Interaction of Gum Arabic with Fatty Acid Studied Using Electron

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Biomacromolecules 2010, 11, 1398–1405

Interaction of Gum Arabic with Fatty Acid Studied Using Electron Paramagnetic Resonance Yapeng Fang,*,† Saphwan Al-Assaf,† Glyn O. Phillips,†,‡ Katsuyoshi Nishinari,† and Peter A. Williams§ Glyn O. Phillips Hydrocolloid Research Center, Glyndwr University, Plas Coch, Mold Road, Wrexham LL11 2AW, United Kingdom, Phillips Hydrocolloid Research Ltd., 45 Old Bond Street, London W1S 4AQ, United Kingdom, and Centre for Water-Soluble Polymers, Glyndwr University, Plas Coch, Mold Road, Wrexham LL11 2AW, United Kingdom Received February 27, 2010; Revised Manuscript Received March 25, 2010

Electron paramagnetic resonance (EPR) is here used to study the interaction between gum arabic and a fatty acid. The EPR spectra of 5-doxyl stearic acid (5-DSA), a spin-labeled fatty acid analog, displayed increasingly anisotropic line features upon addition of gum arabic, indicating a strong immobilization of the nitroxyl moiety when the fatty acid is bound to gum arabic. To understand the nature of the interaction, EPR measurements were carried out at different pHs and using two fractions of gum arabic separated by hydrophobic interaction chromatography (HIC). 5-DSA bound favorably to the hydrophobic fraction, which contains mainly glycoprotein, and a small amount of high molecular weight arabinogalactan protein (AGP). Binding occurred to a less extent to the hydrophilic fraction, which contains essentially arabinogalactan (AG). Such a hydrophobic binding mechanism is further supported by a sharp drop in the binding when pH is raised above the pKa value of 5-DSA (∼pH 5). This is because the ionization of carboxylic groups would lead to increased polarity and hydrophilicity of the fatty acid. A secondary effect involving the formation of ionic hydrogen bonds between carboxylic groups in fatty acid and lysine residues in gum arabic might also contribute. This is consistent with the reduction in binding ability when the pH was elevated above the pKa value of lysine residue (∼pH 10). The biological significance of these findings is considered.

1. Introduction Gum arabic is a natural gum collected as the exudates from two species of the acacia trees, Acacia senegal and Acacia seyal, which grow across the Sahelian belt of Africa, principally in Sudan.1 The use of gum arabic has been a long history, dating back at least to the second millennium BC among ancient Egyptian who used it as an adhesive and binding medium for inks, watercolors, and dyes. Today the properties and functionalities of gum arabic have been extensively explored and developed, resulting in its utilization in a wide range of industrial sectors, including lithography, painting, glue, cosmetics, pharmaceutics, encapsulation, food, and so on.2,3 This is particularly true in the food industry where it is widely applied as a stabilizing, thickening, and emulsifying agent.2 Although gum arabic is consumed in our daily food and beverage, there is no definite scientific consensus about its possible biological effects and health-related value.4,5 Gum arabic has been claimed to have the following biological effects: (1) antioxidant property;6-9 (2) positive contribution to renal disease;10,11 (3) positive cardiovascular influence;11 (4) effect on the gastrointestinal tract;12,13 and (5) effect on lipid metabolism.5 Among these effects, gum arabic altering lipid metabolism merits special comments as it is of more relevance to the present work. Studies by Ross et al.14 on normal human subjects and by Sharma15 on men with hypercholesterolemia reported reduction of total serum cholesterol level by 6 and 10.4%, respec* To whom correspondence should be addressed. Tel.: +44 (0) 1978 29 3330. E-mail: [email protected]. † Glyn O. Phillips Hydrocolloid Research Center, Glyndwr University. ‡ Phillips Hydrocolloid Research Ltd. § Centre for Water-Soluble Polymers, Glyndwr University.

