Gelation and interfacial behaviour of vegetable proteins

Recent studies on gelation and interfacial properties of vegetable proteins are reviewed. Attention is focused on legume proteins, mainly soy proteins, and on wheat proteins. The rheological properties of vegetable protein gels as a function of heating time or temperature is discussed as well as the interfacial gelation upon adsorption of soy and wheat proteins at the air/water interface. It is shown that modification of proteins improves protein functionality and application.     

界面扩张流变法研究C_(12)mimBr对Gemini12-2-12在空气/水界面聚集行为的影响

采用界面扩张流变技术研究了季铵盐偶联表面活性剂C12-(CH2)2-C12·2Br(Gemini12-2-12)及其与离子液体表面活性剂溴化1-十二烷基-3-甲基咪唑(C12mim Br)复配体系的动态界面张力、扩张流变性质和界面弛豫过程等,探讨了C12mim Br对C12mim Br/Gemini12-2-12混合体系界面性质的影响及C12mim Br对Gemini12-2-12界面聚集行为影响的机制.结果表明,随着离子液体表面活性剂的不断引入,体系界面吸附达到平衡所需的时间逐渐缩短,扩张模量和相角明显降低,界面吸附膜由粘弹性膜转变为近似纯弹性膜;同时,界面及其附近的弛豫过程也发生显著变化,慢弛豫过程消失,快弛豫过程占主导地位,且离子液体浓度越高,快弛豫的贡献越大.这些界面性质的变化主要归因于离子液体表面活性剂C12mim Br参与界面形成及两表面活性剂在界面竞争吸附的结果.少量离子液体表面活性剂C12mim Br的加入可以填补疏松的Gemini12-2-12界面上的空位,形成混合界面吸附膜.随着C12mim Br含量的增加,嵌入界面的C12mim Br分子数不断增多,导致界面上相互缠绕的Gemini12-2-12烷基链"解缠",在体相和界面分子扩散交换的过程中"解缠"的Gemini12-2-12分子从界面上解吸回到体相,与此同时,C12mim Br分子相对较小的空间位阻及较强的疏水作用促使其优先扩散至界面进而取代Gemini12-2-12分子,最终界面几乎完全被C12mim Br分子所占据.     

Interfacial rheology of protein–surfactant mixtures

The distribution of proteins and surfactants at fluid interfaces (air–water and oil–water) is determined by the competitive adsorption between the two types of emulsifiers and by the nature of the protein–surfactant interactions, both at the interface and in the bulk phase, with a pronounced impact on the interfacial rheological properties of these systems. Therefore, the interfacial rheology is of practical importance for food dispersion (emulsion or foam) formulation, texture, and stability. In this review, the existence of protein–surfactant interactions, the mechanical behaviour and/or the composition of emulsifiers at the interface are indirectly determined by interfacial rheology of the mixed films. The effect on the interfacial rheology of protein–surfactant mixed films of the protein, the surfactant, the interface and bulk compositions, the method of formation of the interfacial film, the interactions between film forming components, and the displacement of protein by surfactant have been analysed. The last section tries to understand the role of interfacial rheology of protein–surfactant mixed films on food dispersion formation and stability. The emphasis of the present review is on the interfacial dilatational rheology.     

Interfacial shear rheology of protein-surfactant layers

The shear rheology of adsorbed or spread layers at air/liquid and liquid/liquid phase boundaries is relevant in a wide range of technical applications such as mass transfer, monolayers, foaming, emulsification, oil recovery, or high speed coating. Interfacial shear rheological properties can provide important information about interactions and molecular structure in the interfacial layer. A variety of measuring techniques have been proposed in the literature to measure interfacial shear rheological properties and have been applied to pure protein or mixed protein adsorption layers at air/water or oil/water interfaces. Such systems play for example an important role as stabilizers in foams and emulsions. The aim of this contribution is to give a literature overview of interfacial shear rheological studies of pure protein and protein/surfactant mixtures at liquid interfaces measured with different techniques. Techniques which utilize the damping of waves, spectroscopic or AFM techniques and all micro-rheological techniques will not discuss here.     

