Article pubs.acs.org/JAFC
Impact of Cationic Surfactant on the Self-Assembly of Sodium Caseinate Marko Vinceković,*,† Marija Ć urlin,‡ and Darija Jurašin*,§ †
Department of Chemistry, Faculty of Agriculture, and ‡Department of Histology and Embryology, School of Medicine, University of Zagreb, Zagreb, Croatia § Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia ABSTRACT: The impact of a cationic surfactant, dodecylammonium chloride (DDACl), on the self-assembly of sodium caseinate (SC) has been investigated by light scattering, zeta potential, and rheological measurements as well as by microscopy (transmission electron and confocal laser scanning microscopy). In SC dilute solutions concentration-dependent self-assembly proceeds through the formation of spherical associates and their aggregation into elongated structures composed of connected spheres. DDACl interacts with SC via its hydrophilic and hydrophobic groups, inducing changes in SC self-assembled structures. These changes strongly depend on the surfactant aggregation states (monomeric or micellar) as well as concentration ratio of both components, leading to the formation of soluble and insoluble complexes of nano- to microdimensions. DDACl monomers interact with SC self-assembled entities in a different way compared to their micelles. Surfactant monomers form soluble complexes (similar to surfactant mixed micelles) at lower SC concentration but insoluble gelatinous complexes at higher SC concentration. At surfactant micellar concentration soluble complexes with casein chains wrapped around surfactant micelles are formed. This study suggests that the use of proper cationic surfactant concentration will allow modification and control of structural changes of SC self-assembled entities. KEYWORDS: casein, cationic surfactant, self-assembling, complexation
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INTRODUCTION Biopolymers and surfactants are often used simultaneously in the food, pharmaceutical, and cosmetics industries, production of agrochemicals, etc. Understanding their interactions at the molecular level is therefore of great importance in tailoring the final properties of manufactured products. Intensive research on intermolecular interactions between biopolymers (proteins or polysaccharides) and low molecular weight surfactants has provided many new insights into the processes and mechanisms of interactions; however, there is no universal theory describing their complex interactions.1−3 Understanding the behavior of mixtures containing amphiphilic molecules, such as proteins and low molecular weight surfactants, is not straightforward because these species self-assemble in bulk solution and at interfaces by different mechanisms, leading to competitive association and adsorption processes.2 Although much is known about the interactions between surfactants and proteins, knowledge of their molecular structure/reactivity relationship in relationship to their functionality in complex systems is still rather limited, and empirical approaches continue to be required in tackling the problem of improvement and optimization of processing conditions. Depending on aqueous conditions (pH, ionic strength, temperature) and features of the molecular structures of both proteins and surfactants as well as on their concentration and molar ratio, the overall character of their interactions may be significantly different. Addition of small-molecule surfactants to protein solutions modifies the protein functional properties both at the interfaces and in bulk. It was found that hydrophobic and electrostatic interactions as well as hydrogen bonding are the main factors governing protein and surfactant © XXXX American Chemical Society
complexation, resulting in specific surface properties of complexes and conformational changes of proteins.3 This is of special concern in controlled release application, such as plant protection. There are increasing concerns about the level of plant protection active agents found in soil and plant materials causing environmental and health problems. In line with global efforts toward sustainable agriculture, encapsulation with biopolymer matrices has been recognized as an effective method for controlled release of an active agent used for plant protection.4 The development of systems for controlled release strongly depends on adequate physicochemical properties of biopolymers and on economically sustainable production. Milk proteins are widely available, inexpensive, and generally recognized as safe raw materials with many structural properties and functionalities that make them highly suitable natural vehicles for bioactives.5 The presence of anionic phosphoserine and other anionic amino acid residues makes caseins available for cation binding, which is important for the delivery of bioactive cations (copper, iron, etc.) to plants in acceptable form. The main objective of the present work was to explore the possibility of guiding the self-assembly of sodium caseinate (SC) with the model cationic surfactant dodecylammonium chloride (DDACl) and consequently to enhance the suitability of SC self-assembled structures for loading bioactive cations. Most of the studies dealing with the impact of surfactants on the supramolecular organization of the main milk proteins, Received: April 8, 2014 Revised: June 18, 2014 Accepted: July 31, 2014
