- Email: [email protected]
Characterization and properties of Acacia senegal (L.) Willd. var. Senegal with enhanced properties (Acacia (sen) SUPER GUM™): Part 5. Factors affecting the emulsification of Acacia senegal and Acacia (sen) SUPER GUM™
ARTICLE IN PRESS
FOOD
HYDROCOLLOIDS Food Hydrocolloids 21 (2007) 353–358 www.elsevier.com/locate/foodhyd
Characterization and properties of Acacia senegal (L.) Willd. var. TM Senegal with enhanced properties (Acacia (sen) SUPER GUM ): Part 5. Factors affecting the emulsification of Acacia senegal and Acacia (sen) SUPER GUMTM Hiromitsu Aokia,b, Tsuyoshi Katayamaa, Takeshi Ogasawaraa, Yasushi Sasakia, Saphwan Al-Assafb, Glyn O. Phillipsb,c, a San-Ei Gen F.F.I., Inc., 1-1-11 Sanwa-cho, Toyonaka, Osaka 561-8588, Japan Glyn O. Phillips Hydrocolloids Research Centre, The North East Wales Institute, Plas Coch, Mold Road, Wrexham LL11 2AW, UK c Phillips Hydrocolloid Research Ltd., 45 Old Bond Street, London W1S 4AQ, UK
b
Received 19 December 2005; accepted 13 April 2006
Abstract Acacia senegal is the major gum arabic species used in commercial emulsification for the production of beverages and flavor concentrates. This paper examines the effect of gum concentration, molecular weight parameters and homogenization conditions on the droplet size and stability of emulsions prepared using a conventional commercial A. senegal product and a series of A. senegal test gums that were prepared by a controlled maturation process, including a new commercial product produced by this process (Acacia (sen) SUPER GUMTM EM2). Pressure homogenization after vigorous stirring produced the best results. At concentrations below 20%, commercial gum arabic weight average molecular weight MW ca. 6 105 Da produced emulsions which were not stable under accelerated stress conditions. When the weight average MW was increased to a range 1–2.5 106 Da, the droplet size was reduced and the stability was greatly increased, even at a 5% gum concentration. After pressure homogenization, sub-micron droplet sizes could be produced which exhibited complete stability during stress testing at 60 1C and after longer-term storage. A model is proposed to account for the greater effectiveness of the matured gums, whereby the arabinogalactan protein complex unfolds as a result of pressure homogenization and occupies a greater volume around the oil droplet, which in turn produces greater stabilization. r 2006 Elsevier Ltd. All rights reserved. Keywords: Acacia senegal; SUPER GUMTM; Molecular weight; Emulsification; Pressure homogenization
1. Introduction Acacia senegal (gum arabic) is an important naturally occurring oil-in-water emulsifier, which is in regular use in the food industry. The emulsions are prepared by homogenizing the oil and water phases in the presence of gum arabic, preferably under pressure. The gum arabic forms a Corresponding author. Glyn O. Phillips Hydrocolloids Research Centre, The North East Wales Institute, Plas Coch, Mold Road, Wrexham LL11 2AW, UK. Tel.: +44 29 20 843298; fax: +44 29 20 843145. E-mail address: [email protected] (G.O. Phillips).
0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.04.014
protective layer around the oil droplets that prevents the droplets from aggregating (flocculating and/or coalescing). It also reduces the oil–water interfacial tension, thereby facilitating the disruption of emulsion droplets during homogenization (Walstra, 2003). In reviewing the behavior of various hydrocolloid emulsifiers, Dickinson (2003) stated that ‘‘much of the reported emulsifying capability of polysaccharides is explicable in terms of complexation or contamination with a small fraction of surface-active protein’’. This is certainly true of gum arabic. Randall, Phillips, and Williams (1988, 1989) showed that A. senegal was a polydisperse mixture of components and that it was
ARTICLE IN PRESS 354
H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
the arabinogalactan protein (AGP) component which is responsible for the emulsification properties. Several polysaccharide units are linked to a common protein core in this high molecular weight component (MW ca. 2.5 106 Da). The methods of fractionation and quantification of these individual components have been described previously (Aoki, Katayama, Al-Assaf, & Phillips, 2006). The AGP-rich fraction of A. senegal gum is adsorbed on the oil–water interface. The hydrophobic protein component of the AGP firmly anchors the protein–polysaccharide moiety at the interface, and the protruding hydrophilic carbohydrate blocks which are attached to this chain provide a strong steric barrier against flocculation and coalescence (Dickinson, 1995; Islam, Phillips, Sljivo, Snowden, & Williams, 1997; Randall et al., 1989). Whilst charged groups provide the basis for some electrostatic contribution to the colloidal stabilization, the relatively low value of the (negative) zeta potential, 10 to 20 mV under beverage emulsion conditions (Jayme, Dunstan, & Gee, 1999; Ray, Bird, Iacobucci, & Clark, 1995), indicates that the primary stabilization mechanism is steric in character. The level of surface activity is rather low in comparison with typical food protein emulsifiers. Therefore, to generate stable sub-micron-sized droplets, it is necessary to use a rather high gum-to-oil weight ratio, approximately 1:1, as compared with 1:10 for equivalent protein-stabilized emulsions (Dickinson, 2003; McNamee, O’Riordan, & O’Sullivan, 1998). Once formed by adsorption on to the macroscopic oil–water interface, there is little effect on the high surface shear viscosity of the gum arabic film by large dilutions of the aqueous sub-phase (Dickinson, Elverson, & Murray, 1989). The film viscoelasticity is, therefore, maintained even when a major proportion of the hydrocolloid has been removed from the aqueous phase in
contact with the adsorbed layer. Thus, only a small proportion of gum arabic used in emulsion preparation is actually involved in the stabilization process. The objective of this investigation is to create more favorable conditions for emulsification using A. senegal by increasing both the amount and the molecular weight of the AGP component present in the gum. Here, the emulsification performance of the enhanced Acacia gums will be compared with a conventional gum arabic having an average molecular weight of ca. 5–6 105. The objective is to reduce the amount of gum required and still produce a consistent and stable emulsion.
