Crystallization of Organic Compounds in Reversed Micelles. III

While the aspartame solubility in the water pools was comparable in all the investigated MEs, the aspartame/surfactant molar ratio at the w/o interfac...
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Crystallization of Organic Compounds in Reversed Micelles. III. Solubilization of Aspartame Helga Fu¨redi-Milhofer,* Alexey Kamishny, Junko Yano,† Abraham Aserin, and Nissim Garti Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received December 1, 2002. In Final Form: April 2, 2003 The artificial sweetener, aspartame, is a dipeptide, consisting of a hydrophobic phenylalanine methyl ester entity and a hydrophilic aspartyl residue. In this work, its solubilization in five different types of water in oil (w/o) microemulsions (MEs) was investigated. The stabilizing surfactants and cosurfactants of the MEs were ME 1, sodium di-2-ethylhexyl sulfosuccinate (AOT) and no cosurfactant; ME 2, maleic anhydride R-olefin copolymer and dimethyl amino ethanol; ME 3, sucrose ester monostearate and n-butanol; ME 4, L-R-phosphatidylcholine and n-butanol; and ME 5, mono- and diglycerides of fatty acids and L-Rphosphatidylcholine. The maximum amount that could be dissolved (boundary concentration) was determined by adding powdered aspartame to a heated ME and cooling to a specified temperature under controlled conditions. The solutions that remained clear for at least 4 days were regarded as stable, and those of the stable solutions with the highest aspartame concentrations were taken as having the boundary concentrations. From the solubility data, the distribution of the aspartame molecules between the w/o interface and the water pools was calculated, and the results were correlated with the molecular structure and ionic state of the surfactant. The results show that aspartame can be solubilized in all the investigated MEs to an extent, exceeding by far its solubility in pure water, and that overall solubilization is most efficient in water/isooctane MEs stabilized with AOT (ME 1). While the aspartame solubility in the water pools was comparable in all the investigated MEs, the aspartame/surfactant molar ratio at the w/o interface was found to decrease with decreasing polarity of the stabilizing surfactant at the interface. In addition to the solubilization studies, the effect of aspartame on some properties of ME 1 was investigated. It was found that aspartame lowers the interfacial tension at the water/isooctane/AOT interface and, under certain conditions, stabilizes unstable mixtures of water, isooctane, and AOT. The results of small-angle X-ray scattering measurements show that the ME droplet sizes increase in the presence of aspartame molecules and their shapes change from near spherical to ellipsoidal. It was concluded that aspartame acts as a cosurfactant in water/isooctane MEs stabilized with AOT.

1. Introduction Reversed micelles [water in oil (w/o) microemulsions (MEs)] are thermodynamically stable, macroscopically homogeneous dispersions of nanosized domains of water in an apolar liquid (oil phase). The droplets are encased in and stabilized by a shell, consisting of a monolayer of surfactant, or, more frequently, a surfactant and cosurfactant.1-3 The w/o MEs found numerous applications based on their stability, nanosized water pools, and capacity to solubilize slightly soluble compounds. Some interesting applications include4 tertiary oil recovery, lubrication with corrosion inhibition, detergency, dye solubilization, drug delivery systems,5-7 and synthesis of small particles and inorganic clusters for applications in optoelectronics, photosynthesis, the preparation of semiconductors, and catalysis.8-10 Applications of w/o MEs in * Corresponding author. E-mail: [email protected]. † Present address: Lawrence Berkley National Laboratory, Berkley, U.S.A. (1) Overbeek, J. T. G. Faraday Discuss. Chem. Soc. 1978, 65, 7. (2) Luisi, P. L., Straub, B. E., Eds. Reverse Micelles; Plenum Press: New York, 1984. (3) Auvray, L. In Micelles, Membranes, Microemulsions and Monolayers; Gelbart, W. M., Ben Shaul, A., Roux, D., Eds.; Springer: Berlin, 1994; pp 347-393. (4) Langevin, D. In ref 2, pp 287-303. (5) Corswant, C.; Thoren, P. E. G. Langmuir 1999, 15, 3710. (6) Corswant, C.; So¨derman, O. Langmuir 1998, 14, 3506. (7) Kawakami, K.; Yoshikawa, T.; Moroto, Y.; Kanaoka, E.; Takahashi, K.; Nishihara, Y.; Masuda, K. J. Controlled Release 2002, 81, 65. (8) Pileni, M. P., Ed. Structure and Reactivity in Reverse Micelles; Elsevier: Amsterdam, 1989.

