Analysis of the Interactions of a Cationic Surfactant (Lauric Arginate

Dec 9, 2008 - Lauric arginate (LAE), a cationic surfactant, is a highly potent food-grade antimicrobial that is active against a wide range of food pa...
17 downloads 11 Views 449KB Size
Download PDF
116

Langmuir 2009, 25, 116-122

Analysis of the Interactions of a Cationic Surfactant (Lauric Arginate) with an Anionic Biopolymer (Pectin): Isothermal Titration Calorimetry, Light Scattering, and Microelectrophoresis D. Asker, J. Weiss, and D. J. McClements* Department of Food Science, UniVersity of Massachusetts, Amherst, Massachusetts 01003 ReceiVed September 16, 2008. ReVised Manuscript ReceiVed October 27, 2008 Lauric arginate (LAE), a cationic surfactant, is a highly potent food-grade antimicrobial that is active against a wide range of food pathogens and spoilage organisms. In compositionally complex environments, the antimicrobial activity of cationic LAE is likely to be impacted by its interactions with anionic components. The purpose of this study was to characterize the interactions between cationic LAE and an anionic biopolymer (high methoxyl pectin, HMP) using isothermal titration calorimetry (ITC), microelectrophoresis (ME), and turbidity measurements. ITC and ME measurements indicated that LAE bound to pectin, while turbidity measurements indicated that the complexes formed could be either soluble or insoluble depending on solution composition. In the absence of pectin, the critical micelle concentration (CMC) of LAE determined by ITC at 25 °C was 0.21% (w/v). The amount of LAE bound per unit amount of pectin decreased with increasing pectin concentration (from 1.5 to 0.5 g/g for 0.05 to 0.5 wt % pectin) and with increasing temperature (from 1.7 to 1.3 g/g for 15 to 40 °C). The binding contribution to the LAE-pectin interaction was exothermic and was attributed to electrostatic attraction between the cationic surfactant and anionic biopolymer. This study demonstrates that lauric arginate can form either soluble or insoluble complexes with anionic biopolymers depending on the composition of the system.

Introduction R

Lauric arginate (N -lauroyl-L-arginine ethyl ester monohydrochloride, LAE), a cationic surfactant, is a derivative of lauric acid, L-arginine, and ethanol.1 It has been approved as generally recognized as safe (GRAS) within the United States for certain food applications.2 LAE is one of the most potent food antimicrobial agents to have come onto the market in recent years due to its broad activity against a wide range of food pathogens and spoilage organisms.2-4 The high antimicrobial activity of LAE has been attributed to its action on the cytoplasmic membranes of microorganisms, where it alters their metabolic processes without causing cellular lysis.3 In addition, it has a low oil-water equilibrium partition coefficient (KOW < 0.1),2 which means it tends to concentrate in the water phase of products, where most bacterial activity occurs. LAE is hydrolyzed in the human body by chemical and metabolic pathways, which quickly break the molecule into its natural components: lauric acid and 1 L-arginine. Its low toxicity and high antimicrobial activities make it a valuable tool for controlling or preventing microbial growth in food products. On the other hand, its application within the food industry may be limited for a number of reasons: (i) its potency as an antimicrobial may be affected if it interacts with anionic components within the food matrix; (ii) it may bind to anionic biopolymers in the mouth, leading to perceived bitterness; and (iii) it tends to precipitate from solution at pH > 4.5 and at high ionic strength. Both the beneficial (antimicrobial) and detrimental (perceived bitterness, precipitation) properties of LAE, are likely to depend * To whom correspondence should be addressed. Telephone: (413) 5451019. Fax: (413) 545-1262. E-mail: [email protected]. (1) Ruckman, S. A.; Rocabayera, X.; Borzelleca, J. F.; Sandusky, C. B. Food Chem. Toxicol. 2004, 42, 245–259. (2) Bakal, G.; Diaz, A. Food Qual. 2005, 12, 54–61. (3) Rodriguez, E.; Seguer, J.; Rocabayera, X.; Manresa, A. J. Appl. Microbiol. 2004, 96, 903–912. (4) Luchansky, J. B.; Call, J. E.; Hristova, B.; Rumery, L.; Yoder, L.; Oser, A. Meat Sci. 2005, 71, 92–99.

