobic gold surface. The ADSA-P set-up was built in 90° cross-beamed set-up around the prism. By combining the results for the adsorbed amounts and the interfacial tension changes over the two hour adsorption period, two stages in the adsorption process could be distinguished. In the first stage, the adsorbed amounts increase in correspondence with the interfacial tension changes, indicating that the interfacial tension changes are caused by adsorption, whereas in the second stage interfacial tension changes continue despite the adsorbed amounts being constant. Consequently, the second stage must be associated with conformational changes of the adsorbed proteins. For HSA and Fb, the conformational contribution to the interfacial tension changes (7.8 and 5.3 mJ m⁻², respectively) were approximately 2-fold the adsorption contribution, while for IgG both were equal around 3 mJ m⁻².     

Separation of keto–enol tautomers in β-ketoesters: a gas chromatography–mass spectrometric study

The gas chromatographic behaviour for some β-ketoesters was studied. Additionally, the feasibility of the gas chromatographic separation of the corresponding tautomer forms was examined. In this work mass spectrometric detection allowed identification of both keto and enol forms and an estimation of their relative amounts for methylacetoacetate, -chloromethylacetoacetate, ethylacetoacetate and -chloroethylacetoacetate. This finding demonstrates slow tautomerisation kinetics permitting the chemical identity maintenance of the tautomers.     

Competitive adsorption of -α-lysophosphatidylcholine/β-lactoglobulin mixtures at the interfaces of foams and foam lamellae

The stability of foams formed with the protein β-lactoglobulin as a function of increasing concentration of the lipid analogue -α-lysophosphatidylcholine were investigated using a microconductivity technique. The drainage, surface diffusion and thickness properties of thin liquid films (foam lamallae) were also studied using optical microscopy including epi-illumination, fluorescence recovery after photobleaching and film interferometry techniques. In addition, the surfactant binding properties of the protein were examined. The addition of small quantities of -α-lysophosphatidylcholine to β-lactoglobulin (molar ratio, R < 7:1) increased the foam stability, whereas a slightly higher concentration of surfactant in the mixture (R = 10) caused foam destabilisation. The explanation of these observations is based on changes in the composition and structure of the adsorbed interfacial layers of the thin films caused by competitive displacement of the protein by the surfactant.     

The NICS (Nucleus-Independent Chemical Shift) as a probe of the relative stability of β-chalcogenovinylaldehydes stabilized through intramolecular chalcogen–chalcogen interactions

Density functional calculations, at the B3LYP/6-311+G(3df,2p) level, have been carried out for the complete series of β-chalcogenovinylaldehydes, CH(X)–CHCH–YH (X, Y=O, S, Se, Te), to estimate the strength of H–XY or XY–H intramolecular chalcogen–chalcogen interactions, through the use of appropriate homodesmotic reactions. For the same set of compounds the value of the nucleus-independent chemical shift (NICS), on points 1 Å above the corresponding ring critical point, has been obtained at the B3LYP/6-311+G(3df,2p) level. For non-stabilizing chalcogen–chalcogen interactions the NICS value is positive, while the opposite is found when the interaction is stabilizing. In general, there is a good linear correlation between both magnitudes and therefore, we can conclude that NICS value is a reliable probe of the strength of intramolecular chalcogen–chalcogen interactions in this set of compounds.     

Protein-coated β-ferric hydrous oxide particles: An electrokinetic and electrooptic study

Using microelectrophoresis and electric light scattering techniques, we investigated the adsorption characteristics, surface coverage and surface electric parameters of superstructures from two isoforms of plastocyanin, PCa and PCb, in an oxidized state adsorbed on β-ferric hydrous oxide particles. The surface electric charge and electric dipole moments of the composite particles and the thickness of the protein adsorption layer are determined in a wide pH range, at different ionic strengths and concentration ratios of PC to β-FeOOH. The adsorption of the two proteins was found to shift the particles’ isoelectric point and to alter the total electric charge and the electric dipole moments of the oxide particles to different extent. A “reversal” in the direction of the permanent dipole moment is observed at lower pH for PCb- than for PCa-coated oxide particles. Strict correlation is found between the changes in the electrokinetic charge of the composite particles and the variation in their “permanent” dipole moments. Data suggest that the adsorption of the proteins is driven by electrostatic and/or hydrophobic interactions with the oxide surfaces dependent on pH. The adsorption behaviour is consistent with the involvement of the “eastern” and “northern” patches of the plastocyanin molecules in their adsorption on the oxide surfaces that are differently charged depending on pH.     

