Lipid self-assembled structures for reactivity control in food.

Lipid self-assembled structures (SASs) have recently gained considerable interest for their potential applications, especially for sustained nutrient release and protein crystallization. An additional property, which is underexploited, is their ability to control chemical reactions in food products. Here, we concentrate on SASs formed by phospholipids (PLs) and monoglycerides (MGs), those compounds being the most natural surfactants and therefore, the best compatible with food products, in view of providing new functionalities through the formation of SASs. In this work, the phase behaviour of these amphiphiles when mixed with oil and water is described and compared. Subsequently, we address the influence of these structures to the oxidation and Maillard-type reactions. Finally, we show that SASs formed by MGs can strongly increase the yield of key aroma impact compounds generated by Maillard-type reactions when compared with the reaction performed in aqueous precursor solutions. Various SASs are compared. In particular, addition of oil to a reversed bicontinuous structure formed by MG leads to a reversed microemulsion, which, considering its low viscosity, is particularly suitable for food products and act as a very efficient reactor system. The influence of oil and precursors on phase behaviour is discussed and related to the efficiency of the Maillard reactions.This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'.

Adsorption of octanoic acid at the water/oil interface.

Fats are widely present in a large variety of food and represent the main source of energy for the body. In the current study we investigate the behaviour of fatty acids at liquid-liquid interfaces, mimicking some steps of the very complex digestion process. Octanoic acid is used as an example of middle chain fatty acids. For the oil phase we choose sunflower oil as an industrial product and hexane as pure oil. The influence of the fatty acid concentration and the pH of the aqueous phase on the interfacial tension is determined by profile analyse tensiometry (PAT), which allows to examine the way of adsorption and transition of the fatty acids from one phase to the other. Predominantly, the pH affects the dissociation and thereby the strength of the hydrophilic character of the fatty acid. The adsorption behaviour indicates the different interfacial activity of the studied octanoic acid.

Thermodynamics, adsorption kinetics and rheology of mixed protein-surfactant interfacial layers.

Depending on the bulk composition, adsorption layers formed from mixed protein/surfactant solutions contain different amounts of protein. Clearly, increasing amounts of surfactant should decrease the amount of adsorbed proteins successively. However, due to the much larger adsorption energy, proteins are rather strongly bound to the interface and via competitive adsorption surfactants cannot easily displace proteins. A thermodynamic theory was developed recently which describes the composition of mixed protein/surfactant adsorption layers. This theory is based on models for the single compounds and allows a prognosis of the resulting mixed layers by using the characteristic parameters of the involved components. This thermodynamic theory serves also as the respective boundary condition for the dynamics of adsorption layers formed from mixed solutions and their dilational rheological behaviour. Based on experimental studies with milk proteins (beta-casein and beta-lactoglobulin) mixed with non-ionic (decyl and dodecyl dimethyl phosphine oxide) and ionic (sodium dodecyl sulphate and dodecyl trimethyl ammonium bromide) surfactants at the water/air and water/hexane interfaces, the potential of the theoretical tools is demonstrated. The displacement of pre-adsorbed proteins by subsequently added surfactant can be successfully studied by a special experimental technique based on a drop volume exchange. In this way the drop profile analysis can provide tensiometry and dilational rheology data (via drop oscillation experiments) for two adsorption routes--sequential adsorption of the single compounds in addition to the traditional simultaneous adsorption from a mixed solution. Complementary measurements of the surface shear rheology and the adsorption layer thickness via ellipsometry are added in order to support the proposed mechanisms drawn from tensiometry and dilational rheology, i.e. to show that the formation of mixed adsorption layer is based on a modification of the protein molecules via electrostatic (ionic) and/or hydrophobic interactions by the surfactant molecules and a competitive adsorption of the resulting complexes with the free, unbound surfactant. Under certain conditions, the properties of the sequentially formed layers differ from those formed simultaneously, which can be explained by the different locations of complex formation.

Lipases at interfaces: a review.