on its interactions with other molecules in the systems in which it is used. In particular, this cationic surfactant may interact with anionic biopolymers through electrostatic interactions, which may either enhance or reduce its activity. It is therefore important to systematically characterize the interactions between LAE and anionic biopolymers so as to gain a better understanding of how to control the functionality of LAE in compositionally complex food products. When surfactant molecules are mixed with a solution of polymer molecules, they may exist in either a free or a bound form.5-8 In either of these forms, the surfactants may be present as individual molecules or as molecular clusters (e.g., micelles). The partitioning of surfactant molecules between different environments depends on the concentration and molecular characteristics of the polymer and surfactant, as well as on the prevailing environmental conditions such as temperature, pressure, and solvent composition.9 A variety of physicochemical mechanisms may either favor or oppose binding, including electrostatic interactions, hydrophobic interactions, hydrogen bonding, and various entropic effects.10,11 The relative importance of these mechanisms depends on the precise nature of the polymer-surfactant system as well as the prevailing environmental conditions, and therefore, it usually has to be established experimentally. In this study, we use a variety of complementary analytical methods to characterize the interactions between cationic lauric arginate and anionic pectin. Pectin is an anionic heteropolysaccharide of partially esterified R-1,4 linked D-galacturonides, (5) Taylor, D. J. F.; Thomas, R. K.; Penfold, J. AdV. Colloid Interface Sci. 2007, 132, 69–110. (6) Semenova, M. G. Food Hydrocolloids 2007, 21, 23–45. (7) Thunemann, A. F. Prog. Polym. Sci. 2002, 27, 1473–1572. (8) Barany, S. Macromol. Symp. 2001, 166, 71–92. (9) Goddard, E. D.; Ananthapadmanabhan, K. P. Interactions of surfactants with polymers and proteins; CRC Press: Boca Raton, FL, 1993. (10) Singh, S. K.; Caram-Lelham, N. J. Colloid Interface Sci. 1998, 203, 430–446. (11) Tam, K. C.; Wyn-Jones, E. Chem. Soc. ReV. 2006, 35, 693–709.

10.1021/la803038w CCC: $40.75  2009 American Chemical Society Published on Web 12/09/2008

Pectin-Cationic Surfactant Interactions

Langmuir, Vol. 25, No. 1, 2009 117

containing varying amounts of covalently attached rhamnose and branches of L-arabinose, D-galactose, D-xylose, and Lrhamnose.12 Pectin functions as a gelling, thickening, and stabilizing agent in foods. This functionality is related to the molecular weight, degree of esterification (% DE), distribution of ester groups on the backbone, presence of nonuronide components, and other structural characteristics.13-15 Pectin has carboxylic acid side groups that are negatively charged across a wide range of pH values (pKa (3.5)). The charge density and charge distribution along the backbone determines the functionality of pectin in foods. High-methoxy (HM) pectin (DE > 50%) has been widely used as a stabilizer in acidified milk drinks (pH 3.6-4.3), as it aids in preventing flocculation of milk proteins.16,17 Interactions of charged pectins with the cationic groups of proteins play a major role in the stabilizing effect of pectins in acidified dairy drinks and emulsions.18-20 Ultimately, we believe that a better understanding of the interactions between cationic LAE and anionic pectin molecules in aqueous solutions will facilitate the rational design of lauric arginate delivery systems with improved functionality in foods and other applications, for example, enhanced antimicrobial activity and/or reduced bitterness.