the food industry are very important, such as the delivery of water-soluble nutrients, flavors, and flavor enhancers to lipid-based foods.11 In recent years, it has been shown that these systems can also be used for the control of the crystallization of organic compounds, including the control of the crystallizing polymorph.12-14 In most of the above applications, knowledge of the solubilization of the specific organic compounds in reversed micelles is indispensable. There has been a particular interest in the solubilization of proteins,15-19 polypeptides,20 and amino acids21-25 in (9) Fendler, J. H. Membrane Mimetic Approach to Advanced Materials. Advances in Polymer Science 113; Springer: Berlin, 1994. (10) Sager, W. F. C. Curr. Opin. Colloid Interface Sci. 1998, 3, 276. (11) El-Nokaly, M.; Hiler, G., Sr.; McGrady, J. In Microemulsions and Emulsions in Food; El-Nokaly, M., Cornell, D., Eds.; ACS Symposium Series 448; American Chemical Society: Washington, DC, 1991. (12) Yano, J.; Fu¨redi-Milhofer, H.; Wachtel, E.; Garti, N. Langmuir 2000, 16, 10005. (13) Fu¨redi-Milhofer, H.; Garti, N.; Kamishny, A. J. Cryst. Growth 1999, 198/199, 1365. (14) Fu¨redi-Milhofer, H.; Garti, N.; Kamishny, A. Aspartame Crystals and Process of the Preparation Thereof. U.S. Patent 6,294,686 B1, Sept 25, 2001. (15) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209. (16) Leser, M. E.; Luisi, P. L. Chimia 1990, 44, 270. (17) Garti, N.; Lichtenberg, D.; Silberstein, T. Colloids Surf., A 1997, 128, 17. (18) Kawakami, L. E.; Dungan, S. R. Langmuir 1996, 12, 4073. (19) Sun, Y.; Gu, L.; Tong, X. D.; Bai, S.; Ichikawa, S.; Furusaki, S. Biotechnol. Prog. 1999, 15, 506. (20) Gierasch, L. M.; Thompson, K. F.; Lacy, J. E.; Rockwell, A. L. In Reverse Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum Press: New York, 1984; pp 265-277.

10.1021/la026933l CCC: $25.00 © 2003 American Chemical Society Published on Web 06/18/2003

Aspartame Chart 1. Molecular Structure of Aspartame, N-L-r-Aspartyl-L-phenylalanine Methyl Ester

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studies of the solubilization of other amphiphilic dipeptides in w/o MEs. Because aspartame is a food ingredient and many other water-soluble food ingredients also contain amino acids and peptides, it was of interest to study the solubilization of aspartame in w/o MEs stabilized with food-grade surfactants. For this purpose, we developed a simple method that enables us to calculate the distribution of the solute between the water pools and the w/o interface of the ME droplets from solubility data. We then studied the solubilization of the dipeptide in a set of MEs, most of which were stabilized with food-grade surfactants of different polarities, and compared the results with those obtained in AOT-stabilized w/o MEs. We have shown that while interfacial association strongly depends on the polarity of the stabilizing surfactant, solubilization in the water pools was more or less independent of the surfactant but about six times higher than that in an equivalent amount of bulk water.

w/o MEs because of its importance in the understanding of processes at biological membranes and biotechnological applications, such as enzyme catalysis, protein separation, and delivery of water-soluble food ingredients to lipidbased foods. Several interesting studies on the solubilization of amino acids in w/o MEs, stabilized with sodium di-2-ethylhexyl sulfosuccinate (AOT), are available.21-25 It has been shown that the location of the guest molecules within the ME depends on their hydrophobicities.21,23,25 Thus, hydrophilic amino acids are solubilized within the water pools, whereas hydrophobic amino acids are distributed between the water pools and the w/o interface, the degree of association at the interface depending on the hydrophobicity of the particular molecule. Several authors used phenylalanine as a model to probe the action of hydrophobic amino acids as cosurfactants.22,24,25 Interfacial association has been suggested on the basis of evidence of indirect analysis22 and was later confirmed by smallangle X-ray scattering (SAXS) measurements,25 which show that the molecule profoundly influences the size and shape of the ME droplets. The addition of alcohols to the micellar phase has no influence on the phenylalanine partitioning between the micellar and the aqueous phases but changes the rate of incorporation of the hydrophobic amino acid molecules.24 It appears that the most important studies on the solubilization of proteins and amino acids have been carried out in w/o MEs stabilized with AOT, whereas only scarce information is available on the solubilization of amino acids or peptides in MEs stabilized with food-grade surfactants, which are mostly nonionic (such as sucrose esters) or zwitterionic (such as phosphatidylcholine). Aspartame (N-L-R-aspartyl-L-phenylalanine methyl ester) is widely used as an artificial sweetener because of its high sweetening power (150-200 times sweeter than sucrose), no aftertaste, and relatively good compatibility with human consumption.26 However, there are problems associated with its use, such as poor dissolution kinetics in aqueous systems and unfavorable morphological characteristics of its crystals. We have recently developed new crystallization procedures involving the solubilization and recrystallization of aspartame from w/o MEs. These procedures yielded new crystal forms of aspartame, one of them with significantly improved dissolution kinetics.13,14 In view of this interesting result and because of the above documented interest in the solubilization of proteins and amino acids, the solubilization of aspartame in w/o MEs appeared to be of special interest. As is shown in Chart 1, the aspartame molecule is a dipeptide, composed of a highly hydrophilic aspartyl residue and a hydrophobic phenylalanine methyl ester entity.26 As such, the molecule could be a useful model for