tively, when subjects were given 25 and 30 g/day supplements of gum arabic. Using gum arabic supplemented apple juice, Mee and Gee16 observed a drop of total serum cholesterol by 10% in a group of men with mild hypercholesterolemia. Such a drop was considered more significant as the level of fiber supplementation was modest compared with previous studies. However, when gum arabic was evaluated against plasma cholesterol level, inconsistent effects have been found.17-20 Decreased cholesterol levels were found in some but not in all cases. Different mechanisms have been proposed to explain the cholesterol-lowering effect of gum arabic. One mechanism was that gum arabic can bind or sequester bile acids, diminishing their reabsorption in the ileum and giving rise to their increased excretion in the feces.5 This mechanism, however, cannot be confirmed because other studies revealed no significant increase in fecal bile acid excretion.19,20 Another mechanism was related to lipid digestion and absorption. Pasquier et al.21 studied the process of intragastric emulsification and lipolysis of triacylglycerols. They found that the addition of gum arabic markedly reduced the extent of triacylglycerols lipolysis. Their interpretation was that the protein moiety of gum arabic can strongly interact with the lipid droplet surface, possibly blocking the available interface of gastric lipase or, more likely, preventing the release of free fatty acids generated at the droplet surface. Fatty acid is an important category of lipids, which can be produced by hydrolysis of the ester linkages of triacylglycerols in a fat/oil. The uptake, transport, and metabolism of fatty acid in biological bodies were found to closely involve a family of proteins referred to as fatty acid-binding proteins (FABPs).22 FABPs are present in various tissues and the most typical and studied FABP is serum albumin.23-28 A common structural

10.1021/bm100219n  2010 American Chemical Society Published on Web 04/07/2010

Interaction of Gum Arabic with Fatty Acid

motif found in FABPs consists of 10-antiparallel β-strands (AJ) and two short antiparallel R-helices (RI and RII) positioned over one end of the β-structure.24 This structural arrangement allows the formation of a large internal cavity that serves as a binding pocket for fatty acid. Although the cavity is lined primarily by hydrophobic amino acid side chains, it also contains some polar side groups, permitting the formation of ionichydrogen bonding interaction between FABPs and fatty acids.24 Various techniques, including X-ray crystallography,23 highperformance affinity chromatography,24 nuclear magnetic resonance,23 and EPR,25-29 have been employed to characterize the binding of fatty acids to FABPs. Among these, EPR provided a fast and easy means to measure bound and free fatty acids simultaneously and allowed their dynamics to be estimated. One controversial point with this technique was that spin labeling of fatty acid may perturb the microenvironment surrounding the binding sites. Such an effect, however, proved negligible in many studies.25,26 In contrast to the abundant literature on FABPs, few studies have reported polysaccharides binding to fatty acids. This might generally be expected because polysaccharides are usually hydrophilic. Here we report the ability of gum arabic to interact with fatty acids. Such ability can be attributed to the structural characteristics of this gum arabic, specifically the association of proteinaceous moieties with some of its components. Gum arabic is highly heterogeneous material. By using gel permeation chromatography (GPC) and HIC,30,31 three major fractions, AGP, AG, and GP, were identified. They have a molecular weight of 1-2 × 106, 2.5 × 105, and 2 × 105, respectively, and account for ∼10, ∼ 90, and ∼1% of the total gum and ∼10, 20% of the total protein.32,33 The AG fraction has a highly branched and compact carbohydrate structure and was visualized as a disk-like morphology under transmission electron microscopy and atomic force microscopy.34 The AGP fraction is generally accepted to have a “wattle blossom” type structure constituted of branched carbohydrate blocks commonly attached to a polypeptide chain.32 In this paper, the EPR evidence of gum arabic binding fatty acid is presented and possible biological implications of the binding are considered.

2. Experimental Section Material. Two gum arabic samples typical of the Acacia senegal and Acacia seyal species in spray-dried form were obtained from San Ei Gen F.F.I, Inc. (Japan) and Kerry Ingredients (U.K.). 5-DSA was purchased from Sigma-Aldrich (U.K.) and was used as received. All the other chemicals used in the study were purchased from Fisher Scientific (U.K.) and were of analytical grade. Removing Divalent Cations from Acacia senegal. Gum arabic usually contains a considerable amount of divalent cations, particularly Ca2+ and Mg2+. These ions were shown to influence some properties of gum arabic, for instance, its stability against colorants.35 To identify the contribution of the divalent ions to the interaction between gum arabic and fatty acid, a gum arabic sample free of Ca2+ and Mg2+ was prepared. Specifically, the gum Acacia senegal was subjected to treatment using Amerlite IR-120 H+-type exchange resin (SigmaAldrich, U.K.). A total of 100 mL of 10% gum solution was mixed with 300 g of the resin and stirred continuously for 4 h. The mixture was then left at quiescence for the resin to sediment. The supernatant was decanted and filtered through a MF200 glass fiber paper (Fisher Scientific, U.K., 0.8 µm pore size). The filtered solution was adjusted to pH 4.5 (the natural pH of gum arabic solution) using 0.1 M NaOH followed by freeze-drying on a PowerDry LL300 (Heto, U.K.). The Ca and Mg content, as determined by an inductively coupled plasma optical emission spectrometry (ICP-OES) system (Varian Inc., U.S.A.),