Dynamics of protein and mixed protein/surfactant adsorption layers at the water/fluid interface

The adsorption behaviour of proteins and systems mixed with surfactants of different nature is described. In the absence of surfactants the proteins mainly adsorb in a diffusion controlled manner. Due to lack of quantitative models the experimental results are discussed partly qualitatively. There are different types of interaction between proteins and surfactant molecules. These interactions lead to protein/surfactant complexes the surface activity and conformation of which are different from those of the pure protein. Complexes formed with ionic surfactants via electrostatic interaction have usually a higher surface activity, which becomes evident from the more than additive surface pressure increase. The presence of only small amounts of ionic surfactants can significantly modify the structure of adsorbed proteins. With increasing amounts of ionic surfactants, however, an opposite effect is reached as due to hydrophobic interaction and the complexes become less surface active and can be displaced from the interface due to competitive adsorption. In the presence of non-ionic surfactants the adsorption layer is mainly formed by competitive adsorption between the compounds and the only interaction is of hydrophobic nature. Such complexes are typically less surface active than the pure protein. From a certain surfactant concentration of the interface is covered almost exclusively by the non-ionic surfactant. Mixed layers of proteins and lipids formed by penetration at the water/air or by competitive adsorption at the water/chloroform interface are formed such that at a certain pressure the components start to separate. Using Brewster angle microscopy in penetration experiments of proteins into lipid monolayers this interfacial separation can be visualised. A brief comparison of the protein adsorption at the water/air and water/n-tetradecane shows that the adsorbed amount at the water/oil interface is much stronger and the change in interfacial tension much larger than at the water/air interface. Also some experimental data on the dilational elasticity of proteins at both interfaces measured by a transient relaxation technique are discussed on the basis of the derived thermodynamic model. As a fast developing field of application the use of surface tensiometry and rheometry of mixed protein/surfactant mixed layers is demonstrated as a new tool in the diagnostics of various diseases and for monitoring the progress of therapies.     

Correlation between mechanical behavior of protein films at the air/water interface and intrinsic stability of protein molecules

The relation between mechanical film properties of various adsorbed protein layers at the air/water interface and intrinsic stability of the corresponding proteins is discussed. Mechanical film properties were determined by surface deformation in shear and dilation. In shear, fracture stress, sigma(f), and fracture strain, gamma(f), were determined, as well as the relaxation behavior after macroscopic fracture. The dilatational measurements were performed in a Langmuir trough equipped with an infra-red reflection absorption spectroscopy (IRRAS) accessory. During compression and relaxation of the surface, the surface pressure, Pi, and adsorbed amount, Gamma (determined from the IRRAS spectra), were determined simultaneously. In addition, IRRAS spectra revealed information on conformational changes in terms of secondary structure. Possible correlations between macroscopic film properties and intrinsic stability of the proteins were determined and discussed in terms of molecular dimensions of single proteins and interfacial protein films. Molecular properties involved the area per protein molecule at Pi approximately 0 mN/m (A(0)), A(0)/M (M = molecular weight) and the maximum slope of the Pi-Gamma curves (dPi/dGamma). The differences observed in mechanical properties and relaxation behavior indicate that the behavior of a protein film subjected to large deformation may vary widely from predominantly viscous (yielding) to more elastic (fracture). This transition is also observed in gradual changes in A(0)/M. It appeared that in general protein layers with high A(0)/M have a high gamma(f) and behave more fluidlike, whereas solidlike behavior is characterized by low A(0)/M and low gamma(f). Additionally, proteins with a low A(0)/M value have a low adaptability in changing their conformation upon adsorption at the air/water interface. Both results support the conclusion that the hardness (internal cohesion) of protein molecules determines predominantly the mechanical behavior of adsorbed protein layers.     

