Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions
Graphical abstract
Introduction
There has been growing interest in the utilization of nanoemulsions as delivery systems to encapsulate, protect, and release lipophilic active agents due to their high encapsulation efficiency, optical clarity, good physical stability, and high bioavailability [1], [2], [3], [4]. In general, oil-in-water emulsions are thermodynamically unstable systems that consist of spherical oil droplets dispersed within an aqueous continuous phase. By convention, emulsions with droplet diameters in the nanomeric scale (typically between 20 and 200 nm) are referred to as nanoemulsions [2], [3], [5]. In contrast, emulsions containing droplets with larger droplets are referred to as conventional emulsions or macroemulsions. Both nanoemulsions and macroemulsions are thermodynamically unstable systems because the free energy of the separated oil and water phases is lower than that of the emulsion itself [1], [6]. As a consequence, these emulsions typically breakdown over time due to a variety of destabilization mechanisms, e.g., creaming, flocculation, coalescence, and Ostwald ripening [1]. Nevertheless, they can be fabricated to remain metastable for a considerable period by adding appropriate stabilizers, such as emulsifiers, texture modifiers, ripening inhibitors, or weighting agents. Nanoemulsions should not be confused with microemulsions, which are another type of colloidal delivery system containing lipid nanoparticles [7]. Unlike nanoemulsions, microemulsions are thermodynamically stable systems, however, they typically require relatively large amounts of synthetic surfactants to fabricate them, which may limit their use for certain applications [8], [9].
In general, two different approaches can be used to fabricate nanoemulsions: high-energy and low-energy approaches [1], [10]. In high-energy approaches, droplet disruption is mainly achieved by generating large pressure differences within mechanical devices, such as high-shear stirrers, high-pressure homogenizers, or ultrasound generators [6], [11], [12]. In contrast, low-energy approaches rely on the spontaneous formation of small oil droplets at the boundary between the aqueous and organic phases under certain system conditions [2], [13]. The main advantage of low-energy approaches is that they are simple and inexpensive to carry out and do not require the use of specialized homogenization equipment, however, the main disadvantage is that high levels of synthetic surfactant are often required. This limitation is similar to that associated with the formation of microemulsions, however, the total amount of surfactant required to form nanoemulsions by low-energy methods is still less than that required to form microemulsions.
The main objective of the current study was to establish the influence of small chain alcohols on the formation and stability of protein-stabilized nanoemulsions fabricated using a high-energy approach. Previous studies have examined a number of factors that influence the formation of nanoemulsions, such as homogenizer type, operating conditions, sample composition, and the physicochemical properties of the component phases [1], [5], [14]. Typically, the mean particle diameter decreases with increasing homogenization pressure and number of passes [15], and smaller droplets are produced using small-molecule surfactants than using polymeric surfactants [16], [17]. Several studies have also focused on the role of oil and aqueous phase viscosities on droplet disruption within homogenizers [16], [17].
Droplet breakup during homogenization can be described by the Taylor equation in systems with low droplet concentration and low continuous phase viscosity [16]:where γ is the interfacial tension, ηc is the continuous phase viscosity and is the shear rate. This equation highlights the fact that a reduction in the interfacial tension plays a major role in the formation of small-sized droplets. The addition of alcohol to an aqueous phase is known to reduce the oil–water interfacial tension [18], [19], and may therefore be a potential method of further reducing the size of the droplets in nanoemulsions produced by high pressure homogenization.
The aim of the present study was to examine the impact of various aliphatic alcohols (ethanol, 1-propanol, and 1-butanol) on the formation of oil-in-water nanoemulsions stabilized by food-grade protein emulsifiers, i.e. sodium caseinate, whey protein isolate, and fish gelatin. We hypothesized that smaller droplets would be produced during homogenization when alcohol was present in the aqueous phase due to the reduction in interfacial tension. Alcohols with different chain lengths were utilized to examine the influence of their molecular structure on nanoemulsion formation and stability, however it should be noted that only ethanol is suitable for utilization within the food industry.
Section snippets
Materials
Cold water fish skin gelatin (#049K0050) was purchased from Sigma–Aldrich Co., (Steinheim, Germany). Its average molecular weight and pI value were reported to be approximately 60 kDa and pH 6, respectively. Whey protein isolate (#B180214) was donated by Arla Foods Ingredients (Viby, Denmark). The whey protein isolate contained ⩾92% protein, ⩽6% moisture, ⩽4.5% ash, ⩽0.2% fat, and ⩽0.2% lactose, according to the manufacturer’s specification. Sodium caseinate (#L080512201) was purchased from
Influence of homogenization pressure and number of cycles on particle size of base emulsions
We initially examined the influence of homogenization pressure and number of cycles on the particle size of protein-stabilized oil-in-water emulsions containing no alcohol: 10% w/w oil phase (MCT) and 90% w/w aqueous phase (2% w/w emulsifier solution, pH 7). Three emulsifiers varying in structure and molecular weight were used to stabilize these emulsions: caseinate (∼25 kDa) and fish gelatin (60 kDa) have relatively disordered flexible structures, while WPI (∼18 kDa) has a fairly rigid compact
Conclusions
There has been a surge of interest in the development of food-grade nanoemulsions suitable for oral ingestion due to their potential advantages over conventional emulsions, such as increased physical stability, higher optical clarity, and enhanced bioavailability [36], [37], [38], [39]. Recent work has shown that edible nanoemulsions can be formed using both low-energy and high-energy approaches [1], [10]. High-energy approaches, such as microfluidization, have the advantages that lower
Acknowledgments
We would like to thank Rovita GmbH (Engelsberg, Germany) and Arla Foods Ingredients (Viby, Denmark) for generously providing us with protein samples. We also thank the USDA-NRI program (2011-03539 and 2013-03795) for partly funding this research. This manuscript is also a tribute to Prof. Darsh Wasan who has made major contributions to colloid and interface science in general, as well as making important contributions to applying fundamental colloidal principles to understanding food colloids.
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