Chemical investigation of the structural basis of the emulsifying activity of gum arabic

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FOOD

HYDROCOLLOIDS Food Hydrocolloids 21 (2007) 297–308 www.elsevier.com/locate/foodhyd

Chemical investigation of the structural basis of the emulsifying activity of gum arabic$ Madhav P. Yadav1, J. Manuel Igartuburu2, Youchun Yan3, Eugene A. Nothnagel Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 0124, USA Received 7 December 2004; accepted 1 May 2006

Abstract Gum arabic, an exudate from Acacia trees, has a unique combination of excellent emulsifying properties and low solution viscosity. These properties make gum arabic very useful in several industries but especially in the food industry where it is used as a flavor encapsulator and stabilizer of citrus oil emulsion concentrates in soft drinks. Gum arabic is a mixture of principally polysaccharides and proteoglycans, the latter being arabinogalactan proteins (AGPs). Gum arabic also contains trace levels of lipids. Our hypothesis is that these lipids are attached to the gum arabic AGPs as glycosylphosphatidylinositol (GPI) lipids, as found in rose and other AGPs, and that these lipids make important contributions to the emulsifying activity of gum arabic. To test this hypothesis, chemical treatments expected to cleave GPI lipid anchors have been applied to gum arabic, and the resulting effects on structure and emulsifying activity have been examined. Treatment of gum arabic with nitrous acid resulted in diminished emulsifying activity, loss of some glucosamine and nitrogen, but very little effect on the principal carbohydrate composition. Treatment with 50% aqueous HF at 0 1C resulted in diminished emulsion properties but also significant loss of arabinosyl residues. The approximately 1–3% subfraction of gum arabic components that adsorb at the surface of oil droplets has higher abundance of GPI linker components, much higher relative lipid content, much higher nitrogen content, and somewhat higher emulsifying action than the whole gum. These results are consistent with roles of both lipid and protein in the emulsifying activity of the gum. r 2006 Elsevier Ltd. All rights reserved. Keywords: Emulsions; Gum arabic; Emulsion activity; Emulsion stability; Emulsifier; Glycosylphosphatidylinositol (GPI) lipid anchor; GPI linker oligosaccharide

1. Introduction Gum arabic is a naturally occurring exudate collected from Acacia senegal trees and, to a lesser extent, from Acacia seyal $

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. Corresponding author. Tel.: +1 951 827 3777; fax: +1 951 827 4437. E-mail address: [email protected] (E.A. Nothnagel). 1 Present address: Eastern Regional Research Center, US Department of Agriculture, Agricultural Research Service, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA. 2 Present address: Departamento de Quı´ mica Orga´nica, Facultad de Ciencias, Universidad de Ca´diz, Apartado 40, 11510 Puerto Real, Ca´diz, Spain. 3 Present address: Loders Croklaan BV, Lipid Nutrition, P.O. Box 4, 1520 AA Wormeveer, the Netherlands. 0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.05.001

trees (Islam, Phillips, Sljivo, Snowden, & Williams, 1997; Thevenet, 1988). It is one of the oldest and most important industrial gums. The Ancient Egyptians used gum arabic as an adhesive when wrapping mummies and in mineral paints when making hieroglyphs (Verbeken, Dierckx, & Dewettinck, 2003; Whistler, 1993). In modern times, the most important applications of gum arabic have been not as an adhesive but as an emulsifier in the food and pharmaceutical industries. Gum arabic is considered to be the best gum in use in dilute oil-in-water emulsion systems (Garti, 1999), one important example of which is the use of citrus oils as flavoring agents in soft drinks where the oils are converted into a water-dispersible emulsion (Verbeken et al., 2003). The general goal of our research is to understand the structural basis of the excellent emulsifying activity of gum arabic. Gum arabic is predominantly carbohydrate, which is typically 42% (w/w) galactosyl (Gal), 27% arabinosyl

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(Ara), 15% rhamnosyl (Rha), 14.5% glucuronosyl (GlcA), and 1.5% 4-O-methyl-glucuronosyl (4-O-Me-GlcA) residues in gum arabic from Acacia senegal; and 38% Gal, 46% Ara, 4% Rha, 6.5% GlcA, and 5.5% 4-O-Me-GlcA in gum arabic from Acacia seyal (Islam et al., 1997). About 2% of gum arabic is protein which is characteristically rich in hydroxyprolyl, prolyl and seryl residues (Akiyama, Eda, & Kato, 1984; Al-Assaf et al., 2005; Verbeken et al., 2003). Various chromatographic procedures demonstrate that gum arabic is a complex mixture of macromolecules, the bulk of which fall in the range of 250–2000 kilodaltons (kDa) (Al-Assaf et al., 2005; Idris, Williams, & Phillips, 1998). In one particularly relevant study of the uses of gum arabic in the food industry (Randall, Phillips, & Williams, 1988), stabilization of 20% (w/w) orange oil-in-water emulsions was obtained when gum arabic was included at concentrations of 12% (w/w) or higher. Under these conditions, only 1–2% of the applied gum arabic was found adsorbed at the oil-water interface of the oil droplets. This adsorbed material had high molecular mass and a protein content of about 20%, thus accounting for about one-fourth of the total protein in gum arabic. These results indicated that the emulsifying activity of gum arabic is due to minor, relatively protein-rich components. Because treatment of gum arabic with a protease destroyed the emulsifying activity, it was concluded that the protein component is key to emulsification (Randall et al., 1988). A current model of how gum arabic occupies the oil–water interface and stabilizes emulsions (Garti, 1999) suggests that the protein component of gum arabic embeds in the oil while the carbohydrate component extends into the water. This model of emulsion stabilization is also consistent with the observation that pasteurization and demineralization of gum arabic enhance emulsion stability, most feasibly by promoting effective unfolding of the protein moiety at the oil–water interface and enhancing the integrity of the double electrical layer (Buffo, Reineccius, & Oehlert, 2001). Studies by several groups have shown that heterogeneous arabinogalactan proteins (AGPs) are major components of gum arabic (Akiyama et al., 1984; Osman, Menzies, Williams, Phillips, & Baldwin, 1993; Osman et al., 1995). More generally, AGPs are widely distributed throughout the plant kingdom and are present in leaves, stems, roots, floral parts and seeds where they are hypothesized to function as markers of cell identity or as signals in development (Serpe & Nothnagel, 1999). Most AGPs range 60–300 kDa and contain 90–95% carbohydrate, which consists of (1-3)-b-Gal backbones with (1-6)b-Gal side chains that in turn carry large amounts of a-Ara residues and lesser amounts of b-GlcA and other residues. The core of the molecule is a polypeptide that is usually rich in hydroxyprolyl, alanyl, seryl, threonyl, and glycyl residues (Serpe & Nothnagel, 1999). Three research groups (Sherrier, Prime, & Dupree, 1999; Svetek, Yadav, & Nothnagel, 1999; Youl, Bacic, & Oxley, 1998) presented various lines of evidence which showed that many AGPs