2.1. Materials. Commercial, crystalline, and powdered aspartame, obtained courtesy of Nutrasweet Co., Switzerland, was used for all experiments. Phenylalanine was purchased from Sigma-Aldrich and was used as received. The following commercially available surfactants, cosurfactants, and oils were used as received to form MEs. (i) Surfactants: AOT from American Cyanamid or Sigma; Ketjenlube 522 (DAPRAL GE 202, maleic anhydride R-olefin copolymer) from Akzo Nobel Chemicals; Atmos 300 (mono- and diglycerides of fatty acids) from ICI Americas, Inc.; sucrose ester monostearate (SMS 1570) from Mitsubishi-Kagaku Food Corp.; and soybean L-R-lecithin (phosphatidylcholine) from Avanti Polar Lipids, Inc. (ii) Cosurfactants: dimethyl amino ethanol and n-butanol from Sigma-Aldrich. (iii) Oil phases: isooctane and tricapryline from SigmaAldrich; toluene from Frutarom; soybean oil from Shemen Oils Co.; and Neobee M5 (caprylic-capric triglyceride) from Stepan Europe. 2.2. Solubilization Measurements. Five different types of w/o MEs of different compositions were prepared from the ingredients listed in Table 1. The compositions were determined according to the literature data.17,27-32 The MEs were prepared from mixtures of water and the organic component (oil) in which the respective surfactant was dissolved. The ingredients were mixed by vortexing until clear, stable solutions were obtained. The maximum amount of aspartame, which can be solubilized at a specified temperature (boundary concentration), was then determined as follows: The MEs were heated to 65 °C, and different amounts of powdered aspartame were dissolved under constant stirring and sonication. Then the solutions were cooled at the rate of 1/2 °C/min to a predetermined temperature (25, 37, or 45 °C, as is indicated in the results section), and the time necessary for crystallization was observed. Solutions that remained clear for at least 4 days were regarded as stable solutions, and those with the highest concentration of aspartame were taken as the solutions having the boundary concentrations. In one set of experiments (Figure 1), powdered aspartame was dissolved without prior heating in water/isooctane/AOT MEs (ME 1 in Table 1) prepared at room temperature. 2.3. Interfacial Tension Measurements. AOT was dissolved in isooctane and carefully spread over aqueous solutions con-

(21) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6400; 1990, 94, 6411. (22) Leodidis, E. B.; Bommarius, S.; Hatton, T. A. J. Phys. Chem. 1991, 95, 5943. Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1991, 95, 5957 (cosurfactants). (23) Adachi, M.; Harada, M.; Shioi, A.; Sato, Y. J. Phys. Chem. 1991, 95, 7925. (24) Plucinski, P.; Reitmer, J. Colloids Surf., A 1995, 97, 157. (25) Yano, J.; Fu¨redi-Milhofer, H.; Wachtel, E.; Garti, N. Langmuir 2000, 16, 9996. (26) Peterson, M. S., Johnson, A. H., Eds. Encyclopedia of Food Science; Avi Publishing: Westport, CT, 1978; Vol. 3, p 720.