2. Materials and methods 2.1. Materials For this investigation, conventional control A. senegal (FR-2876), test materials (FR-2877, FR-2878 and FR-2879 and Acacia (sen) SUPER GUMTM EM2) which were previously described by Aoki et al. (2006) were used. All samples were produced by the heating maturation process previously described. FR-samples were prepared on a laboratory scale and the sample designated EM2 was produced on a commercial scale (Al-Assaf, Phillips, Aoki, & Sasaki, 2006; Aoki et al., 2006). The molecular parameters are summarized in Table 1. Citric acid, sodium benzoate and middle chain triglyceride (MCT, trade name, ODO, Nisshin Oillio, density ¼ 0.95 g/ml (20 1C)) were provided by San-Ei Gen F.F.I., Inc. Distilled water (Bibby Merit W4000) was used in all experiments.
Table 1 Molecular weight parameters of the control and matured gum arabic obtained by GPC-MALLS analysis (Aoki, Katayama, Al-Assaf, & Phillips, 2006) Sample name
Processing
Molecular weight MW (g/mol)
% mass
Rg (nm)
Control A. senegal FR-2876
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
6.22 105 2.54 106 3.96 105
10.6 89.4
28.5 41.1 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
1.23 106 6.58 106 4.13 105
13.2 86.8
59.0 68.7 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
1.66 106 8.56 106 4.16 105
15.3 84.7
64.2 70.9 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
2.54 106 1.16 107 4.50 105
18.6 81.4
85.1 89.8 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
1.77 106 7.84 106 4.16 105
18.2 81.8
68.4 75.5 —
FR-2877
FR-2878
FR-2879
Acacia (sen) SUPER GUMTM EM2
AGP: arabinogalactan protein; AG: arabinogalactan; GP: glycoprotein.
ARTICLE IN PRESS H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
355
2.2. Preparation of O/W emulsions using a Polytron
3. Results
MCT was selected as the dispersed phase, since the density (0.95 g/ml) is close to that of water; it has no smell, and is stable to oxidization. Therefore, it is suitable for preparing basic emulsions. The test material was dissolved in distilled water. After complete dissolution, citric acid was added to adjust the pH to 4, sodium benzoate was added as a preservative, and MCT was added as the disperse phase. The composition was: test material 5–20 w/w%, citric acid 0.12 w/w%, sodium benzoate 0.13 w/w% and MCT 20 w/w%. In a typical experiment, aqueous solutions of the test material (30 w/w% based on the solid content), 5 w/v% citric acid aqueous solution, and 5 w/v% sodium benzoate aqueous solution were first prepared. Then, 5–20 g of gum arabic solution was mixed with 0.72 ml of the citric acid solution, 0.78 ml of the sodium benzoate solution, 2.5–17.5 g of distilled water, and 6.0 g MCT to give a total 30 g in a 100 ml glass container. The mixture was vortexed vigorously for 1 min. Emulsions were prepared using a Polytron PT-2100 homogenizer at 26,000 rpm for 10 min in an ice water bath. PTDA2112/2EC (9 mm tip diameter, tip speed 10.4 m/s) was used as the dispersing tool.
3.1. Effect of test material concentration on droplet size and stability of the emulsion prepared using the Polytron
The emulsions were also prepared by the high-pressure homogenization method. MCT was added to a premixed aqueous solution of test material, citric acid and sodium benzoate whilst stirring. The pre-emulsified mixture was homogenized in four passes in order to achieve effective disaggregation of the gum using a two-stage high-pressure homogenizer (APV GAULIN 15MR-8TA) at 34 MPa during the first stage and 5 MPa during the second stage. The emulsion composition was: 5–20 w/w% of test material, citric acid 0.12 w/w%, sodium benzoate 0.13 w/w% and MCT 20 w/w%.
2.4. Particle size analysis The particle size distribution of the emulsions was analyzed using a Mastersizer 2000 laser diffractometer (Malvern Instruments). Distilled water was used as the dispersant, and a refractive index of 1.450 was used for MCT. The volume median diameter (VMD) was used to describe the particle size of the emulsions.
3.2. Effect of homogenization on droplet size and stability of the emulsion The Polytron emulsification method alone does not appear effective enough to produce sufficiently small particles. Thus, a high-pressure homogenizer was used to make the emulsions. Using this method, smaller particles can be produced, and the droplet size is also reduced as the molecular weight of the test material is increased (Fig. 2). Fig. 3 is a comparison of the concentrations required for the various gums to produce a droplet size of 0.7 mm. As the molecular weight increased, the droplet size decreased at each of the concentrations used. Thus, homogenization pressure has a profound effect on the emulsion droplet size and results in an improved performance by the higher molecular weight gums. In tests using sample EM2, there was almost a 50% reduction in the gum concentration required to achieve the lowest droplet size under these conditions. The relative stabilities of the various samples during accelerated stress testing are shown in Fig. 4. The maturated samples FR-2877–FR-2879 and Acacia (sen) 8 Volume Median Diameter (µm)
2.3. Preparation of O/W emulsions using a homogenizer
Fig. 1 shows the behavior of the emulsions prepared using only the Polytron, with the test material concentration varied from 5 to 20 w/w% for samples FR-2876–FR2879, Acacia (sen) SUPER GUMTM EM2. There was a decrease in particle size as the concentration of the test material increased. However, there does not appear to be any significant difference in behavior with increasing molecular weight.