are synthesized with a glycosylphosphatidylinositol (GPI) lipid anchor. The lipid portion of the GPI anchor is a ceramide consisting of predominantly tetracosanoic acid (C24:0; similar abbreviations used throughout for fatty acids of other chain lengths) and phytosphingosine (Svetek et al., 1999). Oxley and Bacic (1999) solved the complete structure of the GPI anchor on Pyrus communis AGPs and found it to consist of the same core found in many species (Menon, 1994), modifed in Pyrus by a partial b-D-Gal-(1-4) substitution on the mannosyl (Man) residue adjacent to the glucosaminyl (GlcN) residue (see Serpe & Nothnagel, 1999, for a model of the structure of a GPI anchor on an AGP). The GPI lipid anchor attaches AGPs to the plant plasma membrane and appears to be cut by a phosphatidylinositolspecific phospholipase C or a GPI-specific phospholipase D to release AGPs from the membrane into the cell wall space (Oxley & Bacic, 1999; Svetek et al., 1999). Studies of AGPs in suspension-cultured rose cells showed that approximately 6% of the AGPs secreted into the culture medium still contain the ceramide (Svetek et al., 1999). This observation suggested to us that a small proportion of the AGPs in gum arabic might also still have intact lipid groups, since gum arabic is collected from secretions of Acacia trees rather than directly from the plasma membrane. If an AGP containing an intact lipid group were mixed in an oil-in-water dispersion, the lipid group might play a significant role in localizing the AGP at the surface of the oil droplets. Along with the protein component, the lipid might associate with the oil phase while the large, hydrophilic, carbohydrate component of the AGP extends into the aqueous phase. The present chemical investigation of the structural bases of the emulsifying activity of gum arabic was thus undertaken with particular focus on experiments that would test for the presence of GPI or other lipid groups in gum arabic and the contribution of such groups to emulsification activity. 2. Materials and methods 2.1. Materials Gum arabic samples A and B were obtained from TIC Gums (Belcamp, MD). Sample A was gum arabic spray dry powder ARAB SD#1-European Pharmaceutical, and sample B was gum arabic FT powder. Gum arabic sample C was obtained from Sigma Chemical (St. Louis, MO) and was catalog number G-9752. Cold-pressed orange oil was obtained from Sunkist Growers (Ontario, CA). Hexadecane (99%) was from Aldrich Chemical (Milwaukee, WI). The Tenbroeck tissue grinder (15 ml volume, 0.15 mm clearance between the ground-glass surfaces) was purchased from Wheaton Scientific Products (Millville, NJ), and the EmulsiFlex-B3 high-pressure homogenizer was purchased from Avestin Inc. (Ottawa, Ontario). Amberlite XAD-2010, a large-pore, high-surface area polyaromatic

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resin for hydrophobic interaction column chromatography, was purchased from Sigma Chemical. 2.2. Chemical analyses of gum arabic Sugar and lipid compositions were determined by N-acetylation, methanolysis, re-N-acetylation, trimethylsilylation, and gas chromatography-flame ionization detection or gas chromatography-mass spectrometry analyses as described by Svetek et al. (1999). Elemental analyses, including analysis of P by inductively coupled plasmaatomic emission spectroscopy, were performed by Desert Analytics Inc. (Tucson, AZ). 2.3. Chemical treatment of gum arabic 2.3.1. Nitrous acid deamination Nitrous acid undergoes complex and varied reactions with amines. Applied to a molecule containing a GPI lipid anchor, nitrous acid attacks the GlcN in the oligosaccharide linker, severing the GlcN-inositol linkage and deaminating the GlcN to 2,5-anhydromannose which, after reduction with borohydride, becomes 2,5-anhydromannitol (Ferguson, 1992; Menon, 1994). Nitrous acid deamination and sodium borohydride reduction of gum arabic were carried out by a modification of the procedure of Menon (1994). Gum arabic (sample C, 25 g) was dissolved by slow addition to 160 ml of rapidly stirred water. To the resulting homogeneous solution was added 40 ml of 1 M sodium acetate buffer (pH 3.7) to achieve a 0.2 M acetate buffer concentration. Nitrous acid equivalent to 0.25 M was then generated in the solution by adding 10.5 ml of a solution of 5 M sodium nitrite dissolved in 0.2 M sodium acetate (pH 3.7). The resulting solution was stirred for 5 h at 23 1C, and then the pH was adjusted to 10.3 by adding an aliquot of 5 M NaOH. To this solution was then added 14.1 ml of a solution containing 2.66 g of sodium borohydride dissolved in 0.3 M NaOH, thereby achieving a concentration of 0.27 M sodium borohydride in the combined solution. This reaction mixture was stirred at 23 1C for 12 h, and then the pH was adjusted to 7.0 by adding an aliquot of 1 M HCl. The reaction mixture, totaling approximately 300 ml at this point, was extracted with 1200 ml of 2:1 (v/v) chloroform: methanol (Folch, Lees, & Stanley, 1957). The aqueous upper layer was concentrated on a rotary evaporator to remove methanol, dialyzed (12–14 kDa cutoff tubing) extensively against distilled water at 4 1C and lyophilized. The yield was 16.50 g. A parallel control experiment was performed with another 25 g of sample C gum arabic, except here sodium nitrate replaced sodium nitrite. While sodium nitrite in pH 3.7 buffer generates nitrous acid, sodium nitrate in pH 3.7 buffer generates nitric acid, which is not reactive with amino groups at pH 3.7. The yield of lyophilized material at the end of this reaction was 18.75 g.