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Table 1. Molecular Characteristics of the Gum Arabic Samples Used in the Studya samples Mw (kDa) Mw/Mn Rg (nm) protein (%)

Acacia senegal Acacia seyal 1540 (870) 4.6 (2.6) 28 (26) 2.2

2220 (1910) 3.1 (2.6) 67 (49) 0.9

F1

F2

1680 (920) 4.6 (2.6) 30 (28) 1.8

120 (100) 1.7 (1.5) 15 (13) 3.8

a Note: F1 and F2 are the hydrophilic and hydrophobic fractions of gum Acacia senegal separated by hydrophobic interaction chromatography. The values in brackets were after electronically excluding the contribution of spray dry-induced aggregates using Astra 4.90.08 software.38

were reduced from 7100 to 10 ppm and from 2300 to 30 ppm, respectively, by the treatment. Fractionation of Acacia senegal by Hydrophobic Interaction Chromatography (HIC). Fractionation of gum Acacia senegal, using a previously described method after slight modification,36 was undertaken on a DIAION HP-20 packed column (50 mL, d 22 mm × h 130 mm). This material has been widely used in a variety of applications such as protein absorption and natural product decolorization. It is a highly porous styrenic absorbent resin with pore size of 52 nm and particle size of >250 µm.36 DIAION HP-20 has a strong capacity of holding hydrophobic material. The column was preconditioned with 100 mL of 1% sulfuric acid followed by washing 500 mL of distilled water. A total of 100 mL of 10% Acacia senegal aqueous solution was applied to the column at a rate of 100 mL/h and the eluate was collected immediately. The column was then washed with 100 mL of distilled water. The first 50 mL of the wash was combined to the collected fraction and made a volume of ∼200 mL. This constituted the first hydrophilic fraction designated as F1. The column was washed again with 500 mL of distilled water before introducing 100 mL of 70 v/v% methanol. The methanol eluted material constituted the second hydrophobic fraction (F2). F2 was rotary evaporated at 40 °C to remove methanol and then freeze-dried along with F1. Protein Content Measurements. Protein content of the gum arabic samples and their fractions were determined according to previously reported procedures using the Kjeldahl method.37 A factor of 6.6 was used to convert nitrogen content into protein content. The results were included in Table 1. GPC-MALLS Analysis. Molecular characterization of gum arabic samples was performed on a gel permeation chromatography-multiple angle laser light scattering (GPC-MALLS) system. The system consisted of a series of a Superose 6 10/300GL column (GE Healthcare, U.K.), an Agilent 1100 series UV detector (Agilent Technologies, U.K.) operated at 214 nm, a DAWN EOS multiangle light scattering detector (Wyatt Technology Corporation, U.K.) operated at 690 nm, and an Optilab refractometer (Wyatt Technology Corporation, U.K.). Aqueous NaCl solution (0.2 M) filtered through 0.2 µm Millipore filter was adopted as an eluent and was delivered at a constant rate of 0.45 mL/ min by a KNAUER HPLC pump K-501 (Kinesis, U.K.). The test material was prepared in the same solvent at a concentration of 2 mg/ mL. It was injected into the GPC-MALLS system after filtered using a 0.45 µm Nylon filter. Data was collected and analyzed by Astra 4.90.08 software. Molecular weight parameters of the gum arabic samples in the study are listed in Table 1. EPR Measurements. For the EPR measurements, gum arabic solutions were buffered using 50 mM acetate at pH 5, except those used for pH dependence studies where they were dissolved in distilled water and pH-adjusted properly using 0.1 M HCl or NaOH. A total of 100 µL of 500 µM 5-DSA stock solution in 95% ethanol was pipetted into a glass bottle, and the ethanol was removed with a stream of nitrogen. Gum arabic solution (10 mL) was then added. The final concentration of 5-DSA was 5 µM, which is below its critical micelle concentration and solubility limit.25,26 The solution was roller-mixed overnight before EPR measurement. It was reported that the binding of 5-DSA to protein was fast and completed within 5 min.26 We also confirmed that the EPR spectra obtained at 30 min, overnight and 4

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Figure 2. EPR spectra of 5-DSA in the presence of gum Acacia senegal of various concentrations ranging from 0 to 30 wt %. The concentration of 5-DSA was fixed at 5 µM, and pH was maintained at pH 5 using 50 mM acetate buffer.