7 6 5 4 3 2 1 0 0
2.5. Accelerated temperature stress test The prepared emulsion samples were kept at 60 1C (Gallenkamp, OVA031.XX1.5) for 3 d and the particle size was then re-measured using a Mastersizer 2000.
5
10
15
20
25
Gum arabic (w/w%) Fig. 1. Effect of the molecular weight and concentration of the control and matured Acacia senegal samples on particle size. Emulsions were prepared using the Polytron. (J) FR-2876; (K) FR-2877; (’) FR-2878; (E) FR-2879; (.) Acacia (sen) SUPER GUMTM EM2.
ARTICLE IN PRESS H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
Volume Median Diameter (µm)
356
1.1
10%
1.0
FR-2876 15% 20% 10%
0.9
FR-2877 15% 20%
0.8
10%
0.7
FR-2878 15% 20% 10%
0.6
FR-2879 15% 20%
0.5 0
5
15
10
20
25
Gum arabic (w/w%) Fig. 2. Effect of the molecular weight and concentration of the control and matured Acacia senegal samples on particle size. Emulsions were prepared using the high-pressure homogenizer. (J) FR-2876; (K) FR2877; (’) FR-2878; (E) FR-2879; (.) Acacia (sen) SUPER GUMTM EM2.
10% EM2 15% 20% 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Volume Median Diameter (µm) Fig. 4. Stability of emulsions prepared using the control and matured Acacia senegal. The emulsions were prepared using the high-pressure homogenizer and stored at 60 1C for 3 d. Open bar, initial VMD; closed bar, VMD after accelerated test.
20
Usage of gum arabic (%)
100%
15
77%
72% 61% 53%
10
5
0 FR-2876 FR-2877 FR-2878 FR-2879
EM2
Fig. 3. The reduction in the amount of gum required to produce emulsions with a particle size of 0.7 mm. Data was estimated from Fig. 2. The percentage values represent the amount of gum arabic required to prepare 0.7 mm oil droplets. The amount of control required to make a 0.7 mm droplet is represented as 100%.
SUPER GUMTM EM2 maintained stable emulsions over the test period and were superior to the control gum (FR2876). Thus, a smaller droplet size was obtained by enhancing the maturation process to increase molecular weight and the amount of AGP. 4. Discussion The maturated samples have a greater average molecular weight (2–4 times greater), a greater proportion of AGP (an increase from 10% to 18%) and an increased AGP molecular weight (e.g., from 2.5 to 7.8 106 Da for Acacia (sen) SUPER GUMTM EM2) compared with the control A. senegal, which is a conventional commercial gum arabic.
The first experimental procedure using only the Polytron was disappointing; there was not a great deal of difference in the performance of the various samples tested and this can be attributed to the lack of power obtained using the Polytron alone to make small emulsions. A considerable difference in both the droplet size and the stability of the emulsion with increasing molecular weight was obtained with the high-pressure homogenizer. FR-2877 (MW 1.23 106 Da), FR-2878 (MW 1.66 106 Da), FR-2879 (MW 2.54 106 Da), and Acacia (sen) SUPER GUMTM EM2 (MW 1.77 106 Da) gave stable emulsions. These were superior to the control gum (MW 6.22 105 Da). The controlling parameters, which probably account for the molecular weight effect, which we have observed, are the AGP content and MW of the samples (Table 1). Acacia (sen) SUPER GUMTM EM2 has 18.2% AGP compared with 10.6% for the control gum and it also has a MW of 7.8 106 Da compared with 2.5 106 Da for the control gum. There is a general consensus about the mechanism which enables A. senegal to be such an effective emulsifier (Randall et al., 1988, 1989). The predominantly hydrophobic and protein-rich backbone adsorbs on to the oil interface while the hydrophilic carbohydrate units, containing a small amount of charged groups, protrude into the aqueous solution. In this configuration, it is possible that some electrostatic repulsion from the charged components, along with steric forces, combine to produce electrostatic stabilization (Islam et al., 1997; Jayme et al., 1999). After the adsorption of the gum on to the oil particle surface, the repulsive interaction between two such particles will increase due to the steric hindrance of the polysaccharide moiety. Some accompanying electrostatic
ARTICLE IN PRESS H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
stabilization may arise due to the mutual repulsion of the electrical double layer on both particles. The electrostatic contribution to this stabilization can be quantified by the zeta potential, calculated from the electrophoretic mobility (Hunter, 1981). Jayme et al. (1999) demonstrated the presence of a finite surface charge at the oil droplet surface, which suggests that there is a residual charge at the arabinogalactan (AG)—water interface. This could be due to the uronic acid groups associated with the extremities of the AG structure. Thus, there would be a strong electrostatic barrier for flocculation and coalescence of the oil droplets arising from the polysaccharide blocks protruding towards the emulsion continuous phase from neighboring droplet surfaces, where they are anchored through the more hydrophobic protein moiety of the gum (Dickinson, 2003). A significant result is that pressure homogenization is necessary to produce the smallest droplet size, which in turn results in greater stabilization of the emulsions. Pressure processing (2000 kg/cm2, 196 MPa) of soya bean isolates also led to a reduction in droplet size in emulsions prepared with sunflower oil and increased the percentage of adsorbed protein, possibly due to an increase in protein surface hydrophobicity (Puppo et al., 2005). The interfacial properties of proteins have been shown to be modified by high pressure (Galazka, Dickinson, & Ledward, 2000).