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2.3.2. Aqueous hydrofluoric acid dephosphorylation Organophosphate esters are cleaved by aqueous HF at low temperature. Applied to a molecule containing a GPI lipid anchor, aqueous HF dephosphorylates the GPI linker, severing both the linkage between ethanolamine and mannose and the linkage between inositol and the lipid (Ferguson, 1992; Menon, 1994). Gum arabic (sample C, 5 g) was placed in a 1 liter polyethylene plastic bottle cooled on dry ice. Aqueous 50% HF (100 ml, prechilled at 0 1C) was added to the gum arabic in the bottle, which was then moved to a chamber at 0 1C where it gradually warmed and was allowed to react for 49 h with occasional shaking at 0 1C. The reaction mixture was then slowly added to another polyethylene bottle containing 250 ml of 10 M KOH frozen and held on dry ice. After the resulting neutralization during thawing, the solution was dialyzed extensively against distilled water at 4 1C and lyophilized. The yield was 2.78 g. In addition to cleaving organophosphate esters, aqueous HF can also cleave glycosidic linkages in a very temperature-sensitive manner. Thus, another reaction was performed as described except the 49 h reaction with 50% HF was carried out at
12 1C instead of 0 1C. The yield of lyophilized material at the end of this reaction was 4.06 g. A parallel control experiment was performed with another 5 g of sample C gum arabic, except here aqueous 50% HF was replaced by 88% HCOOH, an acid not noted for dephosphorylation at low temperatures. After the parallel 49 h reaction with 88% HCOOH at
12 1C, the yield of lyophilized material was 4.15 g. 2.4. Assay of emulsifying activity and stability During the course of this project, two methods were used for the preparation of oil-in-water emulsions for testing emulsifier activity and stability. During much of the project, a hand-powered Tenbroeck tissue grinder was used for homogenization. Subsequently, an EmulsiFlex-B3 high-pressure homogenizer with a small volume sample chamber was used. Perhaps indicating a more thorough homogenization, emulsions prepared with the high-pressure homogenizer had a higher optical density than did emulsions prepared with the Tenbroeck tissue grinder at the same oil–gum concentration. Applied to the same set of samples, the two homogenization methods generally led to the same conclusions regarding which samples were the most effective emulsifiers. Thus in the latter phases of the project, the high-pressure homogenizer became our preferred tool for preparation of small-volume emulsions for activity testing, while the Tenbroeck tissue grinder remained our preferred tool for preparation of largevolume emulsions for gum fractionation. Because the two methods likely produced different particle sizes, however, emulsion properties could be compared only between different fractions emulsified with the same technique, rather than between different fractions emulsified with different techniques.

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To start the generation of oil-in-water emulsions with the Tenbroeck tissue grinder for activity testing, 20–30% (w/w) stock solutions of gum arabic in 0.02% sodium benzoate were prepared by adding gum arabic powder and sodium benzoate to water and then gently stirring overnight to produce a homogeneously hydrated solution (Buffo et al., 2001). This gum arabic stock solution and additional water were mixed in various proportions with orange oil (typical overall formulation: 1 g orange oil, 1 g gum arabic, and 3 g water) and then homogenized by 15 strokes in the Tenbroeck tissue grinder. No weighting agent was used. The resulting homogenate was diluted 500  into 0.02% sodium azide solution. The diluted emulsion was measured immediately for optical density (1 cm pathlength cuvette, distilled water as the blank) at 650 nm and then transferred into a separatory funnel for gravity separation at 23 1C without agitation. After 24 h, 37.5% of the total volume of the solution was carefully (to avoid remixing) drained from the bottom of the funnel and discarded. The next 25% of the volume of solution was then carefully drained from the bottom of the funnel. Once collected, this 25% middle fraction was gently swirled to homogeneity and then measured for optical density at 650 nm. This procedure was essentially a scaled-up version of the stability assay of Aoki, Decker, and McClements (2005) where optical density was measured at a height 30% up from the bottom of a cuvette in which an emulsion had been stored for 1 day. In this turbidometric assay of emulsification, the optical density measured immediately after emulsification indicates emulsifying activity, greater optical density corresponding to greater activity. The optical density measured after 24 h indicates emulsion stability (Aoki et al., 2005; Buffo et al., 2001; Pearce & Kinsella, 1978). Since the orange oil was not modified by addition of brominated vegetable oil or other weighting agent, large oil droplets resulting from coalescence in an unstable emulsion rapidly floated up in the separatory funnel and did not contribute to optical density in the middle 25% volume. Only the fine, stabilized droplets floated up slowly enough to still be appreciably present in the middle 25% volume after 24 h. When the EmulsiFlex-B3 high-pressure homogenizer was used, testing for emulsifying activity and stability was conceptually similar to that described above for the Tenbroeck tissue grinder but differed in details. A hydrated gum arabic stock solution was prepared at 2.5% (w/w) gum in 1% sodium benzoate. Typically, 400 mg of this stock solution was mixed with 50 mg of orange oil and sufficient distilled water to make 500 mg. This 500 mg solution was homogenized by 10 passages through the EmulsiFlex-B3 high-pressure homogenizer operating with 340 kPa (50 psi) regulator pressure which, after hydraulic amplification, corresponded to 68 MPa (10,000 psi) homogenization pressure. After ejection of the sample in the tenth passage, the homogenizer sample chamber was rinsed by 4 aliquots of distilled water, each filling the

chamber to capacity (each 3.5 ml; the first 3 aliquots each being passed once through the homogenizer, the fourth being passed through twice). After two additional passages with just air to blow remaining traces of liquid out of the homogenizer, the homogenized sample, water rinses of the chamber, and additional distilled water were pooled to total 125 ml. This diluted emulsion was measured immediately for optical density at 650 nm and then transferred into a separatory funnel for gravity separation at 23 1C without agitation. After 24 h, 50 ml of the solution was carefully drained from the bottom of the funnel and discarded. The next 30 ml of solution was then carefully drained from the bottom of the funnel and measured for optical density at 650 nm. 2.5. Separation of gum arabic subfraction associated with oil droplets in the creamy layer For purification of gum arabic fractions adsorbing to the surface of oil droplets, emulsions were prepared with the Tenbroeck tissue grinder as described above in Section 2.4, with some modifications. Hexadecane was used in place of orange oil. Although the less polar nature of hexadecane might have slightly changed the extent to which gum arabic components absorbed to the oil droplets (Chanamai, Horn, & McClements, 2002), use of orange oil here would have left open the possibility that any fatty acids or long-chain bases subsequently detected in the purified gum arabic fraction were orange oil components that persisted through the planned organic solvent washes. Use of highly purified hexadecane in place of orange oil eliminated this possibility. Another modification from Section 2.4 was that the dilution of the homogenate from the tissue grinder was changed to 20  into 3 M KBr with 0.02% sodium azide, pH 7. Ten batches from the tissue grinder, totaling approximately 1000 ml, were combined in a 1000 ml separatory funnel and allowed to separate under gravity at 23 1C without agitation. Separation was continued for 1–5 days until most of the emulsion had gathered in a creamy layer at the top of the separatory funnel and the lower solution, containing most of the gum arabic, was essentially clear. The clear lower solution was carefully drained from the bottom of the funnel and discarded. The creamy layer was collected and transferred to a centrifuge bottle. The separatory funnel was rinsed with 10 ml water and then several aliquots of isopropanol totaling 200 ml. These water and isopropanol rinses were pooled with the creamy layer in the centrifuge bottle. The isopropanol broke the emulsion and generated a precipitate, which was collected by centrifugation (30 min at 3000 g). The precipitate was further washed by centrifugation with 100 ml isopropanol (2  ), 100 ml hexane (1  ), and 100 ml 2:1 (v/v) chloroform:methanol (2  ). The final pellet was dried, dissolved in water, dialyzed extensively against distilled water, and lyophilized. Recovery of mass as lyophilized material was typically 0.5–3% of the starting mass of gum arabic.