(27) Sager, W. F. C. Langmuir 1998, 14, 6385. (28) Gans, O. Self-assembled microemulsions stabilized by doublecomb-block copolymers. M.S. Thesis, The Hebrew University of Jerusalem, 1999. (29) Maltesh, C.; Xu, Qun; Somasundaran, P.; Benton, W. J.; Nguyen, H. Langmuir 1992, 8, 1511. (30) Fanun, M.; Wachtel, E.; Antalek, B.; Aserin, A.; Garti, N. Colloids Surf., A 2001, 180, 173. (31) Garti, N.; Clement, V.; Fanun, M.; Leser, M. E. J. Agric. Food Chem. 2000, 48, 3945. (32) Geyer, R. P.; Tuliani, V. Nonaqueous Microemulsions for Drug Delivery; U.S. Patent 5,110,606, 1992.

2. Materials and Methods

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Table 1. Ingredients of w/o MEs Employed for the Solubilization of Aspartamea ME

oil phase

surfactant

cosurfactant

reference

1 2 3 4 5

isooctane toluene caprylic-capric triglyceride tricaprilyn soybean oil

AOT Ketjenlube 522; average MW, 20 000 SMS 1570; average MW, 519 L-R-phosphatidylcholine Atmos 300

dimethyl amino ethanol n-butanol n-butanol L-R-phosphatidylcholine

25, 27 28, 29 30, 31 17 32

a

The conditions of preparation were according to the phase diagrams given in the references listed in column 5.

of a ln I(q) versus q2 plot, where q and I(q) are the amplitude of the scattering vector and the scattering intensity. In the Glatter analysis, the pair distance distribution function P(r) was calculated from

∫

P(r) ) (1/2π2) I(q)qr sin(qr) dq

(1)

where r is a distance in real space. This function gives the frequency of the occurrence of vectors of length r, weighted by the electron density at either end of the vector. Then, Rg was obtained from

Rg2 )

∫

∞

0

∫ P(r) dr

P(r)r2 dr/2

∞

0

(2)

When a monodisperse population of cylindrical or ellipsoidal droplets was assumed, the shape parameters were calculated from the relationship Rg2 ) (2a2 + c2)/5, where a is the semiminor axis and c ) Dmax/2 is the semimajor axis. 2.6. Treatment of Solubility Data. The solubility of aspartame in water was calculated from the empirical eq 3:35

S ) 4.36 × 100.017T

Figure 1. Solubilization of aspartame (mg/g of ME) in water/ isooctane/AOT MEs (ME 1 in Table 1) at room temperature: (a) constant concentration (wt %) of AOT and (b) constant concentration (wt %) of water. In part a, the dissolved amount of aspartame in equivalent amounts of pure water is indicated by the dotted line. In part b, the dotted lines 1 and 2 represent the solubilities of aspartame in the amount of pure water equivalent to that in the MEs containing 25 and 2.5 wt % water, respectively. taining various amounts of dissolved aspartame. The interfacial tension of the system was measured after 15 min using a MGW Lauda tensiometer. 2.4. Thermal Stabilization of Water/Isooctane/AOT MEs. A ME with a composition of 19.7 wt % aqueous phase, 70.3 wt % isooctane, and 10.0 wt % AOT, containing different concentrations of aspartame (0-6 g/L), was prepared: aspartame was dissolved in water at room temperature, whereas the required amount of AOT was dissolved in isooctane. The components were then mixed to obtain the above ME composition and cooled to determine the temperature range within which a stable ME could be obtained. Without aspartame, the ME with the above composition was unstable at all temperatures. 2.5. SAXS Measurements. Water/isooctane/AOT MEs loaded with phenylalanine or aspartame as well as controls without the additive were prepared and examined by SAXS [Elliot rotatinganode X-ray generator (1.2 kW) monochromated by a Ni filter and 20-cm Franks mirror; performed at the Faculty of Chemistry, the Weizmann Institute of Science, Rehovot, Israel]. The samples, sealed in a 1.5-mm diameter glass capillary, were inserted into a copper sample holder, and the scattering profiles were measured by a linear position-sensitive detector (48-cm distance) at 25 °C. The scattering from isooctane was subtracted as background from the scattering of the respective ME. Data were analyzed by the Guinier and Fournet method33 and the indirect transformation procedure proposed by Glatter.34 In the Guinier and Fournet analysis, the radius of gyration, Rg, was obtained from the slope

(3)

where S is the solubility in g/L and T is the temperature. From the maximum amount of aspartame that could be solubilized in a ME under a given set of experimental conditions, it was possible to assess the distribution of the dipeptide between the w/o interface and the water pools. The total number of moles of aspartame, nasp(tot), is distributed according to eq 4:

nasp(tot) ) nasp(int) + nasp(w)