357
Compared with the accepted Mark–Houwink constants for Acacia, the matured gums have a Mark–Houwink slope close to zero, corresponding to Mark–Houwink constants of K ¼ 3.36, a ¼ 0.120 (Aoki et al., 2006). This deviation from the conventional behavior indicates that the matured gum Acacia (sen) SUPER GUMTM EM2 has a highly branched structure, so that the aggregating protein forms a more compact structure than would occur if the protein simply extended the polypeptide chain of the basic AGP. Pressure homogenization may unfold this much larger globular structure and so allow the hydrophobic protein moiety to absorb on the surface of the oil droplet and hence provide effective emulsification at a considerably lower concentration. A similar proposal was advanced to explain how pectin is able to produce as fine and stable emulsions as gum arabic but at a much lower dosage (LeRoux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003). Pectin is a semi-flexible polymer, whereas the AGP complex of gum arabic is a coil with a small radius of gyration (Table 1). Due to the more extended conformation of the pectin molecule, it would take up a greater volume around the droplets. The same explanation could account for the lower usage of Acacia (sen) SUPER GUMTM EM2 compared with a standard commercial gum arabic of ca. 5–6 105 (Al-Assaf, Phillips, & William, 2005). The model is illustrated in Fig. 5. Pressure uncoiling of the protein could provide an extended conformation
AG
Matured AGP AGP Protein unfolds with pressure and the conformation changes
Hydrophobic protein chain anchors at the interface
Oil phase
Fig. 5. Proposed model of gum arabic adsorption on to the oil droplet.
ARTICLE IN PRESS 358
H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
with the AGP in an extended form that would occupy a greater space around the oil droplet. The large AGP molecule can interact extensively with the oil droplet and would be thermodynamically more stable than a small AGP molecule, which is able to interact with only a small area of the interface. The small AGP molecule may dissociate from the oil surface during collision of the droplets or heat treatment. This would account for its greater overall efficiency in terms of amount needed to produce the smallest droplet size and the greater stability of the emulsion.
References Al-Assaf, S., Phillips, G. O., & William, P. A. (2005). Studies on Acacia exudate gums, Part I: The molecular weight of Acacia senegal gum exudates. Food Hydrocolloids, 19(4), 647–660. Al-Assaf, S., Phillips, G.O., Aoki, H., Sasaki, Y. (2005). Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUMTM), Part 1: Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier. Food Hydrocolloids, in press, doi:10.1016/j.foodhyd.2006.04.011. Aoki, H., Katayama, T., Al-Assaf, S., Phillips, G.O. (2006). Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUMTM): Part 2. Mechanism of the maturation process. Food Hydrocolloids, in press, doi:10.1016/j.foodhyd.2006.04.002. Dickinson, E. (1995). Emulsion stabilization by polysaccharides and protein–polysaccharide complexes. In A. M. Stephen (Ed.), Food polysaccharides and their applications, (pp. 501–515). New York: Dekker. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17(1), 25–39.
Dickinson, E., Elverson, D. J., & Murray, B. S. (1989). On the filmforming and emulsion-stabilizing properties of gum arabic: Dilution and flocculation aspects. Food Hydrocolloids, 3(2), 101–114. Galazka, V. B., Dickinson, E., & Ledward, D. A. (2000). Emulsifying properties of ovalbumin in mixtures with sulphated polysaccharides: Effects of pH, ionic strength, heat and high-pressure treatment. Journal of the Science of Food and Agriculture, 80(8), 1219–1229. Hunter, R. J. (1981). Zeta potential in colloid science. London: Academic Press. Islam, A. M., Phillips, G. O., Sljivo, A., Snowden, M. J., & Williams, P. A. (1997). A review of recent developments on the regulatory, structural and functional aspects of gum arabic. Food Hydrocolloids, 11(4), 493–505. Jayme, M. L., Dunstan, D. E., & Gee, M. L. (1999). Zeta potentials of gum arabic stabilised oil-in-water emulsions. Food Hydrocolloids, 13(6), 459–465. LeRoux, J., Langendorff, V., Schick, G., Vaishnav, V., & Mazoyer, J. (2003). Emulsion stabilizing properties of pectin. Food Hydrocolloids, 17(4), 455–462. McNamee, B. F., O’Riordan, E. D., & O’Sullivan, M. (1998). Emulsification and microencapsulation properties of gum arabic. Journal of Agricultural and Food Chemistry, 46(11), 4551–4555. Puppo, M. C., Speroni, F., Chapleau, N., de Lamballerie, M., An˜o´n, M. C., & Anton, M. (2005). Effect of high-pressure treatment on emulsifying properties of soybean proteins. Food Hydrocolloids, 19(2), 289–296. Randall, R. C., Phillips, G. O., & Williams, P. A. (1988). The role of the proteinaceous component on the emulsifying properties of gum arabic. Food Hydrocolloids, 2(2), 131–140. Randall, R. C., Phillips, G. O., & Williams, P. A. (1989). Fractionation and characterization of gum from Acacia senegal. Food Hydrocolloids, 3(1), 65–76. Ray, A. K., Bird, P. B., Iacobucci, G. A., & Clark, B. C., Jr. (1995). Functionality of gum arabic: Fractionation, characterization and evaluation of gum fractions in citrus oil emulsions and model beverages. Food Hydrocolloids, 9(2), 123–131. Walstra, P. (2003). Physical chemistry of foods. New York: Marcel Dekker.