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2.6. Hydrophobic interaction chromatography The gum arabic subfraction obtained from the creamy layer as described in Section 2.5 was subjected to further separation by hydrophobic interaction column chromatography on Amberlite XAD-2010, a large-pore, high-surface area polyaromatic resin. Prior to use, the resin was sequentially washed with methanol, water, 1 M NaOH, water, 1 M HCl, water, methanol, chloroform, ethanol, water, and then the starting solvent, which was typically 1 M NaCl, 0.1% TFA. In early experiments, samples of 225–350 mg of gum arabic creamy layer subfraction were loaded on a 45 cm  1.77 cm2 column. In later experiments, samples of 400–425 mg were loaded on a larger 70 cm  3.14 cm2 column. The columns were eluted with two linear gradients. Typically, the initial portion of the elution was with a declining linear gradient from 1 M NaCl, 0.1% TFA to distilled water, and the final portion of elution was with an increasing linear gradient from distilled water to 75% acetonitrile, 0.1% TFA. Details of the elutions, which were sometimes slightly varied in attempts to improve separation, are given in the figure captions. Column output was collected in tubes, 5 or 7 ml each. Aliquots from the tubes were assayed for total carbohydrate by measuring absorbance at 485 nm in the phenol– sulfuric acid assay (Ashwell, 1966). Contents of selected tubes were collected into pooled fractions, dialyzed, and lyophilized. 3. Results and discussion 3.1. Chemical analyses of gum arabic Table 1 presents the results of chemical analyses of carbohydrate, fatty acid, and elemental compositions of three gum arabic samples. Samples A and B were pretested for emulsifying activity by the supplier, since it is well known that the quality of gum arabic depends on environmental factors, the geographic location and Acacia species of collection, and other uncontrollable factors (Idris et al., 1998; Karamalla, Siddig, & Osman, 1998; Whistler, 1993). On the basis of its pretesting, the supplier markets sample B as a material with good emulsifying properties suitable for use in beverages, and sample A as material for pharmaceutical uses. Sample C was purchased from a different supplier and was not, to the authors’ knowledge, pretested for emulsifying activity. All three samples were received as powders. As judged by the lower contents of 4-O-Me-GlcA and higher contents of Rha, samples A and C appear likely to have been collected from Acacia senegal trees, while sample B was probably collected from Acacia seyal trees (Islam et al., 1997; Mocak et al., 1998). All three samples fell within standard specifications for gum arabic (Mocak et al., 1998). All three samples contained inositol (Inos), GlcN, Man, P, and fatty acids (Table 1), the anticipated GPI components. The levels of Inos and Man were roughly

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Table 1 Chemical analyses of carbohydrate, fatty acid, and elemental compositions of three gum arabic samples Component

Sample A

Glycosyl composition (mol%) Ara 30.2 Rha 11.4 GlcA 14.0 4-O-Me-GlcA 1.4 Man 0.11 Gal 41.7 Glc 0.94 GlcN 0.34 Inos 0.017

Sample B

Sample C

47.0 3.6 6.8 7.7 0.056 33.7 0.92 0.19 0.013

34.1 13.2 14.1 1.6 0.069 35.2 1.4 0.30 0.019

Fatty acid composition (nmol fatty acid/g gum arabic) 5.9 10.1 C16:0a C18:0a 5.9 25.5 3.5 4.1 C20:0a C22:0 1.7 2.9 C23:0 1.6 1.2 C24:0 2.0 3.1 C25:0 1.5 1.2 C26:0 2.1 1.6 C28:0 0.4 1.8 C16:0 dioic 0.0 0.0 C18:0 dioic 0.0 0.0

7.2 14.7 14.1 8.1 1.4 14.6 0.87 12.5 6.2 33.4 29.7

Elemental composition (% w/w) C 41.95 H 5.78 N 0.47 Pb 0.003

41.57 6.05 0.37 0.003

41.90 6.03 0.18 0.004

Molar ratios of GPI lipid anchor components to gum arabic (GA) moleculesc (nmol/nmol gum arabic) Inos/GA 0.38 0.29 0.43 GlcN/GA 7.6 4.3 6.7 Man/GA 2.5 1.3 1.5 Fatty acid/GAd 0.009 0.018 0.028 Fatty acid/GAe 0.009 0.018 0.050 a

Data reliability should be considered low due to high background. At limits of sensitivity. c Assuming 350 kDa number-average molecular weight and 98% carbohydrate content for gum arabic (Al-Assaf, Phillips, & Williams, 2005). Using gel permeation chromatography and a multi-angle laser light scattering detector, Al-Assaf et al. (2005) examined 67 gum arabic samples and found each to be mixture of molecules of various sizes. When processed as single peaks, the chromatography profiles of these 67 samples gave number-average molecular weights ranging 185–653 kDa. A size of 350 kDa was arbitrarily selected from this range and was used as the basis for these molar ratio calculations. d Without dioics. e With dioics. b

adequate, and the level of GlcN more than adequate, for one-third to one-half of the molecules in gum arabic to have GPI linker cores (Table 1). If gum arabic molecules are assumed to have an average size of 350 kDa and contain an average of one P atom per molecule, then the expected P content would be 0.0089% (w/w). Because of the heterogeneity of molecular mass among the components in gum arabic, these calculations are only approximations.