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lation of water using a conductivity meter (Metrohm, Herisau, Switzerland). The κ was measured to within 0.01 μS. The surface tension (γ) was determined using the Du Noüy ring method (interfacial tensiometer K100, Krű ss, Hamburg, Germany). The γ was measured to within 0.001 mN m−1. These values then were corrected by using the tables of Huh and Mason. The surface tension of water was measured regularly to provide values for the pure solvent and to check that the technique was being properly carried out. All measurements were performed at 298 K. Light Scattering and Zeta Potential Measurements. Light scattering and electrophoretic measurements were performed using Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The size of particles (d) was determined by means of a dynamic light scattering (DLS) technique using a photon correlator spectrophotometer equipped with a 532 nm “green” laser. DLS measurements were performed at a backscatter angle of 173° to reduce the extent of multiple scattering as well as the effects of dust. Numerous static and DLS studies in SC solutions have shown that variation in their chemical composition resulting from the preparation procedure and methods of sample preparation (centrifugation, filtration, etc.) as well as the experimental conditions led to differences in the reported light scattering data.20−24 Casein self-assembled entities are polydisperse in size, including in some cases the coexistence of several populations as well as a residual lipid material that could not be completely removed upon sample purification. In a polydisperse sample, the scattering intensity of large particles dominates that of smaller ones and the meaning of the average hydrodynamic size is not straightforward; that is, the intensity-weighted size distribution given by DLS overestimates larger aggregates. To distinguish the contribution of SC from that of large particles, DLS data have been analyzed by relying on the volume and number size distributions. The data processing was done by the Zetasizer software 6.32 (Malvern Instruments). The mean diameters derived from volume and number size distributions were always based on six or more measurements with a relative standard deviation of ±20%. The zeta potential (ζ) of particles was calculated from the measured electrophoretic mobility by means of the Henry equation using the Smoluchowski approximation ( f(Ka) = 1.5). Results are reported as an average value of five measurements with a relative standard deviation of ±10%. Samples were prepared by dilution of stock solutions in triple-distilled water, which was previously filtered through a 0.45 μm Millipore filter. All measurements were performed at 298 K. Rheological Measurements. Rheological measurements were employed to obtained the flow behavior and viscosity data. Measurements were performed with a stress-controlled rheometer with a double-gap Couette cell (MC1, Paar Physica, Graz, Austria). Flow curves were determined by recording the steady shear rate (γ) for a series of imposed shear stress (σ). All flow curves revealed that SC solutions behaved as Newtonian fluids. The plots of shear stress versus shear rate gave straight lines, from which viscosity (η) was derived with the linear regression coefficient close to unity for all plots. All measurements were run in triplicate and performed at 298 K. Microscopic Observations. Microscopic phases were identified by transmission electron microscopy (TEM 902A, Zeiss, Thornwood, NY, USA) and confocal laser scanning microscopy (CLSM; TCP SP2, Wetzlar, Germany). The transmission electron microscope (TEM) was operated in bright field mode at an acceleration voltage of 80 kV. Images were recorded with a Canon Power Shot S50 camera attached to the microscope. Samples for TEM were were negatively stained with uranyl acetate (2 wt %). A drop of the sample solution was placed on a carbon-coated mesh copper grid. The sample was allowed to dry for 5 min, and then any excess solution was removed by filter paper; dried sample was stained and allowed to dry. All sample preparations for microscopic observation were performed at room temperature. Samples for CLSM were stained with Rhodamine B (RhB). RhB (0.05 wt %) was dispersed in the SC solution prior to its mixing with DDACl. pH Measurements. pH measurements were performed using a combined electrode connected to an ion meter (Metrohm).
