Stabilization mechanism of oil-in-water emulsions by β-lactoglobulin and gum arabic

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Abstract

Natural biopolymer stabilized oil-in-water emulsions were formulated using β-lactoglobulin (β-lg), gum arabic (GA), and β-lg:GA solutions as an alternative to synthetic surfactants. Emulsions using these biopolymers and their complexes were formulated varying the biopolymer total concentration, the protein-to-polysaccharide ratio, and the emulsification protocol.

This work showed that whereas β-lg enabled the formulation of emulsions at concentration as low as 0.5 (w/w)%, GA allowed to obtain emulsions at concentrations equal to or higher than 2.5 (w/w)%. In order to improve emulsion stability, β-lg and GA were complexed through strong attractive electrostatic interactions. GA solution had to be added to previously prepared β-lg emulsions in order to obtain stable emulsions. Interfacial tension and interfacial rheological measurements allowed a better understanding of the possible stabilizing mechanism. β-lg and GA both induced a very effective decrease in interfacial tension and showed interfacial elastic behaviour. In the mixed system, β-lg adsorbed at the interface and GA electrostatically bound to it, leading to the formation of a bi-layer stabilized emulsion. However, emulsion stability was not improved compared to β-lg stabilized emulsion, probably due to depletion or bridging flocculation.

Graphical abstract

Schematic depiction of the stabilization mechanism of oil-in-water emulsion with gum arabic through electrostatic interactions with previously adsorbed β-lactoglobulin.

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Research highlights

► β-lg and GA form a complex with a very strong affinity at pH 4.2. ► β-lg and GA displays interfacial adsorption and elastic behaviour. ► Both biopolymers enabled the formulation of emulsions. ► In mixed systems, GA electrostatically interacts with the initially adsorbed β-lg.

Introduction

Emulsions are widely used in the formulation of food, pharmaceutical, and cosmetic products. They are not thermodynamically stable (except microemulsions) and can phase separate. Addition of emulsifiers allows the formulation of stable emulsions thanks to the formation of structured interfacial films. The present study aimed at formulating oil-in-water emulsions for dermatologic use. Our objective was to replace synthetic surfactants that are potentially irritating for skin [1] by “natural” emulsifiers and stabilizers in order to answer the increasing demand of natural and biodegradable products [2], [3]. This approach has already been applied in the food industry that currently uses proteins and polysaccharides as texturizing and stabilizing agents [4], [5], [6], [7]. Proteins are less effective than synthetic emulsifiers in decreasing interfacial tension, but they lead to thermodynamically and kinetically more stable emulsions [8], [9]. Indeed, they form a viscoelastic adsorbed layer onto the oil droplets and are able to generate repulsive steric and electrostatic interactions between these droplets [10]. Generally, proteins act as the main stabilizers and polysaccharides contribute to emulsion stability by their thickening and steric stabilizing behaviours [11]. Polysaccharides that do not adsorb at the globule interface would enhance the viscosity of the aqueous phase and thus slow down destabilizing mechanisms [12] such as creaming, flocculation, and coalescence. On the other hand, adsorbing ones would anchor at the interface by their hydrophobic residues, decrease the interfacial tension and form a steric barrier preventing coalescence [13].

Many studies have shown the interest of combining advantages of proteins and polysaccharides via the formation of protein:polysaccharide complexes to emulsify and stabilize emulsions [14], [15], [16], [17]. These complexes can be formed through covalent bonding or electrostatic interactions. In the latter case, the efficiency of a polysaccharide to form an interfacial complex with a protein depends on the distribution of ionized groups onto the protein surface and the stability of the protein structure, but also on the flexibility, charge distribution, and density of the polysaccharide [14], [18]. In this approach it is possible to formulate emulsions stabilized by multilayered interfacial membranes composed of alternatively oppositely charged biopolymers. Under appropriate formulation conditions, this multiple layer deposition has been shown to confer better stability to environmental stresses than conventional oil-in-water emulsions stabilized by a single interfacial biopolymer layer [17], [19], [20], [21], [22].

