Langmuir 2007, 23, 2071-2077
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Olive Oil Microemulsions: Enzymatic Activities and Structural Characteristics Vassiliki Papadimitriou, Theodore G. Sotiroudis, and Aristotelis Xenakis* Institute of Biological Research & Biotechnology, The National Hellenic Research Foundation, 48 Vas. Constantinou AVenue, 11635 Athens, Greece ReceiVed September 6, 2006. In Final Form: NoVember 9, 2006 Microemulsions composed of olive oil, either extravirgin (EVOO) or refined (ROO), as the continuous oil phase, water as the dispersed phase, and a mixture of lecithin-propanol as the emulsifier were prepared and investigated as potential biocompatible media for biotransformations. The area of the microemulsion zone increased considerably by increasing the lecithin to propanol weight ratio in both EVOO- and ROO-based systems. However, the nature of the oil used does not seem to affect the ability of the system to incorporate water. The catalytic activities of two oxidizing enzymes that have been detected in virgin olive oil, namely, tyrosinase and peroxidase, and the activity of a proteolytic enzyme such as trypsin were studied in olive oil microemulsions. In all cases a reduced catalytic activity was observed when ROO was considered as the continuous oil phase. The interfacial properties of lecithin layers were studied by electron paramagnetic resonance spectroscopy employing the nitroxide spin probe 5-doxylstearic acid. By varying the weight ratio of lecithin to propanol and the water content of the microemulsions, the mobility of the probe and the rigidity of the interface were altered. Droplet sizes were measured by dynamic light scattering. At higher water content of the system the size of the droplets was increased. When EVOO was considered as the oil phase, smaller aqueous droplets were formed. Lecithin-based olive oil microemulsions were also characterized with regard to the phenomenon of electrical percolation. At a water content above 3% (w/w) and a lecithin/propanol weight ratio of 2, a sharp increase in conductivity was observed, indicating a structural transition in the bicontinuous form.
Introduction Biocompatible and biodegradable microemulsions are of growing interest to the food, cosmetic, and pharmaceutical industry as solubilization media of hydrophilic, hydrophobic, and amphiphilic reactive molecules.1-5 Microemulsions are spontaneously forming, thermodynamically stable systems consisting of at least three components: a nonpolar solvent, water, and surfactants.6 These systems possess several advantages over their solvent-based counterparts; they can enhance solubilization of otherwise immiscible liquids, they provide a large interface between water and the nonpolar solvent, and they also enable the coexistence of both water-soluble and oil-soluble materials. Microemulsions contain microdomains of different polarities within the same single-phase solution, which can be considered as nanophases for conducting various reactions. Due to their unique properties microemulsions have been used in a variety of technological applications, including cleaning, product formulations, delivery systems, polymerization media, and chemical reaction media.7,8 Soybean lecithin is a naturally occurring nontoxic amphiphile which has been successfully used for over a decade for the construction of water-in-oil (W/O) microemulsions.9-11 However, * To whom correspondence should be addressed. Phone: +302107273762. Fax: +302107273758, E-mail:
[email protected]. (1) Garti, N.; Spernath, A.; Aserin, A.; Lutz, R. Soft Matter 2005, 1, 206-218. (2) Acosta, E.; Nguyen, J., T.; Witthayapanyanon, A.; Harwell, J. H.; Sabatini, D. A. EnViron. Sci. Technol. 2005, 39, 1275-1282. (3) Garti N. Curr. Opin. Colloid Interface Sci. 2003, 8, 197-211. (4) Glatter, O.; Orthaber, D.; Stradner, A.; Scherf, G.; Fanun, M.; Garti, N.; Clement, V.; Leser, M. E. J. Colloid Interface Sci. 2001, 241, 215-225. (5) Watnasirichaikul, S.; Davies, N. M.; Rades, T.; Tucker, I. G. Pharm. Res. 2000, 17, 684-689. (6) Danielson, I.; Lindman, B. Colloids Surf. 1982, 3, 391 (7) Klier, J.; Tucker, C. J.; Kalantar, T. H.; Green, D. P. AdV. Mater. 2000, 12, 1751-1757. (8) Paul, B. K.; Moulik, S. P. Curr. Sci. 2001, 80, 990-1000. (9) Shinoda, K.; Araki, M.; Sadaghiani, A.; Khan, A.; Lindman, B. J. Phys. Chem. 1991, 95, 989-993.