Materials and Methods Materials. The cationic surfactant LAE (C20H41N4O3Cl, MW ) 421.0 g mol-1), available commercially under the trade name MirenatCF (10.5% w/v LAE in 89.5% w/v propylene glycol solvent) was provided by Vedeqsa Grupo LAMIRSA (Terrassa, Barcelona, Spain). High methoxyl pectin (HMP) with a degree of esterification (DE) of approximately 70% (Pectin 1400) was provided by TIC Gums (Belcamp, MD). The mineral content of the pectin was reported by the manufacturer to be 451 mg of sodium, 152 mg of potassium, and 148 mg of calcium per 100 g. Distilled and deionized water was used for the preparation of all solutions. Solution Preparation. Pectin solutions (0.1 wt %) were prepared by dispersing powdered pectin into distilled water, in a sealed bottle, followed by heating in an autoclave at 120 °C under pressure for 15 min and then continuously stirring for 10 min. The pH of this solution was then adjusted to pH 3.5 using HCl or NaOH. Surfactant solution (LAE) was prepared by dispersing Mirenat-CF into deionized water at pH 3.5. Isothermal Titration Calorimetry (ITC). An isothermal titration calorimeter (VP-ITC, Microcal Inc., Northampton, MA) was used to measure enthalpies of mixing at 25.0 °C. Fifty-nine 5 µL aliquots of surfactant solution (1.8 w/v % LAE, pH 3.5) were injected sequentially into a 1480 µL titration cell initially containing either water or 0.1 wt % pectin in water (pH 3.5). Each injection lasted 20 s, and there was an interval of 240 s between successive injections. The solution in the titration cell was stirred at a speed of 315 rpm throughout the experiments. Turbidity, Electrical Charge, and Size. Aliquots of surfactant solution (0-1500 µL, 1.8 w/v % LAE, pH 3.5) were injected into glass vials initially containing either 7.5 mL of water or 0.1 wt % pectin in water (pH 3.5). The resulting solutions were then mixed thoroughly and stored overnight prior to analysis. The electrical charge (ζ-potential) and mean diameter (z-average) of the particles (12) Thakur, B. R.; Singh, R. K.; Handa, A. K. Crit. ReV. Food Sci. Nutr. 1997, 37, 47–73. (13) Oakenfull, D.; Scott, A. J. Food Sci. 1984, 49, 1093–1098. (14) Dea, C. M. Pure Appl. Chem. 1989, 61, 1315–1322. (15) Lofgren, C.; Hermansson, A. M. Food Hydrocolloids 2007, 21, 480–486. (16) Sedlmeyer, F.; Brack, M.; Rademacher, B.; Kulozik, U. Int. Dairy J. 2004, 14, 331–336. (17) Sejersen, M. T.; Salomonsen, T.; Ipsen, R.; Clark, R.; Rolin, C.; Engelsen, S. B. Int. Dairy J. 2007, 17, 302–307. (18) Pereyra, R.; Schmidt, K. A.; Wicker, L. J. Agric. Food Chem. 1997, 45, 3448–3451. (19) Syrbe, A.; Bauer, W. J.; Klostermeyer, H. Int. Dairy J. 1998, 8, 179–193. (20) Wicker, L.; Toledo, R. T.; Moorman, J.; Pereyra, R. Milchwissenschaft 2000, 55, 10–13.

Figure 1. Heat flow versus time profiles resulting from injection of 5 µL aliquots of 1.8%, w/v LAE into a 1480 µL titration cell containing either (a) water at pH 3.5 or (b) 0.1%, w/v pectin at pH 3.5 (25.0 °C).