Figure 1. GPC-MALLLS elution profiles of gum Acacia senegal and fractions thereof: (a) starting material, (b) hydrophilic fraction F1, and (c) hydrophobic fraction F2. Light scattering at 90° (solid lines): RI (dashed lines); UV (dotted lines).

days after mixing were the same for the present system. Therefore, the results reported in the study correspond to equilibrium bindings. Measurements were conducted at 20 °C on an X-band Bruker ESP 300 ESR spectrometer (Bruker Spectrospin, U.K.) using a quartz cell suitable for aqueous solutions. Instrument settings were as follows: microwave frequency, 9.84 GHz; microwave power, 10 mW; center field, 3490 G; sweep width, 100 G; modulation frequency, 100 kHz; modulation amplitude, 0.94 G; receiver gain, 1 × 105; sweep time, 168 s; number of scans, 5.

3. Results HIC Fractionation of Acacia senegal. Figure 1 displays the GPC elution profiles of the gum Acacia senegal and its hydrophilic and hydrophobic fractions (F1 and F2) separated by HIC. General features of the GPC profiles agreed with most previous reports: three components, AGP, AG, and GP, could be separated and identified.39-41 It should be noted that the splitting of the AGP peak into doublet in light scattering signal is typical of spray-dried gum arabic containing a small amount of aggregates formed during the spray-drying process.38 The formation of the aggregates is irreversible and could not be completely removed by filtration through 0.45 µm filters as used in our experiments. It, however, was removable if passing through 0.1 µm filters.38 The hydrophilic fraction F1 (Figure

1b) has almost an identical elution profile to the starting material (Figure 1a). The aggregation shoulder is still present, suggesting that the aggregates survived from freeze-drying. The hydrophobic fraction F2 (Figure 1c), in contrast, has a distinctive profile, containing essentially the GP component with a trace amount of the AGP. Moreover, the RI and UV peaks representing GP in F2 were located at a slightly larger elution volume than the GP-representing UV peak in the staring material. This indicated that the GP contained in F2 was mainly the low molecular weight population of the GP present in the starting material, presumably the low-carbohydrate and high-protein component. Molecular weight analysis showed that F1 had a slightly higher Mw and Rg than those of the starting Acacia senegal (Table 1). This is because the GP has been partially removed. The Mw and Rg of F2 were around 100 kDa and 13 nm, which were considerably lower than the staring material and F1. The HIC fractionation results were in agreement with those published in previous reports.36 EPR Results. The EPR spectra of 5 µM 5-DSA containing various concentrations of gum Acacia senegal are shown in Figure 2. The spectrum in the absence of Acacia senegal exhibited a sharp three lined feature, which is typical of free 5-DSA molecules undergoing rapid isotropic tumbling.42 The rotational correlation time τc, calculated according to the approximation of Zhao et al., was ∼0.2 ns.43 This value is similar to those reported for noninteracting 5-DSA in solutions.28,42 With increasing gum Acacia senegal concentration, the narrow isotropic component gradually disappeared and simultaneously a broad anisotropic component developed. The anisotropic component became completely dominant when the gum concentration was higher than 20%. The line feature of the anisotropic component resembled those observed for 5-DSA bound to bovine serum albumin (BSA)26 and human serum albumin (HSA)27 as well as those recorded at low temperature in frozen solutions.26 It is an indication of immobilization of 5-DSA as a result of binding to Acacia senegal.26,27,29,42 The EPR measurements of 5-DSA in the presence of gum Acacia seyal revealed a similar transition of the spin probe from an isotropic tumbling state to the anisotropic immobilized state (Figure 3). It suggests that the Seyal variety has the ability to bind 5-DSA also. To compare their binding capacities, we calculate the ratio h0,ani/h+1,iso, where h0,ani is the peak height of the central line at 3486 G of the anisotropic component and


Figure 3. EPR spectra of 5-DSA in the presence of gum Acacia seyal of various concentrations ranging from 0 to 30 wt %. The concentration of 5-DSA was fixed at 5 µM, and pH was maintained at pH 5 using 50 mM acetate buffer.