In this work, two biopolymers have been chosen to stabilize oil-in-water emulsions: β-lactoglobulin (β-lg), the major whey protein, and gum arabic (GA), an exudate from Acacia trees.

β-lg is a compact globular protein (monomer molar mass = 18.3 × 103 g/mol) containing 162 amino acid residues with one thiol group and two disulphide bonds. It exists in several genetic variants of which A (Gln59, Asp64, Val118) and B (Gln59, Gly64, Ala118) are the most abundant ones [23]. The isoelectric point (pI) of β-lg is about 5.2. The protein displays a global positive charge below this value and a negative charge above it. Once adsorbed at the interface, there is a structural reorganization of the protein in order to partially unfold and expose hydrophobic residues toward the lipophilic phase [8], [9].

GA is a hybrid polyelectrolyte containing both protein and polysaccharide subunits. It consists mainly of a mixture of arabinogalactan (AG) (80–90% of the total gum in weight), glycoprotein (GP) (2–4% of the total gum in weight), and arabinogalactan-protein (AGP) (10–20% of the total gum in weight) fractions [24]. Molar mass of the whole gum can vary from 3.0 × 105 to 5.8 × 105 g/mol depending on its origin and age [25]. GA carries a net negative charge for pH value above 2.0 conferred by its glucuronic acid residues. The AGP fraction has been considered to be responsible for the emulsifying properties of GA [26], [27], [28], [29]. Indeed, the protein component of the gum would embed in the oil phase while the carbohydrate component would extend into water [28], [30].

The combination of these two biopolymers is of interest to potentially induce a synergistic effect on emulsion stability [31]. Over the last decade, proteins and polysaccharides and their complexes have emerged in the pharmaceutical field mainly for microencapsulation [32], [33], [34]. However, to our knowledge, there is currently no marketed pharmaceutical emulsion stabilized by a combination of protein and polysaccharide. The aim of this study was thus to investigate the potentialities of β-lg, GA, and their mixtures to formulate sweet almond oil-in-water emulsions for dermatologic use. β-lg:GA complexation in solution was first studied by capillary electrophoresis. Affinity capillary electrophoresis (ACE) as a probe for studying molecular interactions has gained increasing popularity in the last few years [35]. The influence of biopolymer concentration, pH, protein-to-polysaccharide ratio, and emulsification process on emulsion stability was then thoroughly studied. In a last step, we have characterized the interactions of both biopolymers with the oil–water interface in order to better understand the emulsion stabilization mechanisms.

Section snippets

Materials

β-lg powder (lot JE 002-8-992) was supplied by Davisco Foods International, Inc. (USA). Its composition was 89.8 (w/w)% protein, 8.8 (w/w)% moisture, and 1.4 (w/w)% ash [36]. GA (INSTANTGUM AA) was a gift from CNI Company (Rouen, France). Its composition contained 10 (w/w)% moisture and 4 (w/w)% ash with a molecular weight of about 350,000 g/mol as reported by the supplier. The protein content was 2.5 (w/w)% as determined by the Kjeldahl analysis (N × 6.66). Sweet almond oil (lot 07120145A) was

Zeta potential measurements

Zeta potential values obtained for the solutions of β-lg, GA, and the β-lg:GA mixtures at both ratios (2:1 and 1:2) and pH 4.2 are gathered in Table 1. These values are in agreement with the pI of β-lg and the pKa of GA. In mixed aqueous dispersions (β-lg:GA), the ζ-potential value was negative and close to that of pure GA solutions. This shows the predominance of the gum, due to its larger size or to the way the biopolymers are complexed with one another: the protein might be hidden inside the

General discussion

The ACE appeared to be a satisfactory method to investigate the affinity between the two biopolymers in aqueous solutions. It showed that β-lg and GA could establish attractive electrostatic interactions with a binding constant in the order of 108 M−1. Interfacial measurements by the WPM showed that β-lg:GA complexes exhibited slightly improved interfacial properties compared to the single biopolymers.