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The results of fatty acid analysis shown in Table 1 are of greatest relevance to the hypothesis that some AGPs in gum arabic contain GPI lipid groups that, along with the protein component, contribute to emulsification activity. It is noteworthy that the fatty acid results of Table 1 were obtained when the gum arabic samples were subjected to three cycles of lipid extraction by the Folch procedure (Folch et al., 1957) prior to methanolysis and analysis of fatty acid methyl esters. Thus, it is highly unlikely that the lipids detected were membrane lipids arising from hypothetical contaminating microorganisms or other hypothetical sources of free lipids in the gum arabic samples. It must also be noted, however, that reliability should be considered low for the data on C20:0 and shorter chain fatty acids in Table 1 and other tables in this report. At the sensitive level of detection required in this project, C20:0 and shorter chain fatty acids were found to be nearly ubiquitous laboratory contaminants, possibly arising from detergents used in cleaning glassware. Even when extremely rigorous cleaning procedures were used (Svetek et al., 1999), some amount of these shorter chain fatty acids could always be detected by gas chromatography-mass spectrometry analysis of solvent-only ‘‘blanks’’. Data on C20:0 and shorter chain fatty acids shown in the tables of this report were obtained by analyzing samples and blanks in parallel and subtracting results for the blanks from the results for the samples. We considered this to be the best feasible approach, but variation in the lipid contents of blanks limited its reliability. In sample B, which was marketed as a good emulsifier, only about 1.8% (or 0.9% if two lipid chains each) of the molecules appeared to carry a lipid. This level was very consistent with the earlier observations (Randall et al., 1988) that only 1–2% of the applied gum arabic was adsorbed at the oil–water interface of the oil droplets in an orange oil emulsion stabilized with gum arabic. Sample A contained only one-half as much lipid as sample B (Table 1). With its higher lipid content, sample C would be predicted to be an even better emulsifier than sample B. A considerable proportion of the total lipid in sample C was due to the presence of hexadecanedioic acid (thapsic acid, C16:0 dioic) and octadecanedioic acid (C18:0 dioic), however, which are not anticipated components of GPI lipid anchors and which were not detected in samples A and B. Long-chain dicarboxylic acids have been found to have fungistatic activity (Brasch & Friege, 1994), and thus it is possible that the C16:0 and C18:0 dioics were present as part of a plant defensive function of the gum (Whistler, 1993).

metric technique remains useful as a qualitative measure for ordering the relative emulsifying properties of different fractions within an experiment (Aoki et al., 2005; EinhornStoll, Ulbrich, Sever, & Kunzek, 2005). As tested by this technique, all three samples exhibited emulsifying activity and emulsification stability (Fig. 1). Although the supplier pretested and certified gum B for use as an emulsifier, gum A was equally good at 0 h and superior at 24 h, as judged by the higher optical densities observed with gum A. Gum C was the best emulsifier at 0 h, as judged by its higher optical densities observed with the qualitative turbidometric technique. Since gum C had the highest content of long chain (C22:0–C28:0) fatty acids (Table 1), its better emulsifier properties seemed to be consistent with a role of GPI lipid anchors in emulsification. 3.3. Emulsification properties change after loss of GPI lipid anchor components Treatment of GPI lipid-containing molecules with NaNO2 at pH 3.7 generates dilute nitrous acid which 0.6 0.5

0 hr After Emulsification

0.4 0.3

Optical Density at 650 nm

302

0.2

Gum Sample A Gum Sample B

0.1

Gum Sample C

0.0 0.12

24 hr After Emulsification

0.10 0.08 0.06 0.04 0.02 0.00 0.2

0.3

0.4

0.5

0.6

0.7

0.8

Gum Arabic-to-Orange Oil Ratio (w/w)

3.2. Emulsifying activities and stabilities of native gum arabic The emulsifying activities of the three gum samples were evaluated by the turbidometric technique (Buffo et al., 2001; Pearce & Kinsella, 1978). Although less informative than characterization of emulsions by particle size analysis with laser light scattering (Aoki et al., 2005), the turbido-

Fig. 1. Emulsifying activities (upper panel) and emulsion stabilities (lower panel) of gum arabic samples A–C of Table 1. Higher optical density indicated greater emulsifying activity (upper panel) or greater emulsion stability (lower panel). See Section 2.4 for details of the assay. When orange oil and water were homogenized without gum arabic, the optical densities were 0.07670.022 at 0 h and 0.000770.0006 at 24 h. Each plotted point was the average of three trials7standard deviation. Emulsions for these experiments were prepared with the Tenbroeck tissue grinder.

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deaminates the GlcN to 2,5-anhydromannose and cleaves the GlcN–Inos glycosidic bond, thereby releasing the Inos–lipid (Ferguson, 1992; Menon, 1994; Schneider & Ferguson, 1995). Substitution of NaNO2 by NaNO3 in a control treatment generates dilute nitric acid which does not deaminate primary amines. Compared to native (as obtained from the supplier) sample C gum arabic, the NaNO2-treated sample C gum arabic was greatly diminished in both emulsion activity (Fig. 2, upper panel) and stabilization (Fig. 2, lower panel), while the NaNO3-treated sample C gum arabic was as effective as native sample C gum arabic in both aspects. Consistent with the hypothesized presence of a GPI anchor in the gum, the NaNO2-treated, but not the NaNO3-treated, gum arabic was diminished in GlcN, Inos, and P content (Table 2). Possible effects of these treatments on other portions of the gum structure must also be considered. The relatively modest pH of 3.7 used with the NaNO2 and NaNO3 treatments is not sufficiently acidic to cleave either peptide bonds or the glycosidic linkages of non-amino sugars (Ferguson, 1992). The observed reten-

0.6

0 hr After Emulsification

303

Table 2 Analysis of chemically treated gum sample Ca Component

Glycosyl composition Ara Rha GlcA 4-O-Me-GlcA Man Gal Glc GlcN Inos

Chemical treatment NaNO3

NaNO2

HF

pH 3.7

pH 3.7


12 1C

0 1C

(mol%) 29.5 19.7 17.7 3.4 NDb 27.9 1.2 0.45 0.052

36.8 15.3 14.1 3.0 0.028 29.5 1.2 0.093 0.010

29.8 15.5 15.5 4.0 0.52 32.8 1.7 0.14 0.036

5.2 21.1 31.4 5.4 ND 29.6 7.3 0.16 0.018

41.38 5.96 0.20 0.001

40.72 6.01 0.38 0.005

ND ND ND ND

Elemental composition (% w/w) C 41.15 H 5.85 N 0.30 Pc 0.003

a See Table 1 for composition of native gum sample C, prior to chemical treatments. b Not determined. c At limits of sensitivity.