Figure 4. Plot of h0,ani/h+1,iso as a function of gum arabic concentration: Acacia senegal (O) and Acacia seyal (0). h0,ani/h+1,iso was calculated from Figures 2 and 3, and its definition was given in the text.

h+1,iso is the peak height of the low field line at 3473 G of the isotropic component. Therefore, qualitatively, the higher the ratio, the more 5-DSA molecules are in bound state. Figure 4 plots the ratio as a function of gum arabic concentration. At the same polymer concentration, Acacia senegal shows a larger value of h0,ani/h+1,iso than Acacia seyal, suggesting a higher binding extent occurring in the former. To estimate the dynamics of 5-DSA in the bound state, a higher concentration of the spin probe were used to enhance the signal-to-noise ratio of the EPR spectra. Figure 5 shows the EPR spectra of 25 µM 5-DSA in the presence of 30% Acacia senegal and Acacia seyal. Both of the spectra corresponded to a powder pattern of 5-DSA with two outer hyperfine extrema clearly visible. The outermost hyperfine splitting (2Az) was 67.8 and 65.2 G for Acacia senegal and Acacia seyal, respectively. Rotational correlation times τc for these spectra could be calculated based on the relationship proposed by Goldman et al using the outermost hyperfine splittings.44 Assuming a Brownian diffusion model and a rigid limit value of 69 G for 2Az,26 τc was estimated to be 130 and 28 ns for Acacia senegal and Acacia seyal, respectively. Such magnitude of the rotational correlation time reflected strongly immobilized 5-DSA by gum arabic.29 The larger value of τc for Acacia senegal than for Acacia seyal might suggest that 5-DSA was bound more tightly to the former. Additionally, τc obtained here was notably larger than those for 5-DSA bound to BSA (∼20-30 ns)26,28 and to R-lactalbumin (∼10 ns).29 This might be due to the difference in molecular size of gum arabic and the globular proteins with which 5-DSA was bound and rotated together as a whole. Simulation of the anisotropic spectra (data not shown), by slow-motional theory using EasySpin 3.0 software,45 yielded


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Figure 5. EPR spectra of 25 µM 5-DSA in the presence of 30% gum arabic: (a) Acacia senegal and (b) Acacia seyal. The pH was maintained at pH 5 using 50 mM acetate buffer. The arrows indicated the positions of outer hyperfine extrema.

comparable values of τc. Moreover, it showed that the rotational diffusion of 5-DSA around the axis parallel to the hydrocarbon chain was about 10 times faster than that around the perpendicular axis. This characteristic was also found in 5-DSA bound to BSA.26 It implies that 5-DSA was somehow confined in a channel structure in gum arabic molecule, where the rotational diffusion around the axis perpendicular to the channel was greatly hindered, whereas that around the axis parallel to the channel was much easier.

4. Discussion 5-DSA Binding to Gum Arabic. As demonstrated in Figures 2, 3, and 5, the presence of gum Acacia senegal and Acacia seyal induced a strong immobilization of 5-DSA molecules in solution. As the gum arabic concentration is increased from 0 to 30%, the rotational correlation time τc of the spin probe increased by a factor of >100 from ∼0.2 ns that is typical of a free rapid isotropic tumbling to >20 ns that is characteristic of a restricted slow anisotropic tumbling. The immobilization of 5-DSA indicated its interaction and binding with gum arabic. Gum arabic as a polyelectrolyte very often contains considerable levels of intrinsic divalent ions, particularly Ca2+ ions.35 It was established that the divalent ions could interact with stearic acid to form low-solubility salt.25 To rule out the possibility of 5-DSA interacting with Ca2+ leading to immobilization, we designed the experiments as shown in Figure 6 to clarify the effect of Ca2+ on EPR spectra. Figure 6b illustrated the effect of addition of 2130 ppm Ca2+ on the EPR spectrum of 5 µM 5-DSA. This level of Ca2+ imitated the intrinsic Ca content present in 30% Acacia senegal. Clearly, the introduction of such a level of Ca2+ did not change the line shape of 5-DSA relative to that without Ca2+ (Figure 6a). This gives direct evidence that Ca2+ ions did not cause immobilization of 5-DSA under the experimental conditions studied. Furthermore, the EPR spectrum of 5-DSA in the presence of 30% decalcified Acacia senegal (Figure 6c) likewise exhibited a broad anisotropic line feature, indicating that 5-DSA was immobilized even by Ca2+-free gum arabic. The spectrum was also nearly identical to that of 30% Acacia senegal without decalcification (Figure 2). It meant that the binding of gum arabic with 5-DSA was not influenced by the removal of divalent cations. These all pointed to the macromolecular components of gum arabic rather than divalent ions to be responsible for immobilizing 5-DSA and, thus, binding of 5-DSA.