FOOD
HYDROCOLLOIDS Food Hydrocolloids 21 (2007) 353–358 www.elsevier.com/locate/foodhyd
Characterization and properties of Acacia senegal (L.) Willd. var. TM Senegal with enhanced properties (Acacia (sen) SUPER GUM ): Part 5. Factors affecting the emulsification of Acacia senegal and Acacia (sen) SUPER GUMTM Hiromitsu Aokia,b, Tsuyoshi Katayamaa, Takeshi Ogasawaraa, Yasushi Sasakia, Saphwan Al-Assafb, Glyn O. Phillipsb,c, a San-Ei Gen F.F.I., Inc., 1-1-11 Sanwa-cho, Toyonaka, Osaka 561-8588, Japan Glyn O. Phillips Hydrocolloids Research Centre, The North East Wales Institute, Plas Coch, Mold Road, Wrexham LL11 2AW, UK c Phillips Hydrocolloid Research Ltd., 45 Old Bond Street, London W1S 4AQ, UK
b
Received 19 December 2005; accepted 13 April 2006
Abstract Acacia senegal is the major gum arabic species used in commercial emulsification for the production of beverages and flavor concentrates. This paper examines the effect of gum concentration, molecular weight parameters and homogenization conditions on the droplet size and stability of emulsions prepared using a conventional commercial A. senegal product and a series of A. senegal test gums that were prepared by a controlled maturation process, including a new commercial product produced by this process (Acacia (sen) SUPER GUMTM EM2). Pressure homogenization after vigorous stirring produced the best results. At concentrations below 20%, commercial gum arabic weight average molecular weight MW ca. 6 105 Da produced emulsions which were not stable under accelerated stress conditions. When the weight average MW was increased to a range 1–2.5 106 Da, the droplet size was reduced and the stability was greatly increased, even at a 5% gum concentration. After pressure homogenization, sub-micron droplet sizes could be produced which exhibited complete stability during stress testing at 60 1C and after longer-term storage. A model is proposed to account for the greater effectiveness of the matured gums, whereby the arabinogalactan protein complex unfolds as a result of pressure homogenization and occupies a greater volume around the oil droplet, which in turn produces greater stabilization. r 2006 Elsevier Ltd. All rights reserved. Keywords: Acacia senegal; SUPER GUMTM; Molecular weight; Emulsification; Pressure homogenization
1. Introduction Acacia senegal (gum arabic) is an important naturally occurring oil-in-water emulsifier, which is in regular use in the food industry. The emulsions are prepared by homogenizing the oil and water phases in the presence of gum arabic, preferably under pressure. The gum arabic forms a Corresponding author. Glyn O. Phillips Hydrocolloids Research Centre, The North East Wales Institute, Plas Coch, Mold Road, Wrexham LL11 2AW, UK. Tel.: +44 29 20 843298; fax: +44 29 20 843145. E-mail address: [email protected] (G.O. Phillips).
0268-005X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.04.014
protective layer around the oil droplets that prevents the droplets from aggregating (flocculating and/or coalescing). It also reduces the oil–water interfacial tension, thereby facilitating the disruption of emulsion droplets during homogenization (Walstra, 2003). In reviewing the behavior of various hydrocolloid emulsifiers, Dickinson (2003) stated that ‘‘much of the reported emulsifying capability of polysaccharides is explicable in terms of complexation or contamination with a small fraction of surface-active protein’’. This is certainly true of gum arabic. Randall, Phillips, and Williams (1988, 1989) showed that A. senegal was a polydisperse mixture of components and that it was
ARTICLE IN PRESS 354
H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
the arabinogalactan protein (AGP) component which is responsible for the emulsification properties. Several polysaccharide units are linked to a common protein core in this high molecular weight component (MW ca. 2.5 106 Da). The methods of fractionation and quantification of these individual components have been described previously (Aoki, Katayama, Al-Assaf, & Phillips, 2006). The AGP-rich fraction of A. senegal gum is adsorbed on the oil–water interface. The hydrophobic protein component of the AGP firmly anchors the protein–polysaccharide moiety at the interface, and the protruding hydrophilic carbohydrate blocks which are attached to this chain provide a strong steric barrier against flocculation and coalescence (Dickinson, 1995; Islam, Phillips, Sljivo, Snowden, & Williams, 1997; Randall et al., 1989). Whilst charged groups provide the basis for some electrostatic contribution to the colloidal stabilization, the relatively low value of the (negative) zeta potential, 10 to 20 mV under beverage emulsion conditions (Jayme, Dunstan, & Gee, 1999; Ray, Bird, Iacobucci, & Clark, 1995), indicates that the primary stabilization mechanism is steric in character. The level of surface activity is rather low in comparison with typical food protein emulsifiers. Therefore, to generate stable sub-micron-sized droplets, it is necessary to use a rather high gum-to-oil weight ratio, approximately 1:1, as compared with 1:10 for equivalent protein-stabilized emulsions (Dickinson, 2003; McNamee, O’Riordan, & O’Sullivan, 1998). Once formed by adsorption on to the macroscopic oil–water interface, there is little effect on the high surface shear viscosity of the gum arabic film by large dilutions of the aqueous sub-phase (Dickinson, Elverson, & Murray, 1989). The film viscoelasticity is, therefore, maintained even when a major proportion of the hydrocolloid has been removed from the aqueous phase in
contact with the adsorbed layer. Thus, only a small proportion of gum arabic used in emulsion preparation is actually involved in the stabilization process. The objective of this investigation is to create more favorable conditions for emulsification using A. senegal by increasing both the amount and the molecular weight of the AGP component present in the gum. Here, the emulsification performance of the enhanced Acacia gums will be compared with a conventional gum arabic having an average molecular weight of ca. 5–6 105. The objective is to reduce the amount of gum required and still produce a consistent and stable emulsion.