Stability of aqueous foam films and foams containing polymers: Discrepancies between different length scales

Polymer-stabilized foams and foam films have received considerable attention during the past years. This review paper gives an overview of recent studies dealing with polyelectrolyte/surfactant mixtures, proteins, and microgels adsorbed at single air/water interfaces, in foam films and in macroscopic foams. These polymeric systems have in common that their structure or shape changes when adsorbing at an air/water interface. These structural changes in comparison to their bulk behavior greatly influence the properties of foam films and foams. Regarding the foam stability, formation of adsorbed layers or aggregates plays an important role. The discrepancy between stabilization of macroscopic foams and destabilization of single foam films might be attributed to the blockage of Plateau borders and, therefore, slowed down drainage. Another important parameter is the interfacial viscoelasticity.     

The interactions of saturated fatty acids at the dodecane/water interface and their sodium salts at the air/water interface

The interfacial tension of lauric, myristic and palmitic acids at the dodecane/water interface and of their sodium salts at the air/water interface were measured using the Du Noüy (ring) method. On the basis of these results the standard free energies of adsorption (ΔG⁰_ad) and of micellization (ΔG⁰_mic) of the above mentioned systems were calculated. According to deviations of the Langmuir isotherm, the corresponding interactions were discussed from the dependence of the standard free energy of adsorption on the interface coverage (Θ).     

SURFACE PROPERTIES OF SOME LOW MOLECULAR WEIGHT PROTEINS

Surface properties of four proteins having molecular weights less than 5,000 are reported at air/water and alumina/water interface at pH 7.0. Reversibility in the adsorption of these proteins at the alumina/water interface is tested. The adsorption on alumina/water interface has been found to be controlled by electrostatic interaction. Positive adsorption was obtained when protein and alumina surface had opposite charges and negative adsorption was obtained when both protein and surface had same charges. Of the four proteins reversibility in adsorption was observed with the one having the lowest molecular weight of 3100. The adsorption behavior apparently had no correlation with their surface hydrophobic!ty. Time dependent changes in air/water interfacial tension was observed for all the four proteins indicating time dependent loosening of compact protein structure and surface unfolding.     

界面扩张流变法研究C12mimBr对Gemini12-2-12在空气/水界面聚集行为的影响

采用界面扩张流变技术研究了季铵盐偶联表面活性剂C₁₂-(CH₂)₂-C₁₂·2Br (Gemini12-2-12)及其与离子液体表面活性剂溴化1-十二烷基-3-甲基咪唑(C₁₂mimBr)复配体系的动态界面张力、扩张流变性质和界面弛豫过程等, 探讨了C₁₂mimBr 对C₁₂mimBr/Gemini12-2-12 混合体系界面性质的影响及C₁₂mimBr 对Gemini12-2-12界面聚集行为影响的机制. 结果表明, 随着离子液体表面活性剂的不断引入, 体系界面吸附达到平衡所需的时间逐渐缩短, 扩张模量和相角明显降低, 界面吸附膜由粘弹性膜转变为近似纯弹性膜; 同时, 界面及其附近的弛豫过程也发生显著变化, 慢弛豫过程消失, 快弛豫过程占主导地位, 且离子液体浓度越高, 快弛豫的贡献越大. 这些界面性质的变化主要归因于离子液体表面活性剂C₁₂mimBr参与界面形成及两表面活性剂在界面竞争吸附的结果. 少量离子液体表面活性剂C₁₂mimBr 的加入可以填补疏松的Gemini12-2-12 界面上的空位, 形成混合界面吸附膜. 随着C₁₂mimBr 含量的增加, 嵌入界面的C₁₂mimBr 分子数不断增多, 导致界面上相互缠绕的Gemini12-2-12烷基链“解缠”, 在体相和界面分子扩散交换的过程中“解缠”的Gemini12-2-12分子从界面上解吸回到体相, 与此同时, C₁₂mimBr 分子相对较小的空间位阻及较强的疏水作用促使其优先扩散至界面进而取代Gemini12-2-12分子, 最终界面几乎完全被C₁₂mimBr分子所占据.     