(4)

The quantity nasp(w) depends on the size of the water pools, which is determined by8

W ) nw/nsurfactant

(5)

In eqs 4 and 5, nasp(int) and nasp(w) are the numbers of moles of aspartame at the w/o interface and in the water pools, respectively, while nw and nsurfactant are the numbers of moles of water and surfactant in the ME. For relatively large water pools, that is, W g 10, nasp(w) is related to the solubility of aspartame in free water and, at a given temperature, should increase linearly with W. Thus, by plotting the nasp(tot)/nsurfactant ratio, Y, as a function of W, we expect a linear relationship:

Y ) Yo + KW

(6)

Extrapolating to W ) 0, we obtain Yo, the aspartame/surfactant molar ratio at the w/o interface, and the slope of the straight line gives the apparent solubility of aspartame in the water pools:

K ) (Y - Yo)/W ) nasp/nw

(7)

At W < 10, the curve deviates from linearity because a large part of the water is bound at the w/o interface.36,37 (33) Guinier, A.; Fournet, G. Small Angle Scattering of X-rays; Wiley: New York, 1955. (34) Glatter, O. In Neutron, X-ray and Light Scattering; Lindner, P., Zemb, T., Eds.; Elsevier Science: New York, 1991; pp 33-60. (35) Kishimoto, S.; Naruse, M. J. Chem. Technol. Biotechnol. 1988, 43, 71. (36) Schulz, P. C. J. Therm. Anal. 1998, 51, 135.

Aspartame

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Figure 3. Solubilization of aspartame in all the MEs listed in Table 1 at 45 °C. The molar nasp(tot)/nsurfactant ratio is plotted as a function of W (as is defined in eq 5). The respective MEs are denoted by the different symbols, as is indicated. The line represents the water/isooctane/AOT MEs.

Figure 2. Solubilization of aspartame (mg/g of ME) in water/ caprylic-capric triglyceride MEs stabilized with SMS 1570 as the surfactant and n-butanol as the cosurfactant (ME 3 in Table 1), at a temperature of 37 °C: (a) constant concentration (25 wt %) of surfactant and (b) constant concentration (15 wt %) of water. In part a, the dissolved amount of aspartame in equivalent amounts of pure water is indicated by the dotted line. In part b, the dotted line represents the solubility of aspartame in the amount of water equivalent to that in the ME containing 15 wt % water.

3. Results 3.1. Solubilization of Aspartame in the MEs. The maximum amount of aspartame that could be solubilized in a set of water/isooctane/AOT MEs, (ME 1 in Table 1), containing different concentrations of AOT, is plotted in parts a and b of Figure 1 as a function of the concentration of water and AOT, respectively. Parts a and b of Figure 2 represent similar data for a nonionic ME (ME 3 in Table 1). It is seen that under all conditions the amount of aspartame that can be dissolved in equivalent amounts of pure water [as was calculated from eq 3 and is represented by the dotted lines] is significantly lower than what could be solubilized in the MEs. Apart from this similarity, an obvious difference between the two systems is apparent: whereas the solubilization in ME 1 is practically independent of the amount of water in the ME (Figure 1a) but sharply increases with the surfactant concentration (Figure 1b), the solubilization in ME 3 increased with both the surfactant and the water concentrations (Figure 2a,b). The solubilizations of aspartame in all the investigated MEs (Table 1) have been compared in Figure 3 by plotting the dependence of the aspartame/surfactant molar ratio, nasp(tot)/nsurfactant, on W, the water/surfactant molar ratio, nw/nsurfactant. We then fitted a straight line to each set of data and calculated the respective intercepts, Yo, and slopes, K, according to eqs 6 and 7. In Table 2, the results are correlated to the molecular structures and ionic states of the surfactants stabilizing the MEs. It is seen from (37) Garti, N.; Aserin, A.; Tiunova, I.; Ezrahi, S. J. Therm. Anal. 1998, 51, 63.

Figure 4. Influence of the temperature and pH on the solubilization of aspartame in water/isooctane/AOT MEs. The molar ratio nasp(tot)/nAOT is plotted as a function of W (as is defined in eq 5). The temperature was 45 °C for line 1 and the squares and 25 °C for line 2 and the circles. The pH of the aqueous phase was 5-6 (squares and circles), 2.6 (triangles), and 8.1 (stars). The respective calculated straight lines are y1 ) 0.00853x + 0.239 (R ) 0.986) and y2 ) 0.00297x + 0.163 (R ) 0.994) for 45 and 25 °C, respectively.