2. Materials and methods 2.1. Materials For this investigation, conventional control A. senegal (FR-2876), test materials (FR-2877, FR-2878 and FR-2879 and Acacia (sen) SUPER GUMTM EM2) which were previously described by Aoki et al. (2006) were used. All samples were produced by the heating maturation process previously described. FR-samples were prepared on a laboratory scale and the sample designated EM2 was produced on a commercial scale (Al-Assaf, Phillips, Aoki, & Sasaki, 2006; Aoki et al., 2006). The molecular parameters are summarized in Table 1. Citric acid, sodium benzoate and middle chain triglyceride (MCT, trade name, ODO, Nisshin Oillio, density ¼ 0.95 g/ml (20 1C)) were provided by San-Ei Gen F.F.I., Inc. Distilled water (Bibby Merit W4000) was used in all experiments.
Table 1 Molecular weight parameters of the control and matured gum arabic obtained by GPC-MALLS analysis (Aoki, Katayama, Al-Assaf, & Phillips, 2006) Sample name
Processing
Molecular weight MW (g/mol)
% mass
Rg (nm)
Control A. senegal FR-2876
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
6.22 105 2.54 106 3.96 105
10.6 89.4
28.5 41.1 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
1.23 106 6.58 106 4.13 105
13.2 86.8
59.0 68.7 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
1.66 106 8.56 106 4.16 105
15.3 84.7
64.2 70.9 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
2.54 106 1.16 107 4.50 105
18.6 81.4
85.1 89.8 —
One peak Two peaks (1, AGP) Two peaks (2, AG+GP)
1.77 106 7.84 106 4.16 105
18.2 81.8
68.4 75.5 —
FR-2877
FR-2878
FR-2879
Acacia (sen) SUPER GUMTM EM2
AGP: arabinogalactan protein; AG: arabinogalactan; GP: glycoprotein.
ARTICLE IN PRESS H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
355
2.2. Preparation of O/W emulsions using a Polytron
3. Results
MCT was selected as the dispersed phase, since the density (0.95 g/ml) is close to that of water; it has no smell, and is stable to oxidization. Therefore, it is suitable for preparing basic emulsions. The test material was dissolved in distilled water. After complete dissolution, citric acid was added to adjust the pH to 4, sodium benzoate was added as a preservative, and MCT was added as the disperse phase. The composition was: test material 5–20 w/w%, citric acid 0.12 w/w%, sodium benzoate 0.13 w/w% and MCT 20 w/w%. In a typical experiment, aqueous solutions of the test material (30 w/w% based on the solid content), 5 w/v% citric acid aqueous solution, and 5 w/v% sodium benzoate aqueous solution were first prepared. Then, 5–20 g of gum arabic solution was mixed with 0.72 ml of the citric acid solution, 0.78 ml of the sodium benzoate solution, 2.5–17.5 g of distilled water, and 6.0 g MCT to give a total 30 g in a 100 ml glass container. The mixture was vortexed vigorously for 1 min. Emulsions were prepared using a Polytron PT-2100 homogenizer at 26,000 rpm for 10 min in an ice water bath. PTDA2112/2EC (9 mm tip diameter, tip speed 10.4 m/s) was used as the dispersing tool.
3.1. Effect of test material concentration on droplet size and stability of the emulsion prepared using the Polytron
The emulsions were also prepared by the high-pressure homogenization method. MCT was added to a premixed aqueous solution of test material, citric acid and sodium benzoate whilst stirring. The pre-emulsified mixture was homogenized in four passes in order to achieve effective disaggregation of the gum using a two-stage high-pressure homogenizer (APV GAULIN 15MR-8TA) at 34 MPa during the first stage and 5 MPa during the second stage. The emulsion composition was: 5–20 w/w% of test material, citric acid 0.12 w/w%, sodium benzoate 0.13 w/w% and MCT 20 w/w%.
2.4. Particle size analysis The particle size distribution of the emulsions was analyzed using a Mastersizer 2000 laser diffractometer (Malvern Instruments). Distilled water was used as the dispersant, and a refractive index of 1.450 was used for MCT. The volume median diameter (VMD) was used to describe the particle size of the emulsions.
3.2. Effect of homogenization on droplet size and stability of the emulsion The Polytron emulsification method alone does not appear effective enough to produce sufficiently small particles. Thus, a high-pressure homogenizer was used to make the emulsions. Using this method, smaller particles can be produced, and the droplet size is also reduced as the molecular weight of the test material is increased (Fig. 2). Fig. 3 is a comparison of the concentrations required for the various gums to produce a droplet size of 0.7 mm. As the molecular weight increased, the droplet size decreased at each of the concentrations used. Thus, homogenization pressure has a profound effect on the emulsion droplet size and results in an improved performance by the higher molecular weight gums. In tests using sample EM2, there was almost a 50% reduction in the gum concentration required to achieve the lowest droplet size under these conditions. The relative stabilities of the various samples during accelerated stress testing are shown in Fig. 4. The maturated samples FR-2877–FR-2879 and Acacia (sen) 8 Volume Median Diameter (µm)
2.3. Preparation of O/W emulsions using a homogenizer
Fig. 1 shows the behavior of the emulsions prepared using only the Polytron, with the test material concentration varied from 5 to 20 w/w% for samples FR-2876–FR2879, Acacia (sen) SUPER GUMTM EM2. There was a decrease in particle size as the concentration of the test material increased. However, there does not appear to be any significant difference in behavior with increasing molecular weight.