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Figure 6. EPR spectra of 5 µM 5-DSA in the absence (a) and presence of 2130 ppm Ca2+ (b) and 30% decalcified Acacia senegal (c). Ca2+ ions were added in the form of CaCl2. The pH was maintained at pH 5 using 50 mM acetate buffer.

Figure 7. EPR spectra of 5 µM 5-DSA in the presence of 5% gum arabic and fractions thereof: (a) control Acacia senegal, (b) hydrophilic fraction F1, and (c) hydrophobic fraction F2. The pH was maintained at pH 5 using 50 mM acetate buffer.

Binding Mechanisms. Gum arabic is a mixture of polysaccharide, glycoprotein and polysaccharide-protein complex.2,34 Although in low content (∼2%), the proteinaceous parts of gum arabic entail great structural complexity in the material.32 They are covalently associated with carbohydrate moieties to render gum arabic with both hydrophilic and hydrophobic characteristics.2 In view of the existence of the proteinaceous parts, the mechanism of gum arabic binding 5-DSA could be similar to FABPs binding to fatty acids. The latter was shown to involve geometrical confinement, hydrophobic interaction, and hydrogen bonding between proteins and fatty acids.24,26 To identify relevant binding mechanisms for the system, gum arabic samples fractionated by HIC with different hydrophilicity/

Fang et al.

hydrophobicity were investigated. Figure 7 displays the EPR spectra of 5 µM 5-DSA interacting with 5% Acacia senegal and its hydrophilic and hydrophobic fractions (F1 and F2). The spectra were all a superposition of a broad anisotropic component and a sharp isotropic component, suggesting the existence of both bound and free 5-DSA. The relative intensity of the anisotropic component to that of the isotropic component, however, was different between the starting materials and its two fractions. Figure 8a compares the values of h0,ani/h+1,iso as defined earlier. This ratio increased in the order of F1 < the starting Acacia senegal < F2, which corresponds to an order of increased binding. This suggests that 5-DSA was bound preferentially to the hydrophobic fraction and less so to the hydrophilic fraction. The starting material as a mean of F1 and F2 had an intermediate binding affinity. The binding of 5-DSA to gum arabic, therefore, seems to correlate with the hydrophobicity of the material, pointing to a hydrophobic binding mechanism. Figure 8b plots the value of h0,ani/h+1,iso as a function of protein content. A clear positive correlation existed between the binding extent and protein content. This indicates that the hydrophobic interaction arises from the proteinaceous parts of gum arabic and the long alkyl chain of 5-DSA, as was often the case for FABPs-fatty acid binding.23,24 The hydrophobic fraction F2, as revealed by GPC-MALLS (Figure 1c), is primarily the GP component that contains a higher amount of protein linked to short carbohydrate segments. The relatively high protein content conferred to this fraction a larger hydrophobicity and more binding sites available for 5-DSA. Moreover, the short carbohydrates segments in GP presumably posed lower steric barrier for 5-DSA to access proteins. These all favored the binding of 5-DSA to F2. It should be noted that SDS-PAGE analysis of gum arabic unveiled the presence of several sharp protein bands with Mw as low as 6 kDa.46 These proteins which may not link to carbohydrate should have also been eluted as F2 and contributed to binding 5-DSA. In comparison, as a result of the loss of GP, F1 should relatively have slightly increased content of AG and AGP, although the change was indiscernible in the GPC elution profiles (Figure 1b). The increased content of AG and AGP, particularly the former being almost purely carbohydrate, enhanced the hydrophilicity of F1, which deterred the binding of 5-DSA. Additionally, the majority of the proteins present in F1 was associated with AGP and embedded as cores in larger carbohydrate blocks as described by the “wattle blossom” model.2 This spatial arrangement of proteins might reduce the accessibility of 5-DSA, as it need overcome a hydrophilic barrier set up by the carbohydrate shell. These together led to a less preferential binding of 5-DSA to F1. In this context, the lower binding of 5-DSA to Acacia seyal relative to Acacia senegal can also be explained by the structural difference between the two species. Acacia seyal was reported to have about half the protein content of Acacia senegal.37 Moreover, Smith degradation studies

Figure 8. (a) Comparison of the binding extent of 5-DSA with different fractions of gum Acacia senegal. (b) Correlation of the binding extent with protein content. h0,ani/h+1,iso was calculated from Figure 7, and its definition was given in the text.