Lipases are acyl hydrolases that play a key role in fat digestion by cleaving long-chain triglycerides into polar lipids. Due to an opposite polarity between the enzyme (hydrophilic) and their substrates (lipophilic), lipase reaction occurs at the interface between the aqueous and the oil phases. Hence, interfaces are the key spots for lipase biocatalysis and an appropriate site for modulating lipolysis. Surprisingly enough, knowledge about the effects of the interfacial composition on lipase catalysis is still limited and only described by the term "interfacial quality". Recent systematic studies based on a biophysical approach allowed for the first time to show the effects of the interfacial microenvironment on lipase catalysis. These studies demonstrate that lipase activity as a function of interfacial composition is more attributed to substrate inaccessibility rather than to enzyme denaturation or inactivation, as it is often hypothesized. A detailed analysis of the interfacial properties of all compounds involved in triglyceride digestion revealed that lipolysis is a self-regulated reaction. This feedback mechanism can be explored as a new avenue to control lipase catalysis. To substantiate this hypothesis, oil hydrolysis in a model gastro-intestinal system was performed, which can be seen as an interfacial engineering approach to enzyme reactivity control. The presented characterization of the interfacial composition and its consequences provide a new approach for the understanding of lipase reactions at interfaces with direct impact on biotechnological and health care applications.

Equilibrium of adsorption of mixed milk protein/surfactant solutions at the water/air interface.

Ellipsometry and surface profile analysis tensiometry were used to study and compare the adsorption behavior of beta-lactoglobulin (BLG)/C10DMPO, beta-casein (BCS)/C10DMPO and BCS/C12DMPO mixtures at the air/solution interface. The adsorption from protein/surfactant mixed solutions is of competitive nature. The obtained adsorption isotherms suggest a gradual replacement of the protein molecules at the interface with increasing surfactant concentration for all studied mixed systems. The thickness, refractive index, and the adsorbed amount of the respective adsorption layers, determined by ellipsometry, decrease monotonically and reach values close to those for a surface covered only by surfactant molecules, indicating the absence of proteins from a certain surfactant concentration on. These results correlate with the surface tension data. A continuous increase of adsorption layer thickness was observed up to this concentration, caused by the desorption of segments of the protein and transforming the thin surface layer into a rather diffuse and thick one. Replacement and structural changes of the protein molecules are discussed in terms of protein structure and surface activity of surfactant molecules. Theoretical models derived recently were used for the quantitative description of the equilibrium state of the mixed surface layers.

Reversibility and irreversibility of adsorption of surfactants and proteins at liquid interfaces.

It is shown experimentally that the desorption of sodium decyl sulphate from the liquid/air interface is purely diffusion controlled, while the desorption of higher surface active surfactants such as the non-ionic surfactants Triton X-100 and tridecyl dimethyl phosphine oxide obeys a mixed mechanism. The desorption kinetics of beta-lactoglobulin (BLG) and beta-casein is, however, determined by a barrier mechanism. From the analysis of the BLG and beta-casein desorption kinetics at different temperatures the activation energy of desorption is calculated. The values obtained are rather close to the free energy of adsorption. The theoretical model of desorption kinetics predicts that these two energetic parameters are similar if the adsorption activation energy is low. This explains why substances with a higher adsorption activity have a lower desorption rate. Adsorption kinetics studies for beta-casein with and without forced convection show the same equilibrium surface tension values. This leads to the conclusion that the protein adsorption at liquid interfaces is thermodynamically reversible, although the slow desorption kinetics would allow to assume it to be an irreversible process.

Surface-pressure isotherms of monolayers formed by microsize and nanosize particles.

The thermodynamic model of a 2D solution developed earlier for protein monolayers at liquid interfaces is generalized for monolayers composed of micro- and nanoparticles. Surface pressure isotherms of particle monolayers published in the literature for a wide range of particles sizes (between 75 microm and 7.5 nm) are described by the theoretical model with one modification. The calculations of surface pressure pi on area A provide satisfactory agreement with the experimental data. The theory also yields reasonable cross-sectional area values of the solvent molecule water in the range between 0.12 and 0.18 nm2, which is almost independent of particle size. Also, the area per particle in a closely packed monolayer obtained from the theory is quite realistic.