7 6 5 4 3 2 1 0 0
2.5. Accelerated temperature stress test The prepared emulsion samples were kept at 60 1C (Gallenkamp, OVA031.XX1.5) for 3 d and the particle size was then re-measured using a Mastersizer 2000.
5
10
15
20
25
Gum arabic (w/w%) Fig. 1. Effect of the molecular weight and concentration of the control and matured Acacia senegal samples on particle size. Emulsions were prepared using the Polytron. (J) FR-2876; (K) FR-2877; (’) FR-2878; (E) FR-2879; (.) Acacia (sen) SUPER GUMTM EM2.
ARTICLE IN PRESS H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
Volume Median Diameter (µm)
356
1.1
10%
1.0
FR-2876 15% 20% 10%
0.9
FR-2877 15% 20%
0.8
10%
0.7
FR-2878 15% 20% 10%
0.6
FR-2879 15% 20%
0.5 0
5
15
10
20
25
Gum arabic (w/w%) Fig. 2. Effect of the molecular weight and concentration of the control and matured Acacia senegal samples on particle size. Emulsions were prepared using the high-pressure homogenizer. (J) FR-2876; (K) FR2877; (’) FR-2878; (E) FR-2879; (.) Acacia (sen) SUPER GUMTM EM2.
10% EM2 15% 20% 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Volume Median Diameter (µm) Fig. 4. Stability of emulsions prepared using the control and matured Acacia senegal. The emulsions were prepared using the high-pressure homogenizer and stored at 60 1C for 3 d. Open bar, initial VMD; closed bar, VMD after accelerated test.
20
Usage of gum arabic (%)
100%
15
77%
72% 61% 53%
10
5
0 FR-2876 FR-2877 FR-2878 FR-2879
EM2
Fig. 3. The reduction in the amount of gum required to produce emulsions with a particle size of 0.7 mm. Data was estimated from Fig. 2. The percentage values represent the amount of gum arabic required to prepare 0.7 mm oil droplets. The amount of control required to make a 0.7 mm droplet is represented as 100%.
SUPER GUMTM EM2 maintained stable emulsions over the test period and were superior to the control gum (FR2876). Thus, a smaller droplet size was obtained by enhancing the maturation process to increase molecular weight and the amount of AGP. 4. Discussion The maturated samples have a greater average molecular weight (2–4 times greater), a greater proportion of AGP (an increase from 10% to 18%) and an increased AGP molecular weight (e.g., from 2.5 to 7.8 106 Da for Acacia (sen) SUPER GUMTM EM2) compared with the control A. senegal, which is a conventional commercial gum arabic.
The first experimental procedure using only the Polytron was disappointing; there was not a great deal of difference in the performance of the various samples tested and this can be attributed to the lack of power obtained using the Polytron alone to make small emulsions. A considerable difference in both the droplet size and the stability of the emulsion with increasing molecular weight was obtained with the high-pressure homogenizer. FR-2877 (MW 1.23 106 Da), FR-2878 (MW 1.66 106 Da), FR-2879 (MW 2.54 106 Da), and Acacia (sen) SUPER GUMTM EM2 (MW 1.77 106 Da) gave stable emulsions. These were superior to the control gum (MW 6.22 105 Da). The controlling parameters, which probably account for the molecular weight effect, which we have observed, are the AGP content and MW of the samples (Table 1). Acacia (sen) SUPER GUMTM EM2 has 18.2% AGP compared with 10.6% for the control gum and it also has a MW of 7.8 106 Da compared with 2.5 106 Da for the control gum. There is a general consensus about the mechanism which enables A. senegal to be such an effective emulsifier (Randall et al., 1988, 1989). The predominantly hydrophobic and protein-rich backbone adsorbs on to the oil interface while the hydrophilic carbohydrate units, containing a small amount of charged groups, protrude into the aqueous solution. In this configuration, it is possible that some electrostatic repulsion from the charged components, along with steric forces, combine to produce electrostatic stabilization (Islam et al., 1997; Jayme et al., 1999). After the adsorption of the gum on to the oil particle surface, the repulsive interaction between two such particles will increase due to the steric hindrance of the polysaccharide moiety. Some accompanying electrostatic
ARTICLE IN PRESS H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
stabilization may arise due to the mutual repulsion of the electrical double layer on both particles. The electrostatic contribution to this stabilization can be quantified by the zeta potential, calculated from the electrophoretic mobility (Hunter, 1981). Jayme et al. (1999) demonstrated the presence of a finite surface charge at the oil droplet surface, which suggests that there is a residual charge at the arabinogalactan (AG)—water interface. This could be due to the uronic acid groups associated with the extremities of the AG structure. Thus, there would be a strong electrostatic barrier for flocculation and coalescence of the oil droplets arising from the polysaccharide blocks protruding towards the emulsion continuous phase from neighboring droplet surfaces, where they are anchored through the more hydrophobic protein moiety of the gum (Dickinson, 2003). A significant result is that pressure homogenization is necessary to produce the smallest droplet size, which in turn results in greater stabilization of the emulsions. Pressure processing (2000 kg/cm2, 196 MPa) of soya bean isolates also led to a reduction in droplet size in emulsions prepared with sunflower oil and increased the percentage of adsorbed protein, possibly due to an increase in protein surface hydrophobicity (Puppo et al., 2005). The interfacial properties of proteins have been shown to be modified by high pressure (Galazka, Dickinson, & Ledward, 2000).