due to lecithin’s strong lipophilicity the addition of short-chained alcohols as cosurfactants was necessary to render the polar solvent less hydrophilic and allow the spontaneous formulation of microemulsions.9 In previous studies, various saturated hydrocarbons such as hexadecane, hexane, isooctane, and cyclohexane were used as organic solvents for the formulation of lecithin-based W/O microemulsions.9,11,12 Microemulsions based on isopropyl myristate, lecithin, and medium-chain alcohols have been reported for drug delivery purposes.13 Recently, Ichikawa and co-workers introduced the use of fatty acids and fatty acid esters to prepare lecithin-based biocompatible microemulsions for application in the pharmaceutical and food industries.14 In the present study the use of olive oil as the nonpolar solvent for the formulation of lecithin-based W/O microemulsions is suggested. Olive oil, one of the oldest known vegetable oils, is a natural, nontoxic, biocompatible, and inexpensive product that can be used as a constituent for the construction of various microemulsions. Virgin olive oil contains endogenous amphiphiles such as free fatty acids and also a variety of minor components such as phospholipids, polyphenols, and partial glycerides.15 Studies from this laboratory have shown the presence of proteins and enzymes in virgin olive oil.16,17 As a consequence (10) Kahlweit, M.; Busse, G.; Faulhaber, B. Langmuir 1995, 11, 1576-1583. (11) Avramiotis, S.; Lianos, P.; Xenakis, A. Biocatal. Biotransform. 1997, 14, 299-316. (12) Marangoni, A. G.; Mccurdy, R. D.; Brown, E. D. J. Am. Oil Chem. Soc. 1993, 70, 737-744. (13) Von Corswant, Ch.; Olsson, C.; Soederman, O. Langmuir 1998, 14, 68646870. (14) Ichikawa, S.; Sugiura, S.; Nakajima, M.; Sano, Y.; Seki, M.; Furusaki, S. Biochem. Eng. J. 2000, 6, 193-199. (15) Boskou, D. OliVe Oil: Chemistry and Technology; AOCS Press: Champaign, IL, 1996. (16) Georgalaki, M. D.; Sotiroudis, T. G.; Xenakis, A. J. Am. Oil Chem. Soc. 1998, 75, 155-159. (17) Georgalaki, M. D.; Bachman, A.; Sotiroudis, T. G.; Xenakis, A.; Porzel, A; Feussner, I. Fett/Lipid 1998, 100, 554-560.
10.1021/la062608c CCC: $37.00 © 2007 American Chemical Society Published on Web 01/03/2007
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virgin olive oil itself can be considered a fine emulsion of a water phase in a continuous nonpolar phase.18 For comparison reasons, refined olive oil was also used as the lipophilic continuous phase for the construction of microemulsions. Refined olive oil is obtained from virgin olive oils that have high acidity levels or/and organoleptic defects by refining methods (either physical or chemical processes). After refining, strong tastes are eliminated and free fatty acids are neutralized. Refined olive oil lacks free fatty acids, proteins, and all the minor components present in virgin olive oil that are considered as natural endogenous amphiphiles. In the present study, four-component W/O microemulsions consisting of extravirgin olive oil (EVOO) or refined olive oil (ROO) as the nonpolar solvent, lecithin as the surfactant, propanol as the cosurfactant, and water were prepared. The choice of the compositions of the microemulsions used was based on the pseudoternary phase diagrams of the four-component system determined at 25 °C for different weight ratios of the components. The activity of two oxidizing enzymes that have been detected in virgin olive oil, namely, tyrosinase and peroxidase, and the activity of a proteolytic enzyme such as trypsin were studied in lecithin-based olive oil microemulsions. Lecithin-based olive oil microemulsions were characterized with regard to the phenomenon of electrical percolation. Measurements of the conductivity of W/O microemulsions have indicated the existence of a percolation threshold, which is dependent not only on the water volume fraction but also on other factors such as temperature, rigidity, and thickness of the surfactant monolayer of the system.19,20 By changing these parameters at constant temperature, the percolation threshold may appear at different water volume fractions, thus providing information on the microemulsion’s structure. Electron paramagnetic resonance (EPR) spectroscopy employing the nitroxide spin probe 5-doxylstearic acid (5-DSA; the spin-labeling method) was undertaken as a valuable spectroscopic method to define the dynamic structure of the surfactant monolayer in W/O microemulsions. The influence of the constituents on the fluidity and structure of the microemulsion membrane can thus be estimated. The droplet size and distribution of olive oil microemulsions have been determined from dynamic light scattering (DLS) experiments. Experimental Procedures Materials. Soybean lecithin (Epicuron 200) containing 96% phosphatidylcholine was supplied from Lucas Meyer, Germany. Extravirgin olive oil was provided by Cretan Unions of Agricultural Cooperatives, Crete, Greece. Refined olive oil was generously donated by ELAIS S.A.-Unilever, Greece. Propanol was from Merck, Darmstadt, Germany. Mushroom tyrosinase was purchased from Sigma (2590 units/mg of solid) and used without further purification. Horseradish peroxidase was from Sigma (987 units/mg of solid). Trypsin from bovine pancreas (40 units/mg of solid) was obtained from Merck, Darmstadt, Germany. Oleuropein was from Extrasynthe`se, Genay, France. 2,2-Azinobis(3-ethylbenzthiazoline-6sulfonic acid) diammonium salt (ABTS; 98%) was from Sigma. Hydrogen peroxide (30%) was from Merck. L-Lysine-p-nitroanilide was from Sigma. The spin-labeled fatty acid 5-doxylstearic acid [5-(1-oxyl)-2,2-dimethyloxazolidine] was purchased from Sigma. High-purity water was obtained by a Millipore Milli-Q Plus water purification system. (18) Bianco, A.; Mazzei, R. A.; Melchioni, C.; Scarpati, M. L.; Romeo, G.; Uccella, N. Olea europea. Food Chem. 1998, 62, 343-346. (19) Lagues, M. J. Phys. Lett. 1979, 40, L331-333. (20) Paul, B. K.; Mitra, R. K. Colloids Surf., A 2006, 273, 129-140.