Crystallography of dispersed liquid crystalline phases studied by cryo-transmission electron microscopy.

Low molecular weight surfactants, for example monoglycerides and phospholipids, form a multitude of self-assembled structures, such as inverted cubic or hexagonal mesophases, if brought into contact with water/oil. These mesophases can be dispersed in water using adequate surface-active materials such as low molecular weight surfactants or surface active polymers. In order to use such mesophase particles for incorporating drugs and aromas, it is essential to determine their internal crystallographic structure and to understand their mechanism of stabilization. Cryo-transmission electron microscopy was used to investigate the internal structure of different dispersed particles at various temperatures and oil contents. It is shown here that cryo-transmission electron microscopy, in combination with fast Fourier transform and tilting experiments, is effective in obtaining information on crystallographic structure, space group and morphology of particles with reversed bicontinuous cubic and hexagonal structures. In particular, using the presence or the absence of the {111} reflections and viewing the same particle under different axes of observation allows one to discriminate between the Im3m and Pn3m space groups. A major advantage of cryo-transmission electron microscopy is the ability to analyse single particles. This allows the identification of particles present at very low concentrations and the coexistence of particles with different internal self-assembly structures. With this technique we have obtained strong evidence for the presence of two cubic internal self-assembly structures with different space groups within the same dispersion. In addition, we found that cryo-transmission electron microscopy combined with tilting experiments enables the analysis of internal particle morphology, allowing the discussion of mechanisms for hexosome stabilization.

Composite interfacial layers containing micro-size and nano-size particles.

Surface layers of micro- and nanoparticles at fluid/liquid interfaces in absence and presence of surfactants are of large importance in the process of re-discovering Pickering systems, i.e. emulsions and foams stabilized by particles. The surface pressure/area isotherms of such layers can provide information about the properties of the used particles (dimensions, interfacial contact angles), the structure of interfacial layers, the interactions between the particles as well as about relaxation processes within the layers. For a correct description of Pi-A isotherms of composite surface layers containing particles the significant difference in size of these particles to that of solvent and surfactant molecules should be taken into account. Corresponding equations can be derived on the basis of the two-dimensional solution theory. The gained equations provide satisfactory agreement with experimental data and predict realistic values for the area of particles at the interface. Also equations of state and of the dilational elasticity for composite surface layers containing particles can be obtained in the framework of the presented methodology.

Kinetics of the desorption of surfactants and proteins from adsorption layers at the solution/air interface.

It is shown by experiments that the DeSNa desorption kinetics is governed by a pure diffusion mechanism, while the desorption of more surface active surfactants such as C13DMPO and Triton X-100 obeys a mixed mechanism. The BLG desorption kinetics, as shown by experiments, is determined by a barrier mechanism. From the analysis of the temperature dependence of the BLG desorption kinetics it is possible to calculate the activation energy of this process, which is quite close to the free energy of BLG adsorption. The theoretical model of desorption kinetics predicts that these two energetic parameters are approximately equal to each other if the adsorption activation energy is low. This can explain the fact that the higher the adsorption activity of a substance is, the lower is its desorption rate.

Surface dilational rheology of mixed beta-lactoglobulin/surfactant layers at the air/water interface.

The general theoretical model by Garrett and Joos proposed in 1976 for the estimation of the dilational elasticity of mixed surfactant solutions, and also the theoretical model proposed by Joos for the limiting elasticity of such mixtures, demonstrate quite satisfactory agreement with experimental results obtained from the oscillating bubble shape method for mixtures of a nonionic surfactant and a protein, that is, beta-lactoglobuline and decyl dimethyl phosphine oxide, C10DMPO.

Dynamic surface tension and adsorption kinetics of beta-casein at the solution/air interface.

