Colloidal properties of sodium caseinate-stabilized nanoemulsions prepared by a combination of a high-energy homogenization and evaporative ripening methods

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Highlights

  • Sodium caseinate nanoemulsions were prepared by a three-step method.

  • Stability behavior was different from that of conventional emulsions.

  • Creaming of individual particles was negligible.

  • When unstable, the main mechanism of destabilization was flocculation.

  • Sedimentation of protein flocs occurred in particular conditions.

Abstract

Nanoemulsions stabilized by sodium caseinate (NaCas) were prepared using a combination of a high-energy homogenization and evaporative ripening methods. The effects of protein concentration and sucrose addition on physical properties were analyzed by dynamic light scattering (DLS), Turbiscan analysis, confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS). Droplets sizes were smaller (~ 100 nm in diameter) than the ones obtained by other methods (200 to 2000 nm in diameter). The stability behavior was also different. These emulsions were not destabilized by creaming. As droplets were so small, gravitational forces were negligible. On the contrary, when they showed destabilization the main mechanism was flocculation. Stability of nanoemulsions increased with increasing protein concentrations. Nanoemulsions with 3 or 4 wt% NaCas were slightly turbid systems that remained stable for at least two months. According to SAXS and Turbiscan results, aggregates remained in the nano range showing small tendency to aggregation. In those systems, interactive forces were weak due to the small diameter of flocs.

Introduction

Nanoemulsions are defined as a thermodynamically unstable colloidal dispersion with a dispersed phase containing small spherical droplets with radius sizes smaller than 100 nm (McClements, 2012a). A conventional emulsion typically has particles with mean radii between 100 nm and 100 μm (McClements & Rao, 2011). Both conventional emulsions and nanoemulsions are metastable systems that break due to a variety of destabilization mechanisms, such as gravitational separation, coalescence, flocculation, and Ostwald ripening (Komaiko & McClements, 2016). For certain applications, it is desirable to prepare systems with very small particles since they have a number of potential advantages over systems containing larger particles (Fryd & Mason, 2012). First, they usually have better stability to physical changes, and second, they contain particles that only scatter light waves weakly, and so they are suitable for incorporation into products that need to be optically clear or only slightly turbid (McClements, 2012b). Nanoemulsions also enhance bioavailability and because of that have been used in many applications involving encapsulation of bioactive compounds (Atrux-Tallau et al., 2014, Komaiko et al., 2016, Vilanova and Solans, 2015).

Nanoemulsions have been prepared by high-energy and low-energy homogenization methods (Silva, Cerqueira, & Vicente, 2012). High-energy methods (such as high-pressure homogenization, microfluidization or sonication) often require a large capital investment in equipment while the major disadvantage for the low-energy methods (such as phase inversion temperature, spontaneous emulsification, or emulsion phase inversion) is the requirement of high amounts of synthetic surfactants (Komaiko & McClements, 2016). Low-energy methods are often more effective at producing small droplet sizes than high-energy approaches, but they are often more limited in the types of oils and emulsifiers that can be used. For example, it is currently not possible to use proteins or polysaccharides as emulsifiers in most of the low-energy approaches used to form nanoemulsions (McClements & Rao, 2011). Although both, high-energy and low-energy homogenization methods have drawbacks, they enable preparing extremely fine particulate nanodispersions when the stabilizer is a small-molecule surfactant (Chu, Ichikawa, Kanafusa, & Nakajima, 2007). In protein systems, a combination of high-energy homogenization and evaporative ripening methods was used to prepare nanoemulsions formulated with corn oil and whey protein (Lee & McClements, 2010). The application of nanotechnology to the food field may allow modifying macroscale properties such as texture, transparency, and stability during shelf life among other characteristics. Therefore, it is of great interest to study physical properties of nanosystems.

Nanoemulsions have been used for applications in the food industry such as beverage (Rao and McClements, 2013, Zhang et al., 2013, Zhang et al., 2015), healthier ice cream, frozen food or they have been designed for performing as carriers or delivery systems for lipophilic compounds (Gulotta et al., 2014, Silva et al., 2012). Recently, formulation of food grade O/W nanoemulsions has been an area of active research (Hategekimana et al., 2015, Komaiko et al., 2016, McClements and Rao, 2011, Ozturk et al., 2015, Tabibiazar and Hamishehkar, 2015), to name a few. Dairy proteins have been extensively used as emulsifiers in conventional emulsions since they adsorb to the oil droplet interface, forming a strong and cohesive protective film that helps prevent droplet aggregation. Sodium caseinate is widely used as an ingredient in the food industry due to its functional properties, which include emulsification, water and fat-binding, thickening and gelation. Maher, Fenelon, Zhou, Haque, and Roos (2011) prepared microfluidized nanoemulsions stabilized by sodium caseinate with droplet sizes between 186 and 199 nm (around 200 nm). Yeramilli and Ghosh used high-pressure homogenizer, obtaining droplets within the same size. In this article, we prepared nanoemulsions using a combination of methods with the aim of obtaining smaller droplets than in Maher et al. (2011) or in Yeramilli and Ghosh (2017) articles. The physical behavior of systems with droplets smaller than 200 nm is expected to be different than the one reported in literature and therefore it is interesting to explore their stability and structure.

The objective of the present study was to investigate the formation of sodium caseinate-stabilized nanoemulsions using a combination of a high-energy homogenization and evaporative ripening methods. The effects of protein concentration and sucrose addition on physical properties were analyzed by dynamic light scattering (DLS), Turbiscan analysis, confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS) with the aim of describing in a deeper way the stability behavior.

Section snippets

Materials

Sucrose (α-d-glucopyranosyl β-d-fructofuranoside) and sodium caseinate (NaCas) were obtained from Sigma (Sigma–Aldrich, St. Louis, Mo., USA) and ethyl acetate from Sintorgan (Sintorgan S.A., Buenos Aires, Argentina). All reagents were analytical grade and they were used without any further purification. HPLC water was used for all experimental work. The oil phase was commercial sunflower seed oil (SFO) which main fatty acids were identified as C16:0 (palmitic), C18:0 (stearic), C18:1 (cis,

Droplet size distribution

To check the ability of the combination of high-energy homogenization and evaporative ripening methods to produce nanoemulsions, samples were analyzed for particle size distribution after ultrasound treatment and after evaporative step. Fig. 1 shows the droplet size distributions of fine emulsions before applying the evaporative step while Fig. 2 reports the same emulsions after evaporating ethyl acetate. Figs. 1a and 2a show the effect of NaCas concentration and Figs. 1b and 2b report the

Conclusions

Nanoemulsions were successfully prepared by a combination of a high-energy homogenization and evaporative ripening methods. This three-step method allowed obtaining droplet sizes smaller than 120 nm. Stability behavior of NaCas-stabilized