Papadimitriou et al. Pseudoternary Phase Diagrams. Four-component systems can be described with pseudoternary phase diagrams. These diagrams were constructed as follows. Olive oil, either extravirgin or refined, was blended with a mixture of surfactant (lecithin) and cosurfactant (propanol) with a determined weight ratio. In the present study surfactant/cosurfactant weight ratios of 0.3 and 2 were used. The mixture of oil, surfactant, and cosurfactant was then titrated with water until it turned turbid. The volume of water added was recorded. The procedure was repeated several times at different olive oil concentrations. Temperature was kept constant, 25 °C. The pseudoternary phase diagram was constructed by plotting the amounts of water (including the traces of water present in the olive oil and lecithin, as determined by Karl Fischer titrations), surfactant/ cosurfactant, and oil used in the experiment. The microemulsion region (transparent solution) was identified as shown in Figure 1. Preparation of Microemulsions. A typical lecithin-based W/O microemulsion was prepared as follows: A stock solution of lecithin in propanol at constant mass ratio was prepared. Appropriate amounts of oil, either EVOO or ROO, and water or aqueous buffer solution were added. Traces of water present in olive oil and lecithin, as determined by Karl Fischer titration, were taken into consideration. The compositions of the microemulsions used were chosen to correspond to the monophasic area of the pseudoternary phase diagrams of the system determined at 25 °C. Enzymatic Activities in Water-in-Olive Oil Microemulsions. Oxidation of Oleuropein by Mushroom Tyrosinase. Enzymatic oxidation of oleuropein by mushroom tyrosinase was started by adding a few milliliters of a concentrated enzyme solution (30 µg/ mL) to an olive oil/lecithin-propanol (weight ratio 0.4) system containing 1.3 × 10-6 M oleuropein.21 The total water content of the system was adjusted by the addition of the appropriate amount of a buffered solution (0.1 M phosphate buffer, pH 7) and kept constant, 1.2% (w/w), throughout the experiment. The final tyrosinase concentration in the reaction medium was 0.08 µg/mL. Oxidation of ABTS by Horseradish Peroxidase. Enzymatic oxidation of ABTS by horseradish peroxidase was started by adding a few milliliters of a concentrated enzyme solution (100 µg/mL) to an olive oil/lecithin-propanol (weight ratio 0.4) system containing 1.2 × 10-4 M ABTS and 1.1 × 10-4 M H2O2. The total water content of the system was adjusted by the addition of the appropriate amount of a buffered solution (0.2 M acetate buffer, pH 4) and kept constant, 1.2% (w/w), throughout the experiment. The final horseradish peroxidase concentration in the reaction medium was 0.16 µg/mL. Hydrolysis of L-Lysine-p-nitroanilide by Trypsin. The reaction was initiated by adding a few milliliters of a concentrated enzyme solution (130 mg/mL) to an olive oil/lecithin-propanol (weight ratio 0.4) system containing 3.8 × 10-4 M LNA.11,22 The final water content of the system, 1.2% (w/w), was adjusted by addition of the appropriate amount of a buffer solution (0.1 M Tris/HCl, pH 8.5). The final trypsin concentration was 0.35 mg/mL. All substrate and enzyme concentrations are expressed with respect to the total volume of the reaction medium, known as “overall” concentrations.23 All measurements were carried out in duplicate. Enzymatic activities were monitored by means of successive absorption spectra for identification of absorbing species. Absorption spectra were recorded in a UV-vis Hitachi U-200 double-beam spectrophotometer. Temperature was controlled at 25 °C using a Julabo UC-F10 circulating bath. Reference cuvettes contained appropriate blank solutions with all the components except the enzyme, ensuring that the color of olive oil does not interfere with the measurements. Conductivity Measurements. The conductivity measurements were performed with a Metrohm 644 conductometer using a thermostated microcell (25 °C). The cell constant, c, was equal to 0.1 cm-1. Water-in-olive oil microemulsions were prepared as (21) Papadimitriou, V.; Sotiroudis, T. G.; Xenakis, A. J. Am. Oil Chem. Soc. 2005, 82, 335-340. (22) Papadimitriou, V.; Xenakis, A.; Evangelopoulos, A. E. Colloids Surf., B 1993, 1, 295-303. (23) Luisi, P. L.; Magid, L. Crit. ReV. Biochem. 1986, 20, 409-474.