A diffusion model is proposed to describe the adsorption kinetics of proteins at a liquid interface. The model is based on the simultaneous solution of the Ward-Tordai equation and a set of recently developed equations describing the equilibrium state of the adsorption layer: the adsorption isotherm, the surface layer equation of state, and the function of adsorption distribution over the states with different molar areas. The new kinetics model is compared with dynamic surface tensions of beta-casein solutions measured with the drop/bubble profile and maximum bubble pressure methods. The adsorption process for low concentrations is governed by the diffusion mechanism, while at large protein concentrations this is only the case in the initial stage. The effective diffusion coefficients agree fairly well with literature data. The adsorption values calculated from the dynamic surface tension data agree very well with the used equilibrium adsorption model.

Effects of non-esterified stanols in a liquid emulsion on cholesterol absorption and synthesis in hypercholesterolemic men.

UNLABELLED: BACKGROUND Numerous studies have shown that dietary plant sterols (phytosterols and phytostanols) and their esters can decrease cholesterol absorption. However, few researchers have examined the effects of plant sterols on cholesterol absorption and synthesis using stable isotope tracers, instead of relying on endogenous pathway precursors. Further, we have worked with non-esterified lecithin-solubilized stanols as opposed to the more frequently studied esterified sterols and stanols. The vehicle was an oil-in-water liquid emulsion rather than the more common spread vehicle typically employed. AIM OF THE STUDY: To determine the effects of relatively low doses of lecithin-solubilized non-esterified stanols in liquid emulsions on cholesterol absorption and synthesis in mildly hypercholesterolemic subjects. METHODS: In a randomized, double blind crossover design, 12 mildly hypercholesterolemic men received either a free phytostanol supplement (3 g/d in 3 servings) or a control treatment for 3 days. Cholesterol endogenous synthesis rate was determined using the rate of incorporation of deuterium from body water into newly formed cholesterol molecules. Cholesterol absorption at the intestinal level was determined using the dual isotope method using 13C cholesterol injected intravenously and 180 cholesterol given orally. RESULTS: Cholesterol absorption was 55.7 +/- 6.5 % for the control and 33.5 +/- 5.3% for the phytostanol treatment. This massive reduction of the cholesterol absorption did not induce, on average, a difference in cholesterol endogenous synthesis which was measured at 0.074 +/- 0.0015 pool/d for plant sterols and 0.0736 +/- 0.0015 pool/d for controls (p > 0.05). CONCLUSIONS: The results demonstrated that lecithin-solubilized stanols administrated during a short period of time (3 days) in an oil-in-water emulsion can dramatically decrease cholesterol absorption, without a consistent, concomitant increase in synthesis, which is highly suggestive of effective LDL cholesterol lowering. The effects of synthesis should be verified in a longer study with more subjects.

Improved oil solubilization in oil/water food grade microemulsions in the presence of polyols and ethanol.

Microemulsions based on five-component mixtures for food applications and improved oil solubilization have been studied. The compositions included water, oil phase [such as R(+)-limonene and medium-chain triglycerides (MCT)], short-chain alcohols (such as ethanol), polyols (propylene glycol and glycerol), and several surfactants and their corresponding mixtures (nonionic, such as ethoxylated sorbitan esters, polyglycerol esters, sugar ester, and anionic, such as phosphatidylcholine). The phase behavior of these systems is discussed with respect to the influence of polyols and short-chain alcohols on the degree of solubilization of oils in the aqueous phase. The alcohol and polyols modify the interfacial spontaneous curvature and the flexibility of the surfactant film, enhancing the oil solubilization capacity of the microemulsions. The solubilization of R(+)-limonene was dramatically improved in the presence of the alcohol and polyols, whereas the improvement of solubilization for triglycerides containing MCT was less pronounced. In some systems high oil solubilization was achieved, and some of them can be easily diluted to infinity both with the aqueous phase and with the oil phase. Viscosity measurements along selected dilution lines [characterized by a single continuous microemulsion region starting from a pseudo binary solution (surfactant/oil phase) to the microemulsion (water/polyol corner)] indicate that at a certain composition the system inverts from a W/O to an O/W microemulsion.