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Figure 10. Plot of h0,ani/h+1,iso as a function of pH for binding of 5-DSA to Acacia senegal. h0,ani/h+1,iso was calculated from Figure 9, and its definition was given in the text.

Figure 9. EPR spectra of 5 µM 5-DSA in the presence of 5% Acacia senegal at different pHs.

showed that most of the proteins in Acacia seyal were located at deeper-seated locations surrounded by more highly branched and compact carbohydrate clusters, in comparison to its counterpart in Acacia senegal.37,47 These circumstances were unfavorable for the binding of 5-DSA to Seyal species. The hydrophobic binding of gum arabic with 5-DSA is reminiscent of its adsorption behavior on air-water and oil-water interfaces. A recent work by Castellani et al. demonstrated that the adsorption of gum arabic fractions to those hydrophobic interfaces increased in the order of AG < AGP < GP, suggesting the highest specific activity for GP.48 Katayama et al. analyzed the oil-adsorbed components of gum arabic in emulsion by GPC-MALLS.49 They demonstrated that the adsorbed fractions of gum arabic were GP and AGP rather than AG. These results are all in line with the preferential interaction of 5-DSA with GP where 5-DSA as a long chain fatty acid has even larger hydrophobicity than air. pH dependence studies of 5-DSA binding to gum arabic were carried out to further explore the binding mechanisms. Figure 9 displays the spectra of 5 µM 5-DSA recorded in the presence of 5% Acacia senegal and at different pHs ranging from 4 to 12. At pH 4 and 5, the EPR spectra were a composite of sharp isotropic and broad anisotropic components. At pH between 5 and 6, the anisotropic component significantly decreased. At pH ) 6-10, although the isotropic component predominated, the anisotropic component was still present as a small shoulder. With further increasing pH to 11 and 12, the anisotropic component was hardly visible. The change of the EPR spectra is more clearly reflected in the plot of h0,ani/h+1,iso against pH as shown in Figure 10. The ratio had a sharp drop when pH was raised across pH 5 and 6, which indicated a marked decrease in the binding extent of 5-DSA. This pH region coincided with the pKa value (∼pH 5) of stearic acid,26 where the carboxylic groups became deprotonated. Deprotonation of the carboxylic groups was expected to reduce the polarity and hydrophobicity of 5-DSA, thus, leading to suppressed hydrophobic binding. This first pH dependence change therefore supported the mechanism of hydrophobic binding. A secondary drop in the binding extent was also identifiable at pH between 10 and 11 (Figure 10), although it was much less pronounced compared with the first. This pH range overlapped with the pKa value (∼pH 10) of the lysine residues in the proteinaceous part of gum arabic,26 which could indicate