Polyelectrolyte/surfactant mixtures in the bulk and at water/oil interfaces

Stabilization of emulsions by mixed polyelectrolyte/surfactant systems is a prominent example for the application in modern technologies. The formation of complexes between the polymers and the surfactants depends on the type of surfactant (ionic, non-ionic) and the mixing ratio. The surface activity (hydrophilic–lipophilic balance) of the resulting complexes is an important quantity for its efficiency in stabilizing emulsions. The interfacial adsorption properties observed at liquid/oil interfaces are more or less equivalent to those observed at the aqueous solution/air interface, however, the corresponding interfacial dilational and shear rheology parameters differ quite significantly. The interfacial properties are directly linked to bulk properties, which support the picture for the complex formation of polyelectrolyte/surfactant mixtures, which is the result of electrostatic and hydrophobic interactions. For long alkyl chain surfactants the interfacial behavior is strongly influenced by hydrophobic interactions while the complex formation with short chain surfactants is mainly governed by electrostatic interactions.     

Protein-lipid interactions at the air/water interface

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.     

X型与线型嵌段聚醚在空气/水和正庚烷/水界面上聚集行为的比较研究

通过阴离子聚合方法合成了环氧乙烷(EO)含量和分子量均相同的线型聚氧丙烯(PEO)-聚氧乙烯(PPO) (LPE)和X型聚氧丙烯-聚氧乙烯(TPE)嵌段聚醚,考察了它们在空气/水及正庚烷/水界面上聚集行为的差异. 界面活性的研究结果表明,TPE降低水、正庚烷界面张力的效率和效能均低于LPE的. 聚醚分子在正庚烷/水界面达到吸附平衡的时间比在空气/水表面短. 由于正庚烷分子插入到聚醚吸附层中,聚醚分子可以在正庚烷/水界面上采取更为直立的状态,因此聚醚分子在正庚烷/水界面扩散较快. 聚醚在正庚烷/水界面的扩张弹性高于空气/水表面的.     

Shear and dilatational relaxation mechanisms of globular and flexible proteins at the hexadecane/water interface

Proteins adsorbed at fluid/fluid interfaces influence many phenomena: food emulsion and foam stability (Murray et al. Langmuir 2002, 18, 9476 and Borbas et al. Colloids Surf., A 2003, 213, 93), two-phase enzyme catalysis (Cascao-Pereira et al. Biotechnol. Bioeng. 2003, 83, 498; 2002, 78, 595), human lung function (Lunkenheimer et al. Colloids Surf., A 1996, 114, 199; Wustneck et al.; and Banerjee et al. 2000, 15, 14), and cell membrane mechanical properties (Mohandas et al. 1994, 23, 787). Time scales important to these phenomena are broad, necessitating an understanding of the dynamics of biological macromolecules at interfaces. We utilize interfacial shear and dilatational deformations to study the rheology of a globular protein, lysozyme, and a disordered protein, beta-casein, at the hexadecane/water interface. Linear viscoelastic properties are measured using small amplitude oscillatory flow, stress relaxation after a sudden dilatational displacement, and shear creep response to probe the rheological response over broad experimental time scales. Our studies of lysozyme and beta-casein reveal that the interfacial dissipation mechanisms are strongly coupled to changes in the protein structure upon and after adsorption. For beta-casein, the interfacial response is fluidlike in shear deformation and is dominated by interfacial viscous dissipation, particularly at low frequencies. Conversely, the dilatational response of beta-casein is dominated by diffusion dissipation at low frequencies and viscous dissipation at higher frequencies (i.e., when the experimental time scale is faster than the characteristic time for diffusion). For lysozyme in shear deformation, the adsorbed protein layer is primarily elastic with only a weak frequency dependence. Similarly, the interfacial dilatational moduli change very little with frequency. In comparison to beta-casein, the frequency response of lysozyme does not change substantially after washing the protein from the bulk solution. Apparently, it is the irreversibly adsorbed fraction that dominates the dynamic rheological response for lysozyme. Using stress relaxation after a sudden dilatational displacement and shear creep response, the characteristic time of relaxation was found to be 1000 s in both modes of deformation. The very long relaxation time for lysozyme likely results from the formation of a glassy interfacial network. This network develops at high interfacial concentrations where the molecules are highly constrained because of conformation changes that prevent desorption.     