Foams and surface rheological properties of β-casein, gliadin and glycinin

Interfacial rheological properties and their suitability for foam production and stability of two vegetable proteins were studied and compared to β-casein. Proteins used ranged from flexible to rigid/globular in the order of β-casein, gliadin and soy glycinin. Experiments were performed at pH 6.7. Network forming properties were characterised by the surface dilational modulus (determined with the ring trough) and the critical falling film length (L_still) at which a stagnant protein film will break. Gliadin had the highest dilational modulus, followed by glycinin and β-casein, whereas glycinin formed the strongest film against fracture in the overflowing cylinder. The rate of decrease in the surface tension was studied at the air–water (Wilhelmy plate method) and the oil–water interface (bursting membrane) and the dynamic surface tension during compression and expansion in the caterpillar. Gliadin had the lowest equilibrium interfacial tensions and β-casein the lowest dynamic surface tension during expansion. Hardly any foam could be formed at a concentration of 0.1 g/l by shaking. At a concentration of 1.4 g/l most foam was formed by β-casein, followed by gliadin and glycinin. It seems that in the first place the rate of adsorption is important for foam formation. For the vegetable proteins, adsorption was slow. This resulted in lower foamability, especially for glycinin.     

NH2SO3H–SiO2 as a new water‐compatible,recyclable heterogeneous catalyst for the synthesis of novel (α,β‐unsaturated) β‐amino ketones via aza‐Michael reaction

NH₂SO₃H–SiO₂/water as a novel catalytic system was used for the synthesis of (α,β‐unsaturated) β‐amino ketones via aza‐Michael reaction at reflux conditions. The methodology was of general applicability and the catalyst exhibited activity up to five cycles. The catalyst was characterized for the first time using FT‐IR, X‐ray diffraction and scanning electron microscopic–energy dispersion analytical X‐ray. The stability of the catalyst was evaluated by differential scanning calorimetry and TGA/differential thermal analysis. High efficiency of the catalyst along with its recycling ability and the rather low loading demonstrated in reactions are the merits of the presented protocol. Copyright © 2013 John Wiley & Sons, Ltd.     

Enhanced epoxidation of soybean oil by novel Al2O3–ZrO2–TiO2 solid acid catalyst

This study aims to develop highly efficient, recyclable solid catalysts for the epoxidation of vegetable oils. An Al₂O₃–ZrO₂–TiO₂ solid acid catalyst was prepared by a co‐precipitation/impregnation method and characterised through scanning electron microscopy, energy‐dispersive spectroscopy, X‐ray diffraction, X‐ray photoelectron spectroscopy, Fourier‐transform infrared and nitrogen adsorption–desorption analyses. The solid acid catalyst with a high surface area and typical slit pore adsorption was successfully synthesised. Al₂O₃–ZrO₂–TiO₂ also exhibits high stability and improved catalytic efficiency in the epoxidation of soybean oil. An oil conversion rate of 86.6%, which is higher than that of conventional catalysts, was obtained with a catalyst loading of 0.8 wt% and was maintained at 76.6% even after recycling the catalyst three times. The performance of the solid catalyst was slightly superior to that of H₂SO₄. Therefore, this novel catalyst may potentially be applicable in catalysing soybean oil epoxidation.     

Selective α-Bromination of β-Keto Esters in Reusable PEG-400 under Mild and Catalyst-free Conditions

An efficient bromination protocol for the synthesis of α-bromo-β-keto esters has been developed. In PEG-400 （poly（ethylene glycol-400））, a variety of β-keto esters were treated with NBS （N-bromosuccinimide） at room temperature to selectively afford the corresponding α-monobromination products in excellent yields. It is noteworthy that the reaction was conducted under mild, environmentally benign and catalyst-free conditions.