Structured fluids as microreactors for flavor formation by the Maillard reaction.

Thermal reactions of cysteine/furfural and cysteine/ribose mixtures were studied in model systems to gain more insight into the influence of structured fluids such as L(2) microemulsions and cubic phases on the generation of aroma compounds. Formation of 2-furfurylthiol from cysteine/furfural was particularly efficient in L(2) microemulsions and cubic phases compared to aqueous systems. The reaction led to the formation of two new sulfur compounds, which were identified as 2-(2-furyl)thiazolidine and, tentatively, N-(2-mercaptovinyl)-2-(2-furyl)thiazolidine. Similarly, generation of 2-furfurylthiol and 2-methyl-3-furanthiol from cysteine/ribose mixtures was strongly enhanced in structured fluids. The cubic phase was shown to be even more efficient in flavor generation than the L(2) microemulsion. It was denoted "cubic catalyst" or "cubic selective microreactor". The obtained results are interpreted in terms of a surface and curvature control of the reactions defined by the structural properties of the formed surfactant associates.

Some characteristics of sugar ester nonionic microemulsions in view of possible food applications.

This study explores some characteristics of microemulsions composed of sucrose monostearate (SMS), medium-chain triglycerides (MCT), or R-(+)-limonene, alcohols, and water. The systems are homogeneous, soft, and waxy solids at room temperature but liquefy and structure into homogeneous microemulsions when heated to >40 degrees C. The amount of solubilized water is enhanced as a function of the alcohol/oil ratio and is inversely proportional to the alcohol chain length. Over 60 wt % water can be solubilized in systems consisting of propanol/MCT/SMS at a weight ratio of 3:1:4 (initial weight ratio). These microemulsions are unique and differ from nonionic ethoxylated-based microemulsions in that their viscosity is very low and is reduced with increasing amounts of solubilized water. The electrical conductivity increases only slightly as a function of the water content and does not show typical bicontinuous or percolated behavior. The water in the core of the microemulsion strongly binds to the headgroups of the surfactant. Only at >15 wt % solubilization of water was free or bulk water detected in the core of the microemulsions. Such unique behavior of the core water might have a possible application in systems requiring monitoring of enzymatic (lipase) reactions carried out in the microemulsions as microreactors.

Reverse micelles in protein separation: the use of silica for the back-transfer process.

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative "back extraction" procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (alpha-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a alpha-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity.

The use of reverse micelles for the simultaneous extraction of oil and proteins from vegetable meal.

A method for the simultaneous extraction of oil and proteins from vegetable meals is presented. The method uses hydrocarbon reverse micelles, so that the oil is extracted directly into the hydrocarbon phase and the proteins are solubilized in the water pools of the reverse micelles. The surfactant used is bis (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane at variable w(0) values (w(0) measures the amount of water in the system, where w(0) = [H(2)O]/[AOT]). A comparison with the usual extraction methods is offered. It is shown that with the micelle system the extraction of oil is as large as with the usual methods, and it is independent of w(0). However the amount and type of proteins extracted depends strongly on w(0). At w(0) values below 6, no protein and only low molecular weight compounds (i.e. chlorogenic acid) are extracted, at larger water content (i.e. by increasing the dimension of the micelle water pool), also proteins are solubilized in a significant amount and with a molecular weight which increases by increasing W(0). The protein solubilized in the microemulsion system can be recovered into an aqueous phase with a back-transfer step.

Application of reverse micelles for the extraction of proteins.

The solubilization of proteins in hydrocarbon solutions of reverse micelles is examined at the aim of establishing whether this process can be utilized for the separation and extraction of proteins. Two techniques of solubilization are considered, the phase transfer of proteins from an aqueous solution into a supernatant micellar solution and the direct extraction of the protein powder into the micellar solution. Basic questions concerning the influence of structural parameters of the proteins, as well as the influence of external parameters (pH, salt concentration) on the specificity of the solubilization process are discussed.