Figure 3 that the overall efficiencies of the investigated MEs in solubilizing aspartame are quite different, with ME 1 (as is defined in Table 1) being by far the most efficient one. It also appears (Table 2) that the differences in interactions of the aspartame molecules with the respective w/o interfaces (i.e., differences in Yo) are far more pronounced than the differences in the apparent solubilities in the water pools of the MEs (K values). It should be noted, however, that the solubility of aspartame in the water pools of any of the investigated w/o MEs is significantly higher than its solubility in pure water (the corresponding value for K is 1.5 × 10-3, as was calculated from eq 3). 3.2. Influence of Solubilized Aspartame on the Properties of the Water/Isooctane/AOT MEs. Because water/isooctane/AOT MEs (ME 1 in Table 1) were highly efficient in solubilizing aspartame, it was of interest to further investigate the underlying interactions. Figure 4 shows the influence of the pH and temperature on the nasp(tot)/nAOT versus W curve. It is seen that although the solubilization is significantly affected by the change in temperature, changing the pH of the water pools to 2.6 or 8 has a much smaller effect only at relatively large water-pool sizes (W g 15). From the respective Yo values obtained at a neutral pH (0.237 and 0.163), the AOT/ aspartame molar ratio at the water/isooctane interface was found to be 6.15 at 25 °C and 4.2 at 45 °C.

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Table 2. Values of Yo and Apparent Solubility, K, in the Water Pools of the MEs (eqs 6 and 7) Correlated to the Molecular Structures and Ionic States of the Surfactants Used to Stabilize the Respective MEsa

a

The MEs are numbered according to Table 1; T ) 45 °C. Table 3. ME Droplet Sizes Obtained by SAXSa MEs purea phenylalaninec aspartamed

Figure 5. Changes of the interfacial tension at the water/ aspartame/isooctane/AOT interface as a function of the concentration of aspartame.

In another experiment (Figure 5), we measured the interfacial tension at a water/isooctane/AOT interface. Clearly, the interfacial tension sharply dropped upon the addition of aspartame to the aqueous phase and further decreased with increasing aspartame concentration. The ability of aspartame to contribute to the stability of the MEs, that is, to act as a cosurfactant, is demonstrated in Figure 6, which shows that a ME composition of water, isooctane, and AOT that is normally unstable at all temperatures can be stabilized within a certain temperature range (between 5 and 15 °C) by introducing

P(r) shape factorsb (nm) W [H2O]/[AOT] Rg (nm) Dmax (nm) a c c/a 4.9 12.2 4.9 12.2 4.9 12.2

1.45 2.28 1.55 2.44 1.90 3.00

4.2 6.4 4.6 6.7 6.1 9.5

1.75 2.81 1.75 3.00 2.09 3.35

2.10 3.20 2.30 3.35 3.05 4.75

1.20 1.14 1.31 1.12 1.46 1.42

a The data were measured from the MEs containing 0.04 mol of AOT. The Rg and Dmax values were obtained from P(r). The Rg values include an error of (0.1 nm. b The semiminor and semimajor axes of the ellipsoidal droplets are represented by a and c and c/a is the semimajor to semiminor axis ratio. c Data is from ref 25. d TheMEs contain the maximum amount of solubilized aspartame.

aspartame. The temperature range of the stability depended on the aspartame concentration (Figure 6). Finally, we determined the influence of solubilized aspartame on the sizes and shapes of the ME droplets in water/isooctane/AOT MEs. In Figure 7 and Table 3, the results of the SAXS measurements characterizing the MEs of two different compositions (W ) 4.9 and 12.2), loaded with aspartame, are compared with the results obtained from the MEs of the same compositions loaded with phenylalanine and controls without solubilizate. The radius of gyration, Rg, was obtained from the pair distance distribution function P(r) (eqs 1 and 2). The shape

Aspartame

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Figure 6. Temperature range of the stable water/isooctane/ AOT MEs as a function of the concentration of aspartame dissolved in the aqueous phase. Without aspartame, the ME with a composition of 19.7 wt % water, 70.3 wt % isooctane, and 10.0 wt % AOT prepared in this experiment is unstable at all temperatures.