357
Compared with the accepted Mark–Houwink constants for Acacia, the matured gums have a Mark–Houwink slope close to zero, corresponding to Mark–Houwink constants of K ¼ 3.36, a ¼ 0.120 (Aoki et al., 2006). This deviation from the conventional behavior indicates that the matured gum Acacia (sen) SUPER GUMTM EM2 has a highly branched structure, so that the aggregating protein forms a more compact structure than would occur if the protein simply extended the polypeptide chain of the basic AGP. Pressure homogenization may unfold this much larger globular structure and so allow the hydrophobic protein moiety to absorb on the surface of the oil droplet and hence provide effective emulsification at a considerably lower concentration. A similar proposal was advanced to explain how pectin is able to produce as fine and stable emulsions as gum arabic but at a much lower dosage (LeRoux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003). Pectin is a semi-flexible polymer, whereas the AGP complex of gum arabic is a coil with a small radius of gyration (Table 1). Due to the more extended conformation of the pectin molecule, it would take up a greater volume around the droplets. The same explanation could account for the lower usage of Acacia (sen) SUPER GUMTM EM2 compared with a standard commercial gum arabic of ca. 5–6 105 (Al-Assaf, Phillips, & William, 2005). The model is illustrated in Fig. 5. Pressure uncoiling of the protein could provide an extended conformation
AG
Matured AGP AGP Protein unfolds with pressure and the conformation changes
Hydrophobic protein chain anchors at the interface
Oil phase
Fig. 5. Proposed model of gum arabic adsorption on to the oil droplet.
ARTICLE IN PRESS 358
H. Aoki et al. / Food Hydrocolloids 21 (2007) 353–358
with the AGP in an extended form that would occupy a greater space around the oil droplet. The large AGP molecule can interact extensively with the oil droplet and would be thermodynamically more stable than a small AGP molecule, which is able to interact with only a small area of the interface. The small AGP molecule may dissociate from the oil surface during collision of the droplets or heat treatment. This would account for its greater overall efficiency in terms of amount needed to produce the smallest droplet size and the greater stability of the emulsion.
References Al-Assaf, S., Phillips, G. O., & William, P. A. (2005). Studies on Acacia exudate gums, Part I: The molecular weight of Acacia senegal gum exudates. Food Hydrocolloids, 19(4), 647–660. Al-Assaf, S., Phillips, G.O., Aoki, H., Sasaki, Y. (2005). Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUMTM), Part 1: Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier. Food Hydrocolloids, in press, doi:10.1016/j.foodhyd.2006.04.011. Aoki, H., Katayama, T., Al-Assaf, S., Phillips, G.O. (2006). Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUMTM): Part 2. Mechanism of the maturation process. Food Hydrocolloids, in press, doi:10.1016/j.foodhyd.2006.04.002. Dickinson, E. (1995). Emulsion stabilization by polysaccharides and protein–polysaccharide complexes. In A. M. Stephen (Ed.), Food polysaccharides and their applications, (pp. 501–515). New York: Dekker. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17(1), 25–39.
Dickinson, E., Elverson, D. J., & Murray, B. S. (1989). On the filmforming and emulsion-stabilizing properties of gum arabic: Dilution and flocculation aspects. Food Hydrocolloids, 3(2), 101–114. Galazka, V. B., Dickinson, E., & Ledward, D. A. (2000). Emulsifying properties of ovalbumin in mixtures with sulphated polysaccharides: Effects of pH, ionic strength, heat and high-pressure treatment. Journal of the Science of Food and Agriculture, 80(8), 1219–1229. Hunter, R. J. (1981). Zeta potential in colloid science. London: Academic Press. Islam, A. M., Phillips, G. O., Sljivo, A., Snowden, M. J., & Williams, P. A. (1997). A review of recent developments on the regulatory, structural and functional aspects of gum arabic. Food Hydrocolloids, 11(4), 493–505. Jayme, M. L., Dunstan, D. E., & Gee, M. L. (1999). Zeta potentials of gum arabic stabilised oil-in-water emulsions. Food Hydrocolloids, 13(6), 459–465. LeRoux, J., Langendorff, V., Schick, G., Vaishnav, V., & Mazoyer, J. (2003). Emulsion stabilizing properties of pectin. Food Hydrocolloids, 17(4), 455–462. McNamee, B. F., O’Riordan, E. D., & O’Sullivan, M. (1998). Emulsification and microencapsulation properties of gum arabic. Journal of Agricultural and Food Chemistry, 46(11), 4551–4555. Puppo, M. C., Speroni, F., Chapleau, N., de Lamballerie, M., An˜o´n, M. C., & Anton, M. (2005). Effect of high-pressure treatment on emulsifying properties of soybean proteins. Food Hydrocolloids, 19(2), 289–296. Randall, R. C., Phillips, G. O., & Williams, P. A. (1988). The role of the proteinaceous component on the emulsifying properties of gum arabic. Food Hydrocolloids, 2(2), 131–140. Randall, R. C., Phillips, G. O., & Williams, P. A. (1989). Fractionation and characterization of gum from Acacia senegal. Food Hydrocolloids, 3(1), 65–76. Ray, A. K., Bird, P. B., Iacobucci, G. A., & Clark, B. C., Jr. (1995). Functionality of gum arabic: Fractionation, characterization and evaluation of gum fractions in citrus oil emulsions and model beverages. Food Hydrocolloids, 9(2), 123–131. Walstra, P. (2003). Physical chemistry of foods. New York: Marcel Dekker.