0.5 0.4

Optical Density at 650 nm

0.3 0.2 0.1 0.0 0.25 0.20

24 hr After Emulsification Gum Treatments: Native HCOOH, -12°C NaNO3

HF, -12°C

NaNO2

HF, 0°C

0.15 0.10 0.05 0.00 0.508

0.773

Gum Arabic-to-Orange Ratio (w/w) Fig. 2. Emulsifying activities (upper panel) and stabilities (lower panel) of gum arabic sample C after application of chemical treatments designed to cleave GPI lipid anchors. See the Fig. 1 caption and Section 2.4 for details of the assay. When oil and water were homogenized without gum arabic, the optical densities were 0.05170.022 at 0 h and 0.001170.0020 at 24 h. Each plotted point was the average of three to six trials7standard deviation. Emulsions for these experiments were prepared with the Tenbroeck tissue grinder.

tion of the molecules during extensive dialysis after the treatments and the comparison of glycosyl compositions with the native material (Table 2, cf. Table 1) are consistent with a lack of gross structural changes. Nitrous acid, as generated in the NaNO2 treatment, undergoes complex and varied reactions with amines, however, so certain amines other than GlcN were probably affected. Among the amines present in proteins, primary and secondary aliphatic amines, but not amides, are reactive with nitrous acid (Allinger et al., 1971). Thus, the polypeptide backbone would not be cleaved by nitrous acid, but the primary amines in the lysine and arginine side chains and at the N-terminus of the polypeptide could react, most likely resulting in replacement of the amino group with an alcohol group (Allinger et al., 1971). Secondary aliphatic amines, such as in the histidine side chain, could be converted to nitrosamines (Allinger et al., 1971). Combined together, lysine, arginine, and histidine account for approximately 8 mol% of the amino acids in gum arabic (Akiyama et al., 1984). The lower N content in the gum after NaNO2 treatment (0.20%, Table 2) than after NaNO3 treatment (0.30%) probably reflects both loss of GlcN (down to 0.093 from 0.45 mol%, Table 2) and some reaction of nitrous acid with primary and secondary amines in the polypeptide. Thus, the observed reduction in emulsifier effectiveness could be due to effects on either a GPI lipid or the polypeptide, or both. Another chemical cleavage approach was tested. Aqueous HF, under appropriate conditions, is reasonably specific for dephosphorylation cleavage at both ends of the GPI linker oligosaccharide, thereby releasing the lipid, the

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linker pentasaccharide, and the protein as separate molecules (Ferguson, 1992; Menon, 1994; Schneider & Ferguson, 1995). After treatment with 50% aqueous HF under the usual conditions of 0 1C for 50–60 h, sample C gum arabic was found to have emulsion activity and stabilization characteristics that were considerably diminished compared to the native gum arabic (Fig. 2). While these results were consistent with the hypothesized presence of a GPI lipid anchor and its contribution to the emulsifying activity of gum arabic, glycosyl composition analysis clearly revealed that the HF treatment at 0 1C altered gum arabic structure in undesirable ways, including loss of most of the Ara residues (Table 2). Although 40–60% aqueous HF at 0 1C is not expected to cleave glycosidic linkages (Knirel, Vinogradov, & Mort, 1989), arabinofuranosyl residues, which are especially susceptible to acid hydrolysis, were apparently cleaved under our conditions. Since HF cleavage of glycosidic linkages is strongly influenced by temperature, the treatment of gum arabic with 50% HF was tested at
12 1C. A parallel control experiment was performed with 88% formic acid, which has similar normality (24 N) as 50% HF (28.9 N) but is not especially reactive with phosphate groups. Neither of these acid treatments had greatly adverse effects on emulsifying activities and stabilities (Fig. 2), and the
12 1C HF-treatment did not have gross effects on either the glycosyl composition or the P content of gum arabic (Table 2). This latter result seemed consistent with observed lack of effect on emulsion characteristics. Apparently the
12 1C was cold enough to prevent both dephosphorylation and Ara cleavage. To obtain a more conclusive result regarding dephosphorylation and its effect on emulsification, it would be necessary to find a temperature between 0 and
12 1C at which dephosphorylation occurs but Ara residues are retained. Thus far, our experiments towards this goal have been unsuccessful. These results involving chemical treatments, especially the nitrous acid treatment, were generally consistent with the hypothesis that GPI lipid anchors are present in gum arabic and make an important contribution to emulsifying activity, but effects on emulsification through modification of the protein portion of the gum cannot be excluded. An excellent, although costly, experiment here would involve use of the enzyme glycosylphosphatidylinositolspecific phospholipase C to cleave GPI lipid anchors (Svetek et al., 1999) in gum arabic. 3.4. Separation and analyses of a gum arabic subfraction associated with oil droplets From earlier work the active emulsifying components were expected to amount to only 1–2% of gum arabic (Randall et al., 1988). Thus, a procedure involving flotation of a creamy layer on 3 M KBr was developed for bulk purification of the gum arabic components that adsorb to lipid droplets. This procedure was applied to gum samples A, B (repeatedly), and C, and in each case the recovery of

gum components from the creamy layer was 0.5–3% of the total gum emulsified with the hexadecane. This range of recovery was very consistent with the results of Randall et al. (1988). For all three gum samples A, B, and C, the gum components adsorbed to the hexadecane droplets in the creamy layer had major glycosyl components at very similar levels to those in the whole gum (Table 3, compare Table 1). The minor glycosyl residues Man and GlcN, which occur in the GPI linker oligosaccharide and in N-glycans of glycoproteins, were more abundant (in some cases more than 10  ) in the gum components from the creamy layer than in the whole gum. Inos also seemed to be somewhat more abundant in the components from the

Table 3 Chemical analyses of carbohydrate, fatty acid, and elemental compositions of gum components from creamy layersa Component

Creamy layer Sample A

Glycosyl composition (mol%) Ara 28.8 Rha 13.2 GlcA 11.8 4-O-Me-GlcA 1.1 Man 0.91 Gal 41.3 Glc 2.5 GlcN 0.64 Inos 0.023 Fatty acid composition C16:0b C18:0b C20:0b C22:0 C23:0 C24:0 C25:0 C26:0 C28:0 C16:0 dioic C18:0 dioic

Sample B

Sample C

39.6 3.6 7.3 5.5 2.1 38.0 1.9 2.2 0.045

30.4 13.7 13.7 1.4 0.83 37.6 2.0 0.73 0.033

(nmol fatty acid/g gum arabic) 149.2 159.6 68.6 275.6 234.3 120.5 20.7 21.5 23.0 7.4 22.5 25.0 4.0 5.5 4.4 11.9 41.7 95.8 1.9 4.5 6.3 8.3 26.9 92.7 7.0 14.7 85.9 0.0 0.0 564.9 0.0 0.0 259.6

Elemental composition (% w/w) C 41.95 H 5.89 N 2.24 Pc 0.005

41.90 5.71 1.79 0.005

40.90 5.69 2.20 0.011

Molar ratios of fatty acids to gum arabic (GA) moleculesd (nmol fatty acid /nmol gum arabic) Fatty acid/GAe 0.170 0.186 0.183 Fatty acid/GAf 0.170 0.186 0.471 a

See Table 1 for composition of native gum samples, prior to fractionation. b Data reliability should be considered low due to high background. c At limits of sensitivity. d Assuming 350 kDa number-average molecular weight for gum arabic (Al-Assaf et al., 2005). e Without dioics. f With dioics.