Sorbitan tristearate layers at the air/water interface studied by shear and dilatational interfacial rheology

The shear and dilatational rheology of condensed interfacial layers of the water-insoluble surfactant sorbitan tristearate at the air/water interface is investigated. A new interfacial shear rheometer allows measurements in both stress- and strain-controlled modes, providing comprehensive interfacial rheological information such as the interfacial dynamic shear moduli, the creep response to a stress pulse, the stress relaxation response to a strain step, or steady shear curves. Our experiments show that the interfacial films are both viscoelastic and brittle in nature and subject to fracture at small deformations, as was supported by in-situ Brewster angle microscopy performed during the rheological experiments. Although any large-deformation test is destructive to the sample, it is still possible to study the linear viscoelastic regime if the deformations involved are controlled carefully. Complementary results for the dilatational rheology in area step compression/expansion experiments are reported. The dilatational behavior is predominantly elastic throughout the frequency spectrum measured, whereas the layers exhibit generalized Maxwell behavior in shear mode within a deformation frequency regime as narrow as two decades, indicating the presence of additional relaxation mechanisms in shear as opposed to expansion/compression. If the transient rheological response from stress relaxation experiments is considered, then the data can be described well with a stretched exponential model both in the shear and dilatational deformations.     

The rheology of lupin seed (Lupinus albus ssp. graecus) protein isolate films at the corn oil–water interface

Lupin seed protein isolates adsorbed at the corn oil–water interface formed, after long ageing times, interfacial films with viscoelastic properties. The viscoelastic parameters of the films, derived by analysis of creep compliance–time curves, were markedly influenced by the aqueous phase protein concentrations, pH, ageing time and isolate preparation methods. Instantaneous elastic modulus, E₀(s), showed maxima at a certain concentration which probably corresponded to monolayer saturation coverage and at pH 5.5, i.e. near to its isoelectric point, where the protein molecules are in a more compact form than at other pH values. The full fat lupin seed protein fractions gave the highest viscoelasticity values under all conditions and this in turn have an effect on the corresponding emulsion/foam stability.     

蛋白质膜界面流变行为研究进展

随着界面流变测量技术以及相关的光学辅助仪器的发展,近10年来界面流变学在食品、化妆品、医药等领域发挥了重要作用。本文介绍了近年来蛋白质膜界面流变行为的研究进展,着重介绍了蛋白质界面流变学在泡沫和乳液方面的研究。本文主要分为3个部分,内容包括:蛋白质膜界面流变学行为与泡沫、乳液稳定性的相互关系,蛋白质-多聚糖的界面流变行为研究,蛋白质-表面活性物质的界面流变行为研究。界面流变学在泡沫和乳液方面取得的较快研究进展不仅促进了人们对蛋白质膜界面流变行为更为深刻的理解,而且为更好地开发和应用蛋白质及其混合物作为表面活性物质和胶体稳定剂而提供重要的理论依据和指导。     