in the solutions were then measured using a commercial instrument capable of electrophoresis and dynamic light scattering measurements (Zetasizer Nano-ZS, model ZEN3600, Malvern Instruments, Worchester, U.K.). The ζ-potential was calculated from the measurement of the electrophoretic mobility of particles in an applied oscillating electric field using laser doppler velocimetry. The mean diameter of the particles was calculated from their Brownian motion via the Stokes-Einstein equation. In some samples, large precipitates were formed that were too large (d > 6 µm) to be within the measurement range of the instrument (d ) 0.6 nm to 6 µm). For this reason, these samples were not analyzed using this instrument. Measurements were conducted on duplicate samples and repeated three times per sample. The optical turbidity (at 600 nm) of the pectin, surfactant, and pectin-surfactant solutions was measured using a UV-visible spectrophotometer (Spectronic 21D, Milton Roy, Rochester, NY) at room temperature. The samples were contained within 1 cm path length optical cells, and distilled water was used as a control. Turbidity measurements were carried out on duplicate samples.

Results and Discussions Solution Properties of LAE. The critical micelle concentration (CMC) of LAE was determined using ITC by measuring the enthalpy change resulting from its titration into acidified water (pH 3.5). Heat flow versus time profiles resulting from sequential injections of 5 µL aliquots of surfactant solution (1.8%, w/v LAE) into a 1480 µL titration cell initially containing water were measured (Figure 1a). The surfactant concentrations in the injector were appreciably above the CMC, so that the injector contained mainly surfactant micelles with some surfactant monomers.

118 Langmuir, Vol. 25, No. 1, 2009

Figure 2. (a) Dependence of enthalpy change per injection on the LAE concentration in the reaction cell when LAE was injected into a reaction cell containing either 0 or 0.1 wt % pectin (pH 3.5, 25 °C). (b) Difference in the enthalpy change per injection of surfactant in the absence and presence of pectin. The interaction regimes I, II, III, and IV are described in the text.

Initially, a series of relatively large endothermic peaks was observed when the surfactant solutions were injected into the reaction cell. These enthalpy changes are the result of micelle dissociation because the surfactant concentration in the reaction cell was initially below the CMC.21 The endothermic nature of these peaks (∆H > 0) indicates that demicellization must lead to an increase in the overall entropy of the system at this temperature, since micelle dissociation is thermodynamically favorable below the CMC (∆G < 0), and therefore, T∆S > ∆H. This entropy increase can be attributed to changes in the structural organization of water molecules around hydrophobic groups, the increase in entropy due to release of surfactant monomers from micelles, and the possible release of counterions associated with the surfactant head groups when micelles break down to monomers.21 After a certain number of injections, there was an appreciable decrease in peak height because the surfactant concentration in the reaction cell exceeded the CMC and so the micelles titrated into the reaction cell no longer dissociated. Above the CMC, the enthalpy change is therefore solely the result of micelle dilution effects.21 The dependence of the enthalpy change per injection on the surfactant concentration in the reaction cell was calculated by integration of the heat flow versus time profiles (Figure 2a). The CMC of the surfactant was determined from the inflection point in the ∆H versus surfactant concentration curves as 0.21 ( 0.01 wt %, which is equivalent to 4.9 ( 0.2 mM LAE. Dynamic light scattering was used to measure the mean particle diameter of the LAE micelles (0.5 w/v %), and the z-average was found to be 10.1 ( 0.4 nm. (21) Bijma, K.; Engberts, J. B. F. N.; Blandamer, B. J.; Cullis, P. M.; Last, P. M.; Irlam, K. D.; Soldi, L. G. J. Chem. Soc., Faraday Trans. 1997, 93, 1579.

Asker et al.