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creamy layers than in the whole gums. The C and H elemental compositions were very similar between the whole gums and the components from the creamy layer, but N was distinctly more abundant in the components from the creamy layer. This increase in N content was larger than could be accounted for by the increase in GlcN content, thus indicating a higher protein content in the components from the creamy layer. This higher protein content in the creamy layer was consistent with the results of others (Flindt, Al-Assaf, Phillips, & Williams, 2005; Randall et al., 1988) and suggests that protein plays a role in the binding of gum arabic molecules to oil droplets. Higher phosphorous contents in the creamy layers would be predicted from the hypothesis that GPI lipid anchors are present in the active gum fraction. The data in Table 3 (compare Table 1) suggest slightly higher phosphorous contents, particularly for sample C, but the phosphorous contents in all samples were near the lower limit of detectability, and thus no certain conclusion could be drawn regarding differences in P abundance. The comparative lipid contents of the whole gums and the components from the creamy layer were of greatest interest. Consistent with the hypothesis that GPI lipid anchors are present in gum arabic and contribute to emulsifying activity, the gum components adsorbed to the oil droplets had markedly higher lipid contents than did the whole gums (Table 3, compare Table 1). When summed over all lipid species listed in Table 3, the enrichment of lipid contents in creamy layer components was 10–20  greater than in the whole gums. Even with the limited reliability of determination of the shorter fatty acids (C:16–C:20, see Section 3.1), it was clear that these lipids, as well as the unusual dicarboxylic fatty acids in sample C, were more abundant in the creamy layers than in the corresponding whole gums. Results for the longer chain (C420) lipids were more highly reliable, since blank corrections here were small or zero, and these results confirmed the greater abundance of lipid groups in the gum components from the creamy layers. Table 4 presents the results of assays of emulsifying activity and stability for the gum components from the creamy layers. Since these emulsions were prepared with the high-pressure homogenizer (the Tenbroeck tissue grinder had been used for the data of Figs. 1 and 2), results of activity assays for the whole gums are also presented in Table 4. For all three gum samples A–C, the emulsifying activity (0 h result) and stability (24 h result) were greater for the components from the creamy layer than for the whole gum. These tests were made at relatively low gum/oil ratios in the expectation that small amounts of a purified active emulsifier would be as effective as large amounts of a crude emulsifier that contained many inactive components. The enhancement of emulsification was not as great as anticipated, however, given that the creamy layer components accounted for only 0.5–3% of the whole gum. The enhancement was only approximately two-fold, as judged by comparing results for 0.2 gum/oil (w/w) emul-

305

Table 4 Emulsifying activities and stabilities of gum arabic samples and their subfractions Gum/oila (w/w)

At 0 hb (OD650)

At 24 h (OD650)

Gum sample A Whole gum Creamy layer Creamy layer

0.2 0.2 0.1

0.98670.099 1.02770.229 0.74770.088

0.25970.034 0.42870.105 0.30070.025

Gum sample B Whole gum Creamy layer Creamy layer HIC fraction Ic HIC fraction II HIC fraction III

0.2 0.2 0.1 0.1 0.1 0.1

0.85970.057 1.16370.039 0.92270.112 1.06970.146 0.72370.151 0.86370.102

0.30870.007 0.46770.173 0.41970.085 0.44170.080 0.29570.073 0.39370.013

Gum sample C Whole gum Creamy layer Creamy layer

0.2 0.2 0.1

0.83370.120 1.05770.022 0.89270.180

0.28270.025 0.42170.028 0.37270.030

Water control

0.0

0.54570.143

0.19970.065

Preparation

a

Gum-to-orange oil ratio (w/w) in emulsion prepared with the highpressure homogenizer. b Optical density (OD at 650 nm) recorded immediately after emulsification (indicating activity) and 24 h later (indicating stability). Averages7standard deviations. c Hydrophobic interaction chromatography (HIC) fractions from Fig. 3(A).

sions for whole gum with the results for 0.1 gum/oil (w/w) emulsions for creamy layer components (Table 4). 3.5. Further separation of creamy layer gum components by hydrophobic interaction chromatography Further separation of the creamy layer gum components was attempted by hydrophobic interaction chromatography. Gum sample B was selected for this work, since its creamy layer components seemed to be slightly better emulsifiers than the creamy layer components from gum samples A and C (Table 4). The elution profile of the sample B creamy components on the hydrophobic interaction column (Fig. 3(A)) initially seemed to indicate a clean separation between components that adhered to the column (Fractions II and III) and those that did not (Fraction I). Chemical analyses of the carbohydrate and fatty acid compositions of these fractions (Table 5) indicated some modest differences between the fractions. The adherent fractions perhaps had slightly greater lipid content, but considerable lipid was also clearly present in the nonadherent Fraction I. Furthermore, the emulsifying action of Fraction I was at least as good, probably better, than the emulsifying actions of Fractions II and III (Table 4). The expectation was that the components adhering to the hydrophobic interaction column would have greater lipid content and better emulsifying action than the nonadherent components.