Partition and water/oil adsorption of some surfactants

Adsorption isotherms have been determined at the water/oil interface for five biphasic systems involving surfactants (non-ionic and ionic) present in both phases at partition equilibrium. The systems studied were polyoxyethylene(23)lauryl ether (Brij35) in water/hexane and four ionic surfactants, hexadecyltrimethylammonium bromide (CTAB), and a series of three tetraalkylammonium dodecylsulfate (TEADS, TPADS, and TBADS) in water/CH 2Cl 2. Interfacial tension measurements performed at the water/air and water/oil interfaces provided all the necessary information for the determination of the adsorption parameters by taking partition into account. These measurements also allowed the comparison of the adsorption properties at both interfaces which showed an increase of the adsorption equilibrium constant and a decrease of the maximum surface concentration at the water/oil interface compared to water/air. The values of the critical aggregation concentration showed, in all cases, that only the surfactant dissolved in the aqueous phase contribute to the decrease of the water/oil interfacial tension. In the case of the four ionic surfactants, the critical aggregation concentration obtained in biphasic conditions were lowered because of the formation of mixed surfactant-CH 2Cl 2 aggregates.     

Protein-lipid interactions at the oil-water interface

The distribution of proteins and lipids in food emulsions and foams is determined by competitive and cooperative adsorption between the two types of emulsifiers at the fluid-fluid interfaces, and by the nature of protein-lipid interactions, both at the interface and in the bulk phase. The existence of protein-lipid interactions can have a pronounced impact on the surface rheological properties of these systems. Therefore, these results are of practical importance for food emulsion formulation, texture, and stability. In this study, the existence of protein-lipid interactions at the interface was determined by surface dynamic properties (interfacial tension and surface dilational modulus). Systematic experimental data on surface dynamic properties, as a function of time and at long-term adsorption, for protein (whey protein isolate (WPI)), lipids (monoglycerides), and protein-lipid mixed films at the oil-water interface were measured in an automated drop tensiometer. The dynamic behaviour of protein+lipid mixed films depends on the adsorption time, the lipid and the protein/lipid ratio in a rather complicated manner. The protein determined the interfacial characteristics of the mixed film as the protein at WPI>/=10(-2)% wt/wt saturated the film, no matter what the concentration of the lipid. However, there exists a competitive or cooperative adsorption of the emulsifier (WPI and monoglycerides), as the concentration of protein in the bulk phase is far lower than that for interfacial saturation.     

Surface adsorption behaviour of milk whey protein and pectin mixtures under conditions of air-water interface saturation

Milk whey proteins (MWP) and pectins (Ps) are biopolymer ingredients commonly used in the manufacture of colloidal food products. Therefore, knowledge of the interfacial characteristics of these biopolymers and their mixtures is very important for the design of food dispersion formulations (foams and/or emulsions). In this paper, we examine the adsorption and surface dilatational behaviour of MWP/Ps systems under conditions in which biopolymers can saturate the air-water interface on their own. Experiments were performed at constant temperature (20 °C), pH 7 and ionic strength 0.05 M. Two MWP samples, β-lactoglobulin (β-LG) and whey protein concentrate (WPC), and two Ps samples, low-methoxyl pectin (LMP) and high-methoxyl pectin (HMP) were evaluated. The contribution of biopolymers (MWP and Ps) to the interfacial properties of mixed systems was evaluated on the basis of their individual surface molecular characteristics. Biopolymer bulk concentration capable of saturating the air-water interface was estimated from surface pressure isotherms. Under conditions of interfacial saturation, dynamic adsorption behaviour (surface pressure and dilatational rheological characteristics) of MWP/Ps systems was discussed from a kinetic point of view, in terms of molecular diffusion, penetration and configurational rearrangement at the air-water interface. The main adsorption mechanism in MWP/LMP mixtures might be the MWP interfacial segregation due to the thermodynamic incompatibility between MWP and LMP (synergistic mechanism); while the interfacial adsorption in MWP/HMP mixtures could be characterized by a competitive mechanism between MWP and HMP at the air-water interface (antagonistic mechanism). The magnitude of these phenomena could be closely related to differences in molecular composition and/or aggregation state of MWP (β-LG and WPC).