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described above. The final concentrations of lecithin-propanol (weight ratios 0.3 and 2) and olive oil were 30% and 70% (w/w), respectively. Increasing amounts of a Tris/HCl (0.2 M) buffer solution were added to the system to obtain the desired final aqueous contents. EPR Measurements. To obtain the desired concentration of 5-DSA in the lecithin-based olive oil microemulsions, 1 g of each microemulsion was added to a tube into which the appropriate amount of 5-DSA had been deposited previously. This was done by placing 10 µL of a stock 5-DSA solution in ethanol (7.8 × 10-3 M) in the tube and by further evaporating the ethanol.24 EPR measurements were carried out at 25 °C, using a Bruker ER 200D spectrometer operating at the X-band. The spectrometer was equipped with a double rectangular cavity ER 4105 DR, and samples were taken up in 734-PQ-8, thin-wall Suprasil, EPR sample tubes (Wilmad Glass Co., Buena, NJ). Typical instrument settings were as follows: center field, 3470 G; scan range, 100 G; gain, 20000; time constant, 500 ms; modulation amplitude, 1 G; phase, 90°; microwave power, 3.1 mW. Data collection was performed using the computerized program DAT-200 (Data Acquisition Program, University of Luebeck, Germany) and analyzed with the GEP (Graphic Evaluation Program, version 1.2) program for personal computers. Interpretation of EPR Data. The rotational correlation time, τR, of the spin probe was calculated from the EPR spectra using the following relationship:25 τR ) (6 × 10-10)[(h0/h+1)1/2 + (h0/h-1)1/2 - 2]∆H0 (s)
(1)
where ∆H0 is the width (G) of the central peak and h+1, h0, and h-1 are the heights of the peaks from the low to the high field, respectively (Figure 4). From the spectral characteristics we have calculated two more parameters: the order parameter, S, and the isotropic hyperfine splitting constant, R0′. S is defined as26 S ) (A| - A⊥)/[AZZ - (1/2)(AXX + AYY)](R0/R0′)
(2)
where A| corresponds to the half-distance of the outer maximum hyperfine splitting, 2Amax (Figure 4), and A⊥ is calculated from the following equations: A⊥ ) Amin + 1.4(1 - Sapp)
(3)
Sapp ) (Amax - Amin)/[AZZ - (1/2)(AXX + AYY)]
(4)
where Amin is equal to the half-distance of the inner minimum hyperfine splitting (Figure 4). R0 is the isotropic hyperfine splitting constant for the nitroxide molecule in the crystal state: R0 ) (AXX + AYY + AZZ)/3
(5)
R0′ is the isotropic hyperfine splitting constant for the spin probe in the membrane: R0′ ) (A| + 2A⊥)/3
(6)
R0′ values are sensitive to the polarity of the environment of the spin probe and are increased when the polarity of the medium is increased. AXX, AYY, and AZZ are the single-crystal values of the spin probe equal to 6.3, 5.8, and 33.6 G, respectively. Dynamic Light Scattering Measurements. Dynamic light scattering measurements of lecithin-based W/O microemulsions were performed using a Malvern Zetasizer Nano ZS light scattering instrument (Malvern Instruments Ltd., Malvern, Worcester, U.K.) at 25 °C. The instrument contains a 4 mW He-Ne laser operating at a wavelength of 633 nm. Olive oil microemulsions were clarified (24) Avramiotis, S.; Cazianis, C. T.; Xenakis, A. Langmuir 1999, 15, 23752379. (25) Kommaredi, N. S.; O’Connor, K. C.; John, V. T. Biotechnol. Bioeng. 1994, 43, 215. (26) Griffith, O. H.; Jost, P. C. Lipid Spin Labels in Biological Membrane. In Spin Labeling, Theory and Applications; Berliner, L. J., Ed.; Academic Press: New York, 1976; pp 454-484.
Figure 1. Pseudoternary phase diagrams for the four-component systems olive oil/lecithin/propanol/water containing either EVOO (9) or ROO (0). The phase separation lines correspond to initially fixed weight ratios of lecithin/propanol of 2/1 (A) and (B) 1/3. Compositions are in weight ratios. The temperature was 25 °C.
by passing them through 0.22 µm Millipore filters prior to the measurements. The droplet size and polydispersity of the dispersed aqueous phase were evaluated with the aid of Malvern DTS software. The microemulsions tested contained 86% (w/w) oil (EVOO or ROO), 12.7% (w/w) lecithin-propanol (weight ratio 0.3), and water (1.2%, w/w).
Results and Discussion Pseudoternary Phase Diagrams. Phase diagrams of the fourcomponent system olive oil/lecithin/alcohol/water were constructed to determine the extent of the monophasic area corresponding to W/O microemulsions. The pseudoternary phase diagrams of olive oil, either extravirgin or refined, water, and a mixture of lecithin and propanol having weight ratios of 2 and 0.3 is shown in parts A and B, respectively, of Figure 1. As can be observed, the area of the microemulsion zone (one-phase region) increased considerably by increasing the lecithin to propanol weight ratio from 0.3 to 2 in both virgin and refined olive oil based systems. However, the nature of the oil used as the continuous phase does not seem to affect the ability of the oil/surfactant/cosurfactant system to incorporate water. Figure 1 clearly shows that the phase boundaries of the monophasic region are practically unchanged when either EVOO or ROO is considered.
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Figure 2. Enzymatic activities in lecithin-based W/O microemulsions formulated with either EVOO (9) or ROO (0). (A) Evolution of oleuropein oxidation products catalyzed by tyrosinase. Conditions were 0.08 µg/mL tyrosinase, 1.3 × 10-6 M oleuropein, and 0.1 M phosphate buffer, pH 7. (B) Evolution of ABTS oxidation products catalyzed by horseradish peroxidase. Conditions were horseradish 0.16 µg/mL peroxidase, 1.2 × 10-4 M ABTS, 1.1 × 10-4 M H2O2, and 0.2 M acetate buffer, pH 4. (C) Evolution of LNA hydrolysis product catalyzed by trypsin. Conditions were 0.35 mg/mL trypsin, 3.8 × 10-4 M LNA, and 0.1 M Tris/HCl buffer, pH 8.5. (C) The temperature was 25 °C.
Enzymatic Activities in Water-in-Olive Oil Microemulsions. Oxidation of Oleuropein by Mushroom Tyrosinase. The enzymatic activity of tyrosinase toward oleuropein was studied in both EVOO- and ROO-based W/O microemulsions. In the case of EVOO microemulsions, formation of oleuropein oxidation products was followed spectrophotometrically at 415 nm for a few minutes (Figure 2A). The enzymatic activity was linear up to 3 min followed by a quick inactivation. The initial rate of the oxidation reaction was 0.006 ((0.001) ∆A415 min-1. As has been reported recently tyrosinase inactivation in lecithin-based EVOO microemulsions can be due either to the denaturating effect of the micellar system or to a possible inhibitory effect of the quinone products.21,27 When lecithin-based ROO microemulsions were used for the oxidation of oleuropein by tyrosinase, no enzymatic activity was observed. The kinetic behavior of tyrosinase is very complex as the oxidation of oleuropein to the corresponding o-quinones occurs (27) Escribano, J.; Tudela, J.; Garcı´a-Carmona, F.; Garcı´a-Ca´novas, F. Biochem. J. 1989, 262, 597-603.
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simultaneously with coupled nonenzymatic reactions that lead to the formation of stable colored polymer products. In the present system, oleuropein is expected to partition in the lecithin interface while the produced o-quinones are accumulated around the protein molecule and within the amphiphile monolayer, causing the observed quick enzyme inactivation.21 Oxidation of ABTS by Horseradish Peroxidase. The enzymatic activity of horseradish peroxidase toward ABTS was studied in both EVOO- and ROO-based W/O microemulsions. Formation of ABTS oxidation products was followed spectrophotometrically at 414 nm for a few minutes (Figure 2B). The initial rate of the oxidation reaction in the case of EVOO microemulsions was 0.004 ((0.000) ∆A414 min-1. When lecithin-based ROO microemulsions were considered for the oxidation of ABTS by horseradish peroxidase, the enzymatic activity was linear up to 6 min followed by a quick inactivation. The initial rate of the reaction was 0.001 ((0.000) ∆A414 min-1. Previous studies have shown that horseradish peroxidase can be solubilized in the aqueous core of various reverse micellar systems and retain catalytic activity. Both horseradish peroxidase and ABTS are water-soluble molecules and are preferably located within the dispersed aqueous droplets. In the case of EVOO microemulsions, horseradish peroxidase was stable during the experiment, exhibiting a satisfactory enzymatic activity. When ROO was used as the continuous oil phase, the activity of the enzyme was decreased. This behavior indicates an increased “sensitivity” of the enzyme to the structural and chemical characteristics of the microenvironment. Hydrolysis of L-Lysine-p-nitroanilide by Trypsin. To study the effect of the reaction medium on the initial rate of LNA hydrolysis by trypsin, W/O microemulsions based on EVOO and ROO were considered. In both cases formation of p-nitroaniline was followed spectrophotometrically at 382 nm for 15 min (Figure 2C). In the case of EVOO microemulsions the initial rate of LNA hydrolysis was 0.004 ((0.000) ∆A382 min-1. When ROO-based microemulsions were used, the initial rate of the hydrolysis was decreased, 0.001 ((0.000) ∆A382 min-1. Proteolytic enzymes such as trypsin have been extensively studied in reverse micellar systems and proved to be stable and active toward many synthetic substrates. The nature of the surfactants, cosurfactants, and solvents as well as the water content of the system always affected the enzymatic behavior, either increasing or decreasing its activity. The above-mentioned findings concerning the enzymatic activities of tyrosinase, peroxidase, and trypsin, in the environment of olive oil microemulsions, indicate a reduced catalytic activity when ROO was considered as the continuous oil phase. The only known difference between the two oils used in the present study for the formation of W/O microemulsions is the existence of free fatty acids, proteins, and minor components which are considered as natural endogenous amphiphiles and are present only in virgin olive oil. To elucidate the differences in the microstructure between the two systems, namely, EVOO- and ROO-based microemulsions, conductivity, EPR, and DLS structural studies were undertaken. Conductivity. The conductivity of lecithin-based olive oil microemulsions was measured at constant temperature, 25 °C, with increasing weight fractions of water. By using an appropriate buffer solution, 0.2 M Tris/HCl, ions were provided in the water phase, rendering it electrically conducting. Nevertheless, W/O microemulsions exhibit small macroscopic conductivity as the surfactant layer and the continuous oil phase separate the water droplets. However, when the water weight fraction is increased
OliVe Oil Microemulsions
Figure 3. Variation of the conductivity of lecithin-based waterin-olive oil microemulsions as a function of the water content (0.2 M Tris/HCl buffer solution) for different systems: EVOO/lecithinpropanol (weight ratio 2)/buffer (9); ROO/lecithin-propanol (weight ratio 2)/buffer (0); EVOO/lecithin-propanol (weight ratio 0.3)/ buffer (b); ROO/lecithin-propanol (weight ratio 0.3)/buffer (O). The temperature was 25 °C.
beyond a certain critical value, a sharp increase in conductivity can be observed.19 In the present study either EVOO or ROO was considered as the nonpolar solvent for the formulation of lecithin-based W/O microemulsions. The weight ratio of lecithin to propanol was either 0.3 or 2. Figure 3 shows the variation of conductivity as a function of the water content in the case of four different microemulsion systems. When the lecithin-propanol ratio was low, 0.3, water incorporation was limited for both EVOO and ROO systems. As a consequence the conductivity was low and the formation of the infinite cluster was not observed. At a higher lecithin-propanol mass ratio, 2, the droplet concentration is increased. Consequently, the interactions between the droplets are increased and the percolation phenomenon is facilitated. At a water content above 3% (w/w), a sharp increase in conductivity was observed, indicating a structural transition in the bicontinuous form. Endogenous emulsifiers in EVOO include free fatty acids (e.g., oleic, linoleic, palmitoleic, palmitic, and stearic acid) and a variety of minor components such as partial glycerides (monoand diglycerides), polyphenols (e.g., oleuropein and lignans), and phospholipids (e.g., R-phosphatidylcholine, R-phosphatidylinositol, and R-phosphatidylserine).15 Those constituents may interfere with lecithin in the reverse micelle formation, thus affecting the conductivity profile of the microemulsions. As can be seen from Figure 3 no change in the conductivity behavior was observed when EVOO and ROO microemulsions were examined. Natural endogenous amphiphiles present in EVOO do not affect the permeability and rigidity of the surfactant monolayer or the attractive interactions between reverse micelles probably due to their low concentration in the system. Because of their amphiphilic nature, these endogenous compounds are considered to participate in the total interface of the system, although such an effect could not be conductometrically evaluated. EPR Studies. The interfacial properties of the surfactant monolayer in olive oil microemulsions were studied by EPR spectroscopy using the spin-labeling technique. The spin-labeled fatty acid 5-DSA is a long amphiphile molecule having a tendency to align with the surfactant molecules. This interface-located fatty acid probe gives EPR spectra reflecting the mobility of the probe and the rigidity of the environment. To express the mobility of the probe and the rigidity of the interface quantitatively, the
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Figure 4. EPR spectra of 5-DSA in (A) EVOO and (B) ROO microemulsions. The ratio of lecithin to propanol was 0.3, and the water content was 1.2% (w/w) in both systems. The rotational correlation time and order parameter are calculated by the indicated heights and splittings, respectively. Table 1. Rotational Correlation Times, τR, Order Parameters, S, and Isotropic Hyperfine Splitting Constants, r0′, of 5-DSA in EVOO/Lecithin/Propanol/Water Microemulsions at Different Compositions lecithin/ propanol
water content (%, w/w)
τR (ns)
S
a0 (G)
0.4 0.4 0.4 0.4 0.4 0.4 0.1 0.2 0.3 0.4 0.2 0.3 0.4 0.5 0.8 1.0
0.2 0.5 0.6 0.9 1.1 1.4 0.4 0.4 0.4 0.4 0.9 0.9 0.9 0.9 0.9 0.9
3.36 2.93 2.78 2.66 2.38 2.36 2.34 2.52 2.53 2.93 2.07 2.46 2.66 3.18 3.34 3.52
0.20 0.20 0.19 0.18 0.18 0.18 0.17 0.20 0.20 0.20 0.16 0.19 0.19 0.21 0.21 0.21
13.60 13.86 13.81 13.73 13.96 14.12 14.12 13.99 13.93 13.34 14.26 14.00 13.73 13.63 13.54 13.34
rotational correlation time, τR, and the order parameter, S, were calculated from the EPR spectra (Figure 4) using eq 1 and eqs 2-6, respectively. In the present study rotational correlation times, τR, order parameters, S, and the isotropic hyperfine splitting constants, R0′ of 5-DSA in both EVOO- and ROO-based microemulsions at different compositions were calculated and are presented in Tables 1 and 2, respectively. The effect of the water content on the rotational correlation times, τR, of 5-DSA in lecithin-based olive oil microemulsions at constant lecithin/propanol ratio, 0.4, is presented (Tables 1 and 2). The lecithin/propanol weight ratio 0.4 is the same ratio used for the enzymatic activity studies. It is obvious that, as long as the water weight fraction increases from 0% to 1.4 % (w/w), the τR values of 5-DSA decrease, indicating an increase in the mobility of the spin probe. Increases of rotational correlation time reflect a more hindered motion of the probe in the interface. The same tendency was observed in both EVOO- and ROObased systems. The effect of the water content on the order parameter, S, at constant lecithin/propanol weight ratio for both EVOO- and ROObased microemulsions is shown in Tables 1and 2, respectively. When the water content is increased from 0% to 1.4% (w/w),
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Table 2. Rotational Correlation Times, τR, Order Parameters, S, and Isotropic Hyperfine Splitting Constants, r0′ of 5-DSA in ROO/Lecithin/Propanol/Water Microemulsions at Different Compositions lecithin/ propanol
water content (%, w/w)
τR (ns)
S
a0 (G)
0.4 0.4 0.4 0.4 0.4 0.4 0.1 0.2 0.3 0.4 0.2 0.3 0.4 0.5 0.8
0 0.4 0.7 0.9 1.0 1.2 0.4 0.4 0.4 0.4 0.9 0.9 0.9 0.9 0.9
3.26 2.60 2.62 2.89 2.45 2.54 2.51 2.63 2.80 2.83 2.29 2.68 2.89 3.11 3.58
0.20 0.19 0.19 0.19 0.18 0.18 0.16 0.20 0.18 0.18 0.16 0.19 0.19 0.20 0.21
13.82 13.85 13.94 13.95 13.89 14.00 14.10 14.07 13.73 13.73 14.10 13.95 13.95 13.62 13.14
the order parameter is slightly decreased in both systems, indicating a small decrease in the rigidity of the interface. This finding is in agreement with the previously observed increase in the probe’s mobility by adding water to the system. Increases of the order parameter reflect increases in the rigidity of the interface. The order parameter varies from 0 to 1, with S ) 1 for the completely ordered state and S ) 0 for the completely random state.26 From the experimental results mentioned above (Tables 1and 2) it can be concluded that in the presence of increasing amounts of water the interface becomes less structured, rendering faster the motion of the spin probe. The micropolarity of the environment of the spin probe as indicated by the R0′ values (eq 6) is increased upon water addition, ranging from 13.60 G at 0.2% (w/w) to 14.12 G at 1.4% (w/w) in EVOO microemulsions (Table 1). Similar behavior was observed in ROO-based microemulsions. Namely, the R0′ values were increased from 13.82 G at 0% to 14.00 G at 1.2% (w/w) water content (Table 2). The interfacial properties of lecithin and bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) based W/O microemulsions have been studied previously using the spin-probing technique.24,28 Namely, the mobility of 5-DSA was studied in lecithin/isooctane/ propanol and AOT/isooctane W/O microemulsions. The behavior of 5-DSA at increasing water contents was examined. In those systems as the water content increased, the mobility of 5-DSA decreased, indicating the formation of curved entities with an aqueous core of reverse micellar type. The greater the amount of water added to the system, the less curved the lecithin layer, thus hindering the mobility of the probe. It has to be mentioned that the water content in those microemulsion systems was far larger than the water content obtained in olive oil microemulsions of the present work. In lecithin-based olive oil microemulsions water does not seem to behave like bulk water. The water molecules added to the system probably serve to hydrate the head groups of the surfactants, thus reducing lecithin packing and consequently the rigidity of the interface. The variation of the calculated correlation times as a function of the lecithin/propanol weight ratio at low (0.4%, w/w) and higher (0.8%, w/w) water contents, in both EVOO- and ROObased microemulsions, is also presented (Tables 1and 2). By increasing the surfactant/cosurfactant weight ratio from 0.1 to (28) Avramiotis, S.; Cazianis, C. T.; Xenakis, A. Prog. Colloid Polym. Sci. 2000, 115, 196-200.
0.4 in the case of the low water content, the rotational correlation time of the probe is increased, indicating a decrease of the mobility of the nitroxide ring of the 5-DSA. The same tendency was also observed at higher water content. The rotational correlation time of the probe is increased by increasing the surfactant/cosurfactant weight ratio from 0.2 to 1, indicating a decrease of the mobility of the probe. The effect of the lecithin/propanol weight ratio on the order parameter at low and higher water contents is also presented (Tables 1 and 2). By increasing this ratio from 0.1 to 0.4, at low water content, the order parameter values are increased in both EVOO- and ROO-based microemulsions. At a higher water content of the system, the same profile is observed upon a lecithin/ propanol weight ratio increase. From the experimental results mentioned above (Tables 1and 2) it can be concluded that in the presence of increasing amounts of lecithin the interface becomes more structured, rendering slower the motion of the spin probe. This finding is in agreement with the previously observed increase in the probe’s mobility as expressed by the τR values in both EVOO and ROO microemulsions upon a lecithin increase. When the lecithin/propanol ratio of the olive oil microemulsions is varied, the micropolarity of the probe’s environment is also varied. The R0′ values are decreased from 14.12 to 13.34 G in EVOO microemulsions (Table 1) and from 14.10 to 13.73 G in ROO microemulsions (Table 2) upon an alcohol decrease (lecithin/propanol ratios from 0.1 to 0.4) at low water content. The same behavior was also observed at higher water contents. The polarity is decreased from 14.26 to 13.34 G in EVOO microemulsions (Table 1) and from 14.10 to 13.14 G in ROO microemulsions (Table 2) upon an alcohol decrease (lecithin/ propanol ratios from 0.2 to 1). By adding more propanol to lecithin-based microemulsions, the polarity of the water is increased, rendering the dispersed phase less hydrophilic and consequently the curvature of the lecithin monolayer more pronounced.9 This can be confirmed by the results of Tables 1 and 2, showing the variation of the calculated isotropic hyperfine splitting constants as a function of the lecithin/propanol weight ratio. The doxyl stearic acids are well-known membrane probes since they are preferably located in the lecithin interface, between the oriented lecithin molecules, rather than in the aqueous or the oil continuous phase. The spin probe’s mobility is hindered from the presence of the neighbor lecithin hydrophobic tails. As a consequence the mobility of 5-DSA is reduced upon lecithin addition to the system at constant water concentrations. At the same time, by increasing the lecithin content of the system, the rigidity of the micellar interface is increased and the polarity of the environment is decreased. The same behavior was observed in both EVOO and ROO W/O microemulsions. The presence of natural endogenous amphiphiles in EVOO microemulsions does not affect the interfacial properties of the systems at least as they can be estimated by using the spin-labeling technique. It is interesting to compare the rotational correlation time, τR, of the probe in both EVOO and ROO microemulsions at constant water content and also constant lecithin/propanol weight ratio (Tables 1 and 2). The τR values of 5-DSA are in general higher in ROO microemulsions in comparison to the τR values in EVOO microemulsions. This increase in τR values can be possibly explained by the formation of larger aqueous droplets when ROO is considered as the continuous nonpolar phase. According to the literature, when lecithin-based W/O microemulsions are considered, the more swelled the dispersed aqueous structures, the
OliVe Oil Microemulsions
less curved the lecithin monolayer, thus hindering the mobility of the spin probe.24,28 To conclude, by varying the weight ratio of lecithin to propanol in the microemulsions, the mobility of the probe and the rigidity of the interface are altered. The greater the amount of lecithin contained in the system, the more immobilized the probe and thus more hindered its motion. At the same time, the polarity of the spin probe’s microenvironment is increased upon propanol addition, indicating some incorporation of alcohol in the polar parts of the lipid layer. When the water content of the system is low (