Characterization of LAE-Pectin Interactions. The enthalpy changes resulting from injection of LAE solution (1.8%, w/v) into 0.1 wt % pectin solutions was also measured using ITC (Figure 1b). The dependence of the enthalpy change per injection on the total surfactant concentration in the reaction cell was determined by integration of the measured heat flow versus time profiles (Figure 2a). The presence of pectin in the reaction cell caused an appreciable alteration in the enthalpy change versus surfactant concentration profile, indicating that there was some form of interaction between the cationic surfactant and anionic biopolymer. It was convenient to divide the enthalpy versus surfactant concentration profile into a number of different regions for the LAE-pectin system depending on the proposed interaction taking place (marked in Figure 2). A schematic representation of the proposed structures found in each of these regions is given in Figure 3. Region I: 0-0.025 wt % LAE. In this region, the enthalpy change was endothermic in the presence of pectin, but it was considerably less endothermic than in the absence of pectin. This indicated that there was an exothermic interaction between the LAE and the pectin. We postulate that the LAE micelles titrated into the reaction cell broke down into monomers, which then bound to the pectin molecules in the form of monomers. It is likely that the positive head groups of the LAE molecules bound to negative carboxylic acid groups on the pectin molecules through electrostatic attraction. Region II: 0.025-0.13 wt % LAE. In this region, the enthalpy change was still endothermic in the presence of pectin and was still considerably less endothermic than in the absence of pectin. Again, this indicated that there was an exothermic interaction between the LAE and the pectin. Nevertheless, the enthalpy difference between the pectin-free and pectin-containing solutions was less than that observed at lower surfactant concentrations (region (I)). We therefore postulate that LAE micelles bound to the pectin molecules in this concentration range. It is likely that micellelike surfactant clusters formed on the polymer so as to reduce the thermodynamically unfavorable contact of the hydrophobic LAE tails with water. At the end of this period, the pectin should be saturated with surfactant molecules, and hence, the surfactant binding capacity of the pectin () Cmicelle/Cpectin) would be about 1.3 g of surfactant per 1 g of pectin. This value was calculated by dividing the surfactant concentration when the pectin was saturated with micelles (Cmicelle) 0.13 wt %) by the total pectin concentration in the solution (Cpectin) 0.1 wt %). Region III: 0.13-0.21 wt % LAE. In this region, the enthalpy change was fairly similar (highly endothermic) in the presence and absence of pectin, indicating that there was little direct interaction between the pectin and surfactant. We postulate that the pectin molecules had become saturated with LAE micelles, and that any additional LAE micelles titrated into the reaction cell dissociated into monomers because the free surfactant concentration in the aqueous phase was below the CMC, thereby leading to a highly endothermic enthalpy change. Region IV: >0.21 wt % LAE. In this region, the enthalpy change became progressively less endothermic with increasing surfactant concentration, which followed a similar trend as that observed in the absence of pectin but at higher surfactant concentrations. We postulate that the concentration of free surfactant in the continuous phase had increased above the CMC, so that any additional LAE micelles titrated into the reaction cell remained as micelles. The relatively small enthalpy changes observed at higher surfactant concentrations can be attributed to enthalpy of dilution effects, that is, alterations in the distance

Pectin-Cationic Surfactant Interactions

Langmuir, Vol. 25, No. 1, 2009 119

Figure 3. Schematic representation of the interaction between cationic LAE and anionic pectin.

Figure 4. Dependence of the ζ-potential of pectin-LAE solutions on the LAE concentration for 0.1 wt % pectin solutions. LAE solution (1.8%, w/v) was titrated into aqueous 0.1 wt % pectin solution (pH 3.5).

(and therefore molecular interactions) between the micelles and other components within the reaction cell. One of the limitations of ITC is that it only measures the overall enthalpy change of a system and it is not possible to directly isolate the contributions of different physicochemical mechanisms, for example, micelle dissociation, monomer binding, micelle binding, biopolymer conformational changes, biopolymer aggregation, and so forth. Ideally, ITC should therefore be used in conjunction with other analytical techniques that can provide complementary information about the system. For this, reason we used turbidity and microelectrophoresis measurements to provide further insights into aggregation and binding interactions. The ζ-potential was negative (ζ ) -12 mV) in the absence of LAE (Figure 4), which can be attributed to the presence of partially charged carboxylic acid groups along the pectin chain (pKa ≈ 3.5). As the LAE concentration was increased, the ζ-potential initially became increasingly less negative and then it became increasingly more positive, with a point of zero charge around 0.22 wt % LAE. This result indicates that the cationic surfactant bound to the anionic pectin and formed a molecular complex, with charge neutralization occurring at a surfactantto-polymer mass ratio of ≈2:1. The increasing positive charge