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80

NaCl ACETONITRILE UREA

15

0.0 1.0

0 100

0.8

80

0.6

10

0.4

60 40

5

0 2.5

0.2

20

0.0 1.0

0

ACETONITRILE (%)

Component

100

2.0

0.8

1.5

0.6

1.0

0.4

0.5

0.2

2

20

0.0 250

0

0

0

50 100 150 200 TUBE NUMBER

6 4

Fraction Ia

Glycosyl composition (mol%) Ara 44.6 Rha 4.8 Fuc 0.0 GlcA 6.3 4-O-Me-GlcA 7.4 Man 0.51 Gal 34.4 Glc 1.3 GlcN 0.73 Inos 0.04

8

0.0 (C)

0.2

Table 5 Chemical analysis of carbohydrate and fatty acid compositions of pooled fractions from the separation of sample B creamy layer by hydrophobic interaction chromatography

80 60 40

ACETONITRILE (%)

0

40 20

Nacl (M)

Fr III

Fr II 10

0.4

Nacl (M)

0.6

50

60

UREA (M)

ABSORBANCE (485 nm)

(B)

0.8

Nacl (M)

ABSORBANCE (485 nm)

(A)

60

Fr I

ABSORBANCE (485 nm)

1.0

ACETONITRILE (%)

306

Fig. 3. Separation of gum arabic sample B creamy layer fraction (Table 3) by hydrophobic interaction chromatography on Amberlite XAD-2010. (A) The column was equilibrated in 1 M NaCl, 0.1% TFA, and the sample was dissolved and loaded in this same solvent. The first portion of column elution was with a declining linear gradient (total volume ¼ 600 ml) from 1 M NaCl, 0.1% TFA to 0 M NaCl, 0.0% TFA (distilled water). The column was then washed with 150 ml of distilled water. The final portion of the elution was with an increasing linear gradient (total volume ¼ 600 ml) from distilled water to 75% acetonitrile, 0.1% TFA. Expressed relative to the mass of sample loaded, recovery of mass from the column was 54.3719.3% in pooled fraction I, 13.574.5% in pooled fraction II, and 8.777.1% in pooled fraction III (averages7standard deviations from 3 experiments). Materials eluting from the column were detected by the phenol–sulfuric acid assay for total carbohydrate (absorbance at 485 nm). (B) Non-adherent, pooled fraction I materials from two chromatography experiments as in (A) were combined and reapplied to the same Amberlite XAD-2010 column. Elution was with linear gradients similar to those in (A), except with the final elution extending to 99.8% acetonitrile, 0.2% TFA. Mass recovery was 65.8% in the non-adherent peak and 15.1% in the adherent peak eluting just after the start of the acetonitrile gradient. (C) Gum arabic sample B creamy layer fraction was dissolved in 1 M NaCl, 8 M urea, 0.2% TFA and applied to the column which was equilibrated with this same solvent. The first portion of column elution was with a declining linear gradient (total volume ¼ 700 ml) from 1 M NaCl, 8 M urea, 0.2% TFA to 0 M NaCl, 0 M urea, 0.2% TFA. The final portion of the elution was with an increasing linear gradient (total volume ¼ 700 ml) from water containing 0.2% TFA to 99.8% acetonitrile, 0.2% TFA.

Fraction II

39.4 4.8 5.5 9.1 8.0 2.2 29.8 1.2 0.16 0.03

Fraction III

42.2 3.9 8.1 6.8 12.1 3.7 15.6 1.1 6.2 0.18

Fatty acid composition (nmol fatty acid/g gum arabic) 0.0 6.3 99.1 C16:0b C17:0b 4.6 4.2 1.9 C18:1b 16.7 41.5 82.9 C18:0b 92.8 68.3 230.2 14.6 0.0 8.0 C19:0b C20:0b 65.3 81.3 58.8 C22:0 27.7 33.6 47.4 C23:0 12.4 81.7 41.8 C24:0 64.5 82.3 94.0 C25:0 9.5 71.6 75.6 C26:0 0.49 85.5 80.9 C28:0 20.4 47.0 22.3 C30:0 14.4 6.5 4.1 Molar ratio of fatty acids to gum arabic moleculesc (nmol fatty acid /nmol gum arabic) Total fatty acid 0.12 0.21 0.30 a See Fig. 3(A) for the column elution profile and identification of the pooled fractions. b Data reliability should be considered low due to high background. c Assuming 350 kDa number-average molecular weight for gum arabic (Al-Assaf et al., 2005).

Further study revealed an anomaly in the separation of the creamy layer gum components on the hydrophobic interaction column. In particular, when nonadherent Fraction I materials were collected and reapplied to the same column, separation into nonadherent and adherent fractions was observed (Fig. 3(B)). Such behavior could indicate column overload, although the expected capacity of the Amberlite XAD-2010 resin was much greater than the loads actually applied in Figs. 3(A and B). When a larger column was prepared and tested, the same chromatographic behavior was observed, thus ruling out overload. These and other data suggested that the creamy layer gum components might have a considerable tendency to self-associate into aggregates. This self-association could put the hydrophobic sites towards the center of the aggregate, so the aggregates could pass through the hydrophobic interaction column without adhering. To test this idea, a search was made to find solvent additives that would more completely solubilize the creamy layer components. Among urea, guanidine hydrochloride, methylsulfoxide, and lithium-3,5-diiodosalicylate (all powerful chaotropic

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reagents), only 8 M urea seemed to hold any promise. When creamy layer components were loaded on the hydrophobic interaction column in the presence of 8 M urea, separation into nonadherent and adherent fractions was again observed (Fig. 3(C)), but here the proportion of adherent material was greater than in Fig. 3(A). Even with this improvement, however, the separation remained unsatisfactory. 4. Conclusions The main findings of this study were the following: 1. Gum arabic contained small amounts of Man, GlcN, Inos, and lipid, the expected components of a GPI lipid anchor. 2. Treatment with nitrous acid, which was expected to cleave the GPI linker oligosaccharide, greatly decreased emulsifying activity, but the nitrous acid likely reacted with some amino acid side chains as well and could have thereby influenced emulsifying activity. 3. Treatment with aqueous HF, which was expected to cleave the GPI linker, reduced emulsifying activity, but this result was compromised by the simultaneous cleavage of Ara and other residues from the gum. 4. The approximately 1–3% of gum arabic components that absorbed at the surface of oil droplets had much higher relative lipid and protein contents than the whole gum and somewhat greater emulsifying action. These results point to the combined importance of the lipid and protein components relative to the emulsifying properties of gum arabic. 5. Overall, the results of the study were consistent with, but did not prove, the hypothesis that GPI lipid anchors are present in gum arabic and contribute to the emulsifying action of the gum. An important remaining point is that the chemical analyses to date have not shown gum arabic to contain phytosphingosine, a long chain base that occurs in GPI lipid anchors of arabinogalactan proteins from rose and pear.

Acknowledgement This work was supported by award number 2001-3550310027 from the NRI Competitive Grants Program/USDA. The authors are pleased to acknowledge the technical assistance of Hue Nguyen in some of the hydrophobic interaction chromatography experiments. References Akiyama, Y., Eda, S., & Kato, K. (1984). Gum arabic is a kind of arabinogalactan-protein. Agricultural and Biological Chemistry, 48, 235–237. Al-Assaf, S., Phillips, G. O., & Williams, P. A. (2005). Studies on acacia exudate gums. Part I. the molecular weight of Acacia senegal gum exudate. Food Hydrocolloids, 19, 647–660.

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