caseins, have been conducted with anionic or nonionic surfactants.3,6 Data on the interaction with cationic surfactants are relatively scarce.7−10 One of the interesting features of the mixture of oppositely charged amphiphilic molecules is that the self-assembled structures and the charge of their electrical double layer can be manipulated by simple variation of concentrations and molar ratio of components. Sodium caseinate is the sodium salt of the main milk proteins, caseins. Caseins are relatively small, amphiphilic, randomly or flexibly structured molecules composed of four main proteins: αs1-, αs2-, β-, and κ-caseins. They are proteins characterized by an open structure and a high degree of hydration. In none of their native states do they adopt random coil conformation, but they contain significant amounts of defined secondary structure with few tertiary folds, but do not, for various reasons, fold into compact globular proteins.11 Casein monomers as complex amphiphilic copolymers exhibit differences in self-association due to the different hydrophobic and hydrophilic regions along the protein chain; β- and κcaseins self-assemble as surfactants, whereas αs-caseins associate in a series of consecutive steps to form open aggregates, which can be rigid worm-like structures.12 Besides expressed selfassociation, casein molecules also interact with each other, forming associated structures with the κ-casein mainly located on the surface and other caseins mainly located in the interior of self-assembled entities.13−16 The microenvironment, such as pH, ionic strength, concentration, temperature, and surfactant, can significantly alter the self-assembled structures of casein molecules.12,17,18 All literature data dealing with casein and oppositely charged surfactant interactions rely on the experiments performed at various surfactant concentrations and constant casein concentration. On the contrary, our data rely on experiments performed at a constant concentration of cationic surfactant and various SC concentrations to show how surfactant aggregation state (monomer or micellar) affects SC selfassembly. The major novelty of our research is the comprehensive focus on the overall changes in the mechanism of SC self-assembly induced by the addition of constant concentration, both monomer and micellar, of model cationic surfactant. We hope that the results obtained will provide better insight into the nature of interactions between caseins and an oppositely charged surfactant, providing an opportunity to control the formation of SC self-assembled structures.
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EXPERIMENTAL PROCEDURES
Materials and Sample Preparation. Commercial food-grade SC containing protein with sodium ≤3% and calcium ∼0.1% (SigmaAldrich, USA) was used throughout without purification and was dried in a vacuum before weighing. Preparation and purification of DDACl were described earlier.19 Stock solutions were prepared by dissolving dried SC or DDACl in deionized water, treated in an ultrasonic bath, filtered through a 0.45 μm filter (Millipore Corp.), and left to stand overnight. Numerous literature data relating to casein self-assembly have generally been obtained at adjusted pH and ionic strength. On the contrary, we prepared samples with various SC concentrations in the presence or absence of constant DDACl concentrations (monomer (0.001 mol dm−3) or micellar (0.03 mol dm−3)) with no pH adjustment or salt added. Samples were left to equilibrate for 24 h in a water bath at 298 K. All samples contained 0.01 wt % NaN3 as a bacteriostatic agent. Methods. Electrical Conductivity and Surface Tension Measurements. Electrical conductivity (κ) measurements were performed in a temperature-controlled double-walled glass container with a circuB
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RESULTS AND DISCUSSION Self-Assembly in SC Dilute Solutions. Sodium caseinate is a heterogeneous mixture of phosphorus-containing proteins containing many ionizable groups on the side chains of their amino acids as well as their amino and carboxyl termini. Depending on solution conditions, functional groups of the side chains belonging to amino acids combined into the protein polypeptide chain can be either protonated or deprotonated.25−27 The polar regions of the caseins are dominated by phosphoseryl residues and thus carry a net negative charge above the pH called the isoelectric point (pI). The average pI of caseins is around 4.6 at 293 K;28 at pI casein is electrically neutral due to the presence of an equal amount of oppositely charged ionizable groups. The increase of the net negative charge beyond the pI arises from the deprotonation of carboxyl groups, loss of positive charge on the histidines and lysines, and increased negative charge on the phosphoseryl residues.29 Figure 1a shows variation of pH with SC concentration. It is evident that at very low concentrations SC aqueous solutions are basic, showing rapid pH decrease with increasing concentration up to the critical concentration, denoted