Figure 5. Dependence of the turbidity (at 600 nm) of pectin-LAE solutions on LAE concentration for 0.1 wt % pectin solutions. LAE solution (1.8%, w/v) was titrated into aqueous 0.1 wt % pectin solutions (0-0.5 wt % pectin) at pH 3.5.

measured at LAE concentrations > 0.22 wt % may have been because additional cationic surfactant was incorporated into the molecular complexes or because of the contribution of free cationic micelles in the continuous phase. The ζ-potential of LAE micelles (0.5 w/v %) in the absence of pectin was +37 ( 4 mV at pH 3.5. The change in turbidity of the pectin solutions when LAE was titrated into them is shown in Figure 5. When the LAE concentration was increased from 0 to 0.04 wt %, the turbidity remained relatively low (2500 nm at 0.15 wt % LAE). The ITC measurements indicated that there was a break in the enthalpy versus surfactant concentration profile at an LAE concentration of about 0.13 wt %, which we attribute to the saturation of the pectin molecules with surfactant micelles (Figure 2a). Above this concentration, it was postulated that any additional surfactant added to the system was present as either monomers or micelles in the continuous phase. The fact that there was a large increase in mean particle diameter around this concentration suggests that the free surfactant promoted complex aggregation, possibly by acting as a bridge between them; that is, more than one pectin molecule was bound to a single micelle. This bridging phenomenon may also account for the fact that the binding capacity of pectin decreased with increasing pectin concentration (see next section). Influence of Biopolymer Concentration on LAE-Pectin Interactions. Further insights into the nature of pectin-LAE interactions were obtained by examining the impact of pectin concentration on the observed enthalpy changes. The enthalpy changes resulting from injection of LAE solution (1.8%, w/v) into pectin solutions of varying concentration (0-0.5%, w/v) were measured using ITC (pH 3.5). The dependence of the enthalpy change per injection on the total surfactant concentration in the reaction cell was determined by integration of the measured heat flow versus time profiles (Figure 6). The amount of pectin initially present in the reaction cell had a pronounced impact on the enthalpy versus surfactant concentration profiles. In addition, the different interaction regimes [(I)-(IV)] listed earlier were clearly distinguishable within each of the profiles. These interaction regimes were characterized in terms of characteristic surfactant concentrations (Figure 2b). Region I: C < Cmonomer. In this regime, the LAE initially added to the reaction cell bound to the pectin molecules as monomers (rather than micelles). The upper surfactant concentration of this limit (Cmonomer) was determined as the point where a break was observed in the enthalpy versus surfactant concentrations at relatively low LAE concentrations (as marked in Figure 2a). Region II: Cmonomer < C < Cmicelle. In this regime, the LAE bound to the pectin molecules as micelles (rather than monomers) until the pectin was saturated with surfactant. The value of Cmicelle was determined as the point where there was a break in the enthalpy versus surfactant concentration at intermediate LAE concentrations (as marked in Figure 2a). The value of Cmicelle

Asker et al.

Figure 7. Influence of initial pectin concentration in the reaction cell on the critical surfactant concentrations (C*CMC, Cmonomer, and Cmicelle) characterizing micellization, monomer binding, and micelle binding.