Figure 7. Pair distance distribution, P(r), of (1) pure, (2) phenylalanine-loaded, and (3) aspartame-loaded MEs (W ) 4.9, 12.2). All the measurements were carried out at 2 wt % AOT.

of the P(r) curve reflects the forms of the ME droplets. The asymmetric shapes of the P(r) curves in Figure 7 indicate ellipsoidal droplet shapes in the water/isooctane/AOT MEs. In the presence of phenylalanine and aspartame, the P(r) curves shifted to larger dimensions and the symmetry was further distorted, indicating more elongated shapes of the ME droplets. These effects were most significant when the MEs were loaded with aspartame (curves 3 in Figure 7). 4. Discussion The above results show that aspartame can be solubilized in all the investigated MEs to an extent exceeding by far its solubility in water. The solubilized guest molecules are distributed between the aqueous phase and the w/o interface, as is shown in Table 2. It is seen that the aspartame/surfactant molar ratios at the w/o interfaces of the different MEs (Yo) are significantly different,

whereas the apparent solubilities within the water pools (K) are comparable for all the MEs but about 4-5 times higher than the solubility in pure water. From the reciprocal values of Yo, (Yo values given in Table 2) indicating the number of surfactant molecules per molecule of aspartame, we conclude that the contribution of the aspartame molecules at the w/o interfaces in MEs 1-3 is quite significant (i.e., the number of surfactant molecules per molecule of aspartame is 4.2, 9, and 10 for MEs 1, 2, and 3, respectively), whereas it decreases sharply for MEs 4 and 5 (19.2 and 41.6 molecules of surfactant per molecule of aspartame, respectively). Clearly, the phenomenon is related to the driving force for interfacial association, which in turn is related to the structure of the w/o interface and, thus, to the molecular structure and ionic state of the surfactant and cosurfactant, stabilizing the respective ME (columns 2 and 3 in Table 2 and column 4 in Table 1). To better understand the underlying mechanisms, we will first consider the most efficient ME, ME 1, in detail and then discuss the Yo and K values obtained from all the other MEs in correlation with the above parameters. When considering the solubilization of aspartame in water/isooctane MEs stabilized by AOT (ME 1), an obvious question that comes to mind is whether changing the charge of the aspartame molecule will change its solubilization behavior. Because aspartame is zwitterionic at the aspartyl end, the isoelectric pH being 5.2,26 in aqueous solution (pH ≈ 6) the molecule would be slightly negative, whereas changing the pH of the environment to 2 and 8 will change its charge to positive and negative, respectively. Figure 4 shows in both cases a slight decrease in the solubility of aspartame, provided the water pools are large (i.e., at W g 15). However, at small water-pool sizes (W < 15) the effects of the pH changes are negligible, indicating that the driving force for interfacial association is related to the tendency of the hydrophobic phenylalanine end of the dipeptide to “escape” the polar environment rather than some specific or charge interactions at the w/o interface. This interpretation is in accordance with previous findings by our group25 and Leodidis and Hatton21 concerning the solubilization of phenylalanine in water/ isooctane/AOT MEs. Next to consider is the available space to fit a relatively large molecule, such as aspartame, between the surfactant molecules at the w/o interface. In this respect, the water/ isooctane/AOT ME is particularly convenient because it does not require a cosurfactant (see Table 1). Thus, it should be possible for the aspartame molecules to fill that role, that is, intersperse between the AOT molecules and act as a cosurfactant. There are several indications that this seems, indeed, to be the case. It has been demonstrated in this work that (i) aspartame is effective in reducing the interfacial tension at the water/isooctane/AOT interface (Figure 5); (ii) the molecule can effectively stabilize MEs that are otherwise unstable mixtures of water, isooctane, and AOT, albeit in a limited temperature range (Figure 6); (iii) the AOT/aspartame molar ratios at the w/o interface are relatively small and do not deviate by more than 2-5% from round numbers (6.15 and 4.2 at 25 and 45 °C, respectively, as were calculated from the respective Yo values (Figure 4 and Table 2); and (iv) as was shown in the preceding paper25 hydrophobic amino acids such as phenylalanine, which are located primarily in the w/o interfacial area, profoundly affect the sizes and shapes of the water/isooctane/AOT ME droplets. The dipeptide aspartame has an even stronger effect in that respect