Formulating emulsions with β-lg, GA, and their complexes, very different stabilities were

Conclusion

This study showed the interest of β-lg, GA, and their complexes for the formulation of pharmaceutical emulsions. Different experimental protocols were screened to understand biopolymers stabilization mechanisms. Two biopolymers, two concentrations, two ratios, and three formulation protocols were investigated. β-lg allowed the stabilization of emulsions at 0.5 (w/w)% and 2.5 (w/w)% concentrations. GA stabilized emulsions at 2.5% concentration but for only two days. The combination of β-lg and

Acknowledgments

We thank Dr. Nicolas Huang for useful discussions on the interfacial rheology measurements and Morgane Beigneux and Ni Zeng for their technical assistance in the experimental work. Dr. Claire Gaiani and Carole Jeandel (LIBio, ENSAIA-INPL) are acknowledged for the Kjeldahl analysis.

References (69)

  • G. Secchi

    Clin. Dermatol.

    (2008)
  • N. Garti

    Colloids Surf., A

    (1999)
  • R. Lutz et al.

    Colloids Surf., B

    (2009)
  • M.L. Jayme et al.

    Food Hydrocolloids

    (1999)
  • S.R. Euston et al.

    Food Hydrocolloids

    (2000)
  • X. Huang et al.

    Food Hydrocolloids

    (2001)
  • D.J. McClements

    Curr. Opin. Colloid Interface Sci.

    (2004)
  • C. Sun et al.

    Food Hydrocolloids

    (2007)
  • I. Capek

    Adv. Colloid Interface Sci.

    (2004)
  • D. Guzey et al.

    Adv. Colloid Interface Sci.

    (2006)
  • P. Thanasukarn et al.

    Food Res. Int.

    (2006)
  • Y.S. Gu et al.

    J. Agric. Food Eng.

    (2007)
  • T. Aoki et al.

    Food Hydrocolloids

    (2005)
  • M.A. Meza-Nieto et al.

    J. Dairy Sci.

    (2007)
  • O.H.M. Idris et al.

    Food Hydrocolloids

    (1998)
  • R.C. Randall et al.

    Food Hydrocolloids

    (1988)
  • L. Picton et al.

    Carbohydr. Polym.

    (2000)
  • E. Dickinson

    Food Hydrocolloids

    (2003)
  • M. Klein et al.

    Colloids Surf., B

    (2010)
  • A. Lamprecht et al.

    Eur. J. Pharm. Biopharm.

    (2000)
  • N. Iki et al.

    J. Chromatogr., A

    (1996)
  • C. Ringard-Lefebvre et al.

    Colloids Surf., B

    (2002)
  • M. El-Mahrab-Robert et al.

    Int. J. Pharm.

    (2008)
  • V. Lemesle-Lamache et al.

    J. Chromatogr. A

    (1996)
  • S. Laplante et al.

    Food Hydrocolloids

    (2005)
  • E. Akiyama et al.

    J. Colloid Interface Sci.

    (2005)
  • B. Demé et al.

    Colloids Surf., B

    (1995)
  • S. Tcholakova et al.

    Adv. Colloid Interface Sci.

    (2006)
  • P. Joos

    Biochem. Biophys. Acta

    (1975)
  • R. Ipsen et al.

    Colloids Surf., B

    (2001)
  • R. Wüstneck et al.

    Colloids Surf., B

    (1999)
  • D.E. Graham et al.

    J. Colloid Interface Sci.

    (1979)
  • A. Gharsallaoui et al.

    Carbohyd. Polym.

    (2010)
  • L. Jourdain et al.

    Food Hydrocolloids

    (2008)
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