c*. The pH decrease with increasing SC concentration indicates a release of hydrogen ions into solution. As the SC concentration increased beyond the c*, the pH continued to decrease to a smaller extent, approaching the pH of milk. The self-buffering power of proteins is diminished in dilute solutions,30 and it seems that small amount of ions commonly present in a commercial sample overcome the SC self-buffering capacity. This effect is compensated at higher SC concentration. Like other amphiphilic molecules, caseins self-assemble in aqueous solutions, thus inducing changes in the behavior of physicochemical properties (such as conductivity, surface tension, light scattering, etc.). Figure 1b shows the plots of specific conductivity and surface tension against SC concentration. The intersection point between two linear parts of the κ-c(SC) curve (denoted c*) indicates a change in the conductivity behavior of the species present, that is, a change in the SC self-assembled structures. The considerable exposure of hydrophobic amino acid residues caused by the open structure of casein molecules underlies the ability of SC to adsorb readily at the air/water interface, thus decreasing the surface tension. It can be seen in the inset that the decrease in surface tension starts immediately at a very low SC concentration. The break point between the steeply descending and fairly constant branch of the γ-c(SC) curve indicates the monolayer formation at the air/water interface and corresponds to the c* obtained by pH and specific conductivity measurements. Constant surface tension beyond the c* indicated that the air/liquid interface is occupied with surface active casein molecules. Compared to a conventional surfactant, SC lowers the surface tension of water to a smaller extent. Our results are in good agreement with literature data; maximum lowering of surface tension is consistent with the equilibrium surface tension obtained for sodium caseinate dissolved in water with 0.1 mol dm−3 NaCl.31 The origin of an abrupt change in measured physicochemical properties of SC solutions was systematically examined by light scattering, zeta potential, and rheological measurements. Literature data based on light scattering measurements revealed polydispersity as a characteristic property of SC solutions. Commonly, a bimodal size distribution with the variable relationship between peak areas (PA) depending on the origin
Figure 1. (a) Variation of pH with SC concentration (c(SC)) for samples without (○) and with constant DDACl concentration (c(DDACl) = 0.001 mol dm−3 (△) and 0.03 mol dm−3 (□)). (b) Variation of specific conductivity (κ) and surface tension (γ) (inset) with SC concentration (c(SC)). All SC samples were dispersed in water without addition of salt or pH adjustment. Critical SC concentration (c*) is denoted by an arrow. Capital letters denote the appearance of samples in various concentration regions at constant monomeric DDACl concentration: C, clear region; T, turbid region; S, sedimentation region. Temperature is 298 K.
of SC sample and the experimental conditions was obtained.15,23,32,33 To avoid overestimation arising from the scattering intensity of large particles (especially of a small weight fraction of complexes containing fat that cannot be removed completely by sample purification),15 we have analyzed DLS data relying on the comparison between volume and number size distributions. Volume and number size distributions were used to estimate the relative amounts of multiple size peaks and are not considered absolute. A typical example of volume size distributions for SC samples below the c* presented in Figure 2a shows the existence of two particle populations. A population of smaller particles with the d value of 25 nm at the peak maximum (PA = 57%; d varies in the range from 16 to 50 nm) along with a population of larger particles with the d value of 191 nm at the peak maximum (PA = 43%; d varies in the range from 70 to 712 nm) were observed. The number size distribution (Figure 2b) revealed the presence of 99.7% of smaller particles and a negligible C
dx.doi.org/10.1021/jf5016472 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Size distribution by volume (a, d) and number (b, e) of casein associates (left) and aggregates (right). SC samples (concentrations as denoted) were dispersed in water with no salt and no pH adjustment. Corresponding images taken with transmission electron microscope (c, f); bars = 100 nm.
particles with c(SC). Two separate concentration regions can be recognized, below and above the c*. The first region is dominated by the presence of spherical associates with the size very close to the formerly reported size of SC in the presence of electrolyte23,32,33 as well as to the value reported for the size of native casein micelle building blocks, called submicelles.34 Like other proteins, casein molecules carry charges according to their amino acid sequence and the aqueous pH in which they are dissolved. Generally, when pH >4.6, caseins have a net negative charge and when pH