should be equivalent to the surfactant concentration where the pectin is saturated with LAE. Region III: Cmicelle < C < C*CMC. In this regime, the pectin was saturated with LAE but the free surfactant concentration in the aqueous phase was still below the critical micelle concentration. Hence, any surfactant micelles added to the reaction cell dissociated and formed monomers in the aqueous phase. The value of C*CMC was determined as the inflection point in the enthalpy versus surfactant concentration profiles at higher LAE concentrations (as marked in Figure 2a). Region IV: C > C*CMC. In this regime, the pectin was saturated with LAE and the free surfactant concentration in the aqueous phase exceeded the CMC. Hence, any surfactant micelles added to the reaction cell remained as micelles, rather than dissociating into monomers. The values of Cmonomer, Cmicelle, and C*CMC are plotted as a function of pectin concentration in the reaction cell in Figure 7. All three characteristic concentrations increased with increasing pectin concentration, which would be expected if surfactant bound to the pectin molecules. Nevertheless, the increases in Cmonomer, Cmicelle, and C*CMC with pectin concentration were not linear. Indeed, the surfactant binding capacity of the pectin () Cmicelle/ Cpectin) appeared to decrease with increasing pectin concentration, being 1.4, 1.3, 0.9, and 0.5 g of surfactant per 1 g of pectin for 0.05, 0.1, 0.2, and 0.5% (w/v) pectin solutions, respectively. This effect may be attributed to interactions between surfactantpectin complexes at higher pectin concentrations, which may have limited the amount of surfactant they could bind. For example, some of the surfactant micelles may have been shared between two or more pectin molecules. In addition, there will be some small ions associated with the pectin molecules (e.g., Na+, K+, and Ca2+; see Materials and Methods section), which may have altered the CMC and interactions of the surfactant in the aqueous phase.22 We also measured the influence of pectin concentration on the formation of LAE-pectin complexes that were large enough to scatter light (Figure 5). For all pectin concentrations studied (0.05-0.5 wt %), there was a slight increase in solution turbidity from 0 to 0.04 wt % LAE, followed by a steeper increase at higher surfactant concentrations. Above about 0.1-0.2 wt % LAE, the turbidity reached a relatively constant value, whose magnitude was fairly similar for the systems containing 0.1-0.5 wt % pectin. In general, the LAE concentration where the plateau region was first reached increased with increasing pectin concentration. These results suggest that only small aggregates (22) Thongngam, M.; McClements, D. J. Food Hydrocolloids 2005, 19, 813– 819.

Pectin-Cationic Surfactant Interactions

Langmuir, Vol. 25, No. 1, 2009 121

Figure 9. Influence of temperature of the reaction cell on the critical surfactant concentrations (C*CMC, Cmonomer, and Cmicelle) characterizing micellization, monomer binding, and micelle binding.

Figure 8. Temperature-dependence of enthalpy change per injection on the LAE concentration in the reaction cell when LAE was injected into a reaction cell containing either (a) no pectin or (b) 0.1 wt % pectin (pH 3.5).

were formed at relatively low surfactant concentrations (0.1-0.2 wt % LAE) suggests that there was little further change in the number and size of the LAE-pectin complexes formed upon further addition of surfactant. There appeared to be a correlation between the LAE concentration where the plateau region was reached in turbidity measurements and the LAE concentration where the pectin became saturated with surfactant determined by ITC (Figure 6). Hence, once the pectin molecules had become saturated with LAE, there appeared to be little further change in the number or size of the aggregates formed. Influence of Temperature on LAE-Pectin Interactions. Practically, it is important to understand how temperature influences the interactions between pectin and LAE, since many commercial products are subjected to temperature changes. The enthalpy changes resulting from injection of LAE solution (1.8%, w/v) into 0 and 0.1 wt % pectin solutions at different holding temperatures (20-50 °C) were therefore measured using ITC. As described previously, the dependence of the enthalpy change per injection on the total surfactant concentration in the reaction cell was then determined by integration of the measured heat flow versus time profiles (Figure 8). Measurements could not be made at lower temperatures, since the LAE precipitated out of solution when it was stored at 10 °C and below. In the absence of pectin, the enthalpy change versus surfactant concentration profiles had a similar form at all temperatures: they were highly endothermic up to a particular LAE concentration and then became progressively less endothermic above this value. The high endothermic enthalpy observed at lower surfactant concentrations is associated with breakdown of the LAE micelles after they are titrated into the reaction cell because the surfactant concentration is initially below the CMC. The progressive decrease in the endothermic enthalpy change above a particular