its participation in binding 5-DSA. A similar behavior was also observed in BSA binding to 5-DSA.26 It has been suggested that the basic and polar amino acid side chains in FABPs could form ionic hydrogen bonding with the carboxyl groups of fatty acids, contributing to their bindings.23,24,26 The mechanism might also be applicable to the present system. Gum arabic was shown to have ∼3 lysine residues per 100 amino acids.32,50 Deprotonation of the lysines could result in the loss of positive charges in side chain amino groups, breaking the salt bridge and hydrogen bonding formed with 5-DSA and leading to a decreased binding. Note that, in addition to lysine, arginine in FABPs was proposed also to participate in the mechanism.26 This amino acid was present in gum arabic at a level of 1-3%.32,50 However, due to its high pKa value (pH > 12), the current pH dependence study was unable to reveal its contribution. The involvement of hydrophobic interaction and ionic hydrogen bonding in gum arabic binding 5-DSA was comparable to FABPs binding fatty acids. In the latter case, the interactions were in coordination with geometric confinement where fatty acid molecules were entrapped in a channel fixed by the secondary structure of the FABPs, particularly, the β-strands and R-helices.23,24 Simulation of EPR by EasySpin software indeed implied that 5-DSA was confined somehow to a channel structure of gum arabic. However, due to the lack of resolved secondary and tertiary structure of gum arabic proteins, it is impossible to geometrically assign the binding sites. Nevertheless, recent circular dichroism and FTIR measurements did reveal the presence of a significant amount of β-sheet structure in the GP and AGP fractions of gum arabic.50 Particularly, the GP molecule contained up to ∼6500 amino acid residues and they were proposed to distribute in several glycoprotein subunits.50 It is speculated that the spatial arrangement of β-strands within a subunit and between the subunits can provide a similar “channel structure” to accommodate fatty acid. Biological Implications. 5-DSA is a typical long chain fatty acid (18:0) labeled with a doxyl group at C5 position. The doxyl group proved to have negligible effect on the properties of fatty acids.25,26 The findings obtained in the study can represent those on real fatty acids. Gum arabic binding fatty acids may have several implications in the biological effects and health-related benefits of gum arabic. First, it supports the proposal of Pasquier et al.21 that gum arabic can reduce dietary lipid digestion. Their results on intragastric emulsification and lipolysis of triacylglycerols showed that the presence of gum arabic markedly reduced the extent of the lipolysis. They attributed this to the interfacial activity of gum arabic which brought gum arabic to the surface of lipid droplets, sterically preventing the access of lipase and the release of lipolysis product fatty acids. Binding with fatty acids might help attract gum arabic toward the surface of lipid

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droplets, further enhancing its interfacial activity. It might also help accumulate fatty acids around the interface, inhibiting the forward reaction of lipolysis. It is therefore likely that not only the steric barrier effect at interface but also the binding with fatty acids would contribute to a reduced lipid digestion by gum arabic. Second, it implies that the subsequent transport and absorption of fatty acids after lipolysis might be altered. A subcategory of FABPs, called intestine-type FABPs, has been identified in the intestine.22 They were believed to play important roles in assisting the transport and assimilation of fatty acids, especially saturated fatty acids.22 Gum arabic as a dietary fiber was primarily indegradable in the intestine until it reached the colon where it fermented.4 One probable outcome of gum arabic present in the small intestine is that it might bind fatty acids and transport them downward the digestive tract, reducing their intake and increasing their excretion. Third, gum arabic has been proposed to lower blood cholesterol by binding bile acids and diminishing their reabsorption in the ileum.5 However, there was no consistent experimental data supporting this. Considering the similarity that fatty acids and bile acids both have a polar carboxylic head and a hydrophobic chain, the binding of gum arabic with fatty acids might also be translatable to bile acids. This may help understand the cholesterol-lowering effect of gum arabic. Large amounts of bile acids are secreted into the intestine after ingestion of high-fat and high-protein diets. Excessive excretion of bile acid, which could be present as the remaining portion of secondary bile acid in the large intestine, might increase the risk of intestinal diseases such as colon cancer. A binding process could reduce this behavior.

5. Conclusion The interaction of gum arabic with fatty acids has been evaluated using spin-labeled stearic acid (5-DSA) by means of EPR technique. Gum arabic, including Acacia senegal and Acacia seyal, was found to be capable of binding 5-DSA, changing its rotational correlation time τc from ∼0.2 ns in the free state to >20 ns in the bound state. Mechanistic studies revealed that the binding arised from the hydrophobic interaction and ionic hydrogen bonding between 5-DSA and the proteinaceous moieties of gum arabic. Geometrical confinement of 5-DSA in a channel structure probably formed by the β-strands of gum arabic proteins might also be involved. The binding phenomenon found in the study suggests potential effects of gum arabic in delaying/reducing lipid digestion, diminishing fatty acid intake, and helping clear excessive bile acids in the human gastrointestinal tract. A future relevant study involving quantitative measurement of thermodynamic parameters of the binding at different temperatures, such as binding constant and stoichiometry by using techniques like isothermal titration calorimetry, would be useful to further elucidate its nature. Acknowledgment. Y.F. acknowledges the financial supports from Phillips Hydrocolloids Research Ltd (U.K.) and San-Ei Gen FFI Inc. (Japan). We thank Professor Kazunari Ushida of Laboratory of Animal Science, Kyoto Prefectural University, Japan, for helpful discussions.

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Interaction of Gum Arabic with Fatty Acid Studied Using Electron

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