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(Figure 7), which is consistent with the assumption that these molecules act as cosurfactants in water/isooctane/ AOT MEs. Next, applying the principles outlined above, we discuss the efficiency of the interfacial interactions of aspartame in all the other investigated MEs. The stabilizing surfactants in these MEs are ionic (ME 2), zwitterionic (ME 4), and nonionic with high (ME 3) and low (ME 5) HLB (hydrophilic/lipophilic balance) values (Table 2). As was expected, the aspartame/surfactant molar ratio at the w/o interface, Yo, which is a measure for the intensity of the interfacial association, decreases with decreasing polarity of the environment (Table 2). Upon first glance, it appears surprising that ME 2 (ionic surfactant) and ME 3 (nonionic surfactant) have almost the same capacity of interfacial association with aspartame (as was evidenced by similar values of Yo in Table 2). To understand this result, we have to consider the molecular structure of Ketjenlube 522 (which is the stabilizing surfactant in ME 2) in greater detail. The molecule is a maleic anhydride R-olefin copolymer with a comblike structure (average molecular weight, 20 000), containing both hydrophobic and hydrophilic side chains (Table 2 and refs 28 and 29). Maltesh et al.29 studied the polarity and aggregation behavior of the surfactant in aqueous solutions and found that the solutions have relatively low polarities. At low polymer concentrations, the polarity was comparable to that of water, whereas at higher surfactant concentrations, it further decreased because of the formation of hydrophobic aggregates. Increasing the pH above pH 4 (i.e., causing the carboxyl groups to dissociate) did not impede aggregation, which was explained by the fact that the long ethoxylated chains could shield the charged COO- groups. When these observations are considered, it is possible to assume that at the w/o interface Ketjenlube behaves like a nonionic surfactant with a high HLB value rather than a fully dissociated ionic surfactant. We also have to consider that spatial constraints, caused by the size of the Ketjenlube molecule, may hinder the association of aspartame at the w/o interface of ME 2. Next, we consider ME 4, which is stabilized by phosphatidylcholine with butanol as the cosurfactant (Tables 1 and 2). The headgroup of the molecule is zwitterionic with an isoelectric pH 6.4-6.7,39 which would render it approximately neutral or slightly positive in an aqueous solution. Thus, the environment at the w/o interface of ME 4 has a low polarity, and, consequently, the driving force for the interfacial association with aspartame should be low. Indeed, according to the respective Yo value (Table 2), the association capacity is about half of those observed (38) Ryotto Sugar Ester Technical Information; Mitsubishi-Kasei Food Corp.: Tokyo, Japan, 1982. (39) Wittcoff, H. The Phosphatides; Reinhold: New York, 1951; p 14.

Fu¨ redi-Milhofer et al.

in the MEs stabilized with Ketjenlube (ME 2) and SMS 1570 (ME 3). The very low interfacial association of aspartame in ME 5 may be explained by two reasons: (i) the low polarity of the headgroups of both the surfactant (Atmos 300; HLB 2.8)40 and the cosurfactant (phosphatidylcholine, discussed above) and (ii) the spatial constraints because both the surfactant and the cosurfactant are relatively large molecules. The relatively high apparent solubility of aspartame in the water pools of all the MEs compared to its solubility in pure water may be explained as follows: The w/o MEs consist of reversed micelles, that is, micelles that solubilize a certain amount of water. At small water pools, most of the water is associated with the surfactant at the interface,36,37 whereas at W >10-15, a part of the water molecules behave as “bulk” water. However, most probably the structure of this “bulk” water is also affected by the proximity of the w/o interface, making it less hydrogenbonded than pure water. This situation reduces the hydrophobic interactions and increases the water solubility of organic substances. Another consideration is the distribution of the stabilizing surfactant molecules between the interface and the water pools. Because dissolved surfactant molecules are always present in the water pools, they may inhibit the crystallization of aspartame by adsorbing at incipient crystal nuclei and inhibiting their growth. In the case of aspartame, this phenomenon seems to be rather nonspecific, hence, the relatively small differences between the effects of the different MEs (column 6 in Table 2). To explain the differences that do exist, further studies of the crystallization of aspartame from aqueous solutions in the presence of the previously mentioned surfactants would be necessary. In conclusion, the above results indicate that the solubilities of slightly soluble organic molecules, such as aspartame, may be greatly enhanced by solubilizing the molecules in w/o MEs. Ionic MEs seem to work best, but even nonionic ones (as are most edible MEs) may be efficient because, in addition to some degree of interfacial association, the surfactant molecules that are present in the water pools may act as inhibitors of the crystallization of the guest molecules. Acknowledgment. The help by Dr. E. Wachtel from the Faculty of Chemistry, the Weizman Institute of Science, Rehovot, Israel, in measuring and interpreting the SAXS spectra is gratefully acknowledged. LA026933L (40) McCutcheon’s Detergents & Emulsifiers, North American Edition; MC Publishing Co.: Glen Rock, NJ, 1980.