surfactant concentration is due to the fact that the CMC is exceeded so that any additional LAE micelles added no longer break down. The magnitude of the endothermic enthalpy change associated with micelle breakdown, as well as the value of the CMC, did depend on temperature. Micelle breakdown became more endothermic with increasing temperature (Figure 8). This phenomenon can be attributed to the fact that the enthalpy change associated with the transfer of a hydrophobic substance from a hydrophobic environment into water becomes increasingly endothermic as the temperature is increased.22,23 In our study, the nonpolar LAE tails will move from the hydrophobic core of the LAE micelles into water when they break down after titration into the reaction cell. The CMC of the LAE was determined as a function of temperature by finding the inflection point from the enthalpy versus surfactant concentration profiles (Figure 8a). The CMC (CCMC) increased from around 0.18 wt % at 15 °C to around 0.23 wt % at 40 °C (Figure 9). Hence, the thermodynamic driving force for micelle formation decreased slightly with increasing temperature. Possible explanations for this phenomenon are an increase of the entropy of mixing with increasing temperature, or temperature-dependent changes in the tail-tail and/or head-head surfactant interactions. In the presence of 0.1 wt % pectin, the enthalpy change versus surfactant concentration profiles also had a similar form at all temperatures (Figure 8b), indicating that similar physicochemical phenomena occurred at all temperatures, for example, micelle dissociation, monomer binding, and micelle binding. However, the magnitude of the enthalpy changes and the characteristic concentrations where specific events occurred were temperaturedependent. In general, the enthalpy changes were more highly endothermic at higher temperatures, which again can be attributed to the characteristic temperature-dependence of the transfer enthalpy of nonpolar groups from hydrophobic to aqueous environments mentioned above. The values of Cmonomer, Cmicelle, and C*CMC were calculated from the enthalpy versus surfactant concentration profiles as described above (Figure 9). All three characteristic concentrations increased slightly with increasing temperature, suggesting that slightly more LAE bound to the pectin at higher temperatures. The surfactant binding capacity of the pectin () Cmicelle/Cpectin) changed from around 1.7 to 1.3 g/g when the temperature was increased from 15 to 40 °C.

Conclusions This study has shown that isothermal titration calorimetry can provide valuable information about interactions between a cationic (23) Silverstein, K. A. T.; Haymet, A. D. J.; Dill, K. A. J. Chem. Phys. 1999, 111, 8000–8009.

122 Langmuir, Vol. 25, No. 1, 2009

surfactant (LAE) and an anionic biopolymer (pectin). The technique was able to detect enthalpy changes associated with the dissociation of micelles in biopolymer-free solutions and with the binding of surfactant to pectin in solutions containing biopolymer. The enthalpy versus surfactant profiles highlighted that different physicochemical phenomena occurred in the surfactant-biopolymer solutions depending on surfactant and biopolymer concentration, for example, micelle dissociation, monomer binding, and micelle binding. The turbidity and microelectrophoresis measurements confirmed that a binding interaction had occurred and that either soluble or insoluble complexes could be formed. Binding of LAE to pectin was attributed to electrostatic attraction between the cationic surfactant and the anionic pectin. At relatively low surfactant concentrations,

Asker et al.

the LAE-pectin complexes were relatively small (low turbidity, slow sedimentation), but at higher concentrations the complexes were relatively large (high turbidity, rapid sedimentation). The interaction of cationic LAE with anionic pectin may have important implications for the application of lauric arginate as a functional ingredient in food and other industrial applications. For example, these complexation interactions may impact the ability of LAE to act as an antimicrobial or they may impact its sensory attributes. Acknowledgment. This material is based upon research work supported by Vedeqsam Group LAMIRSA (Terrassa, Barcelona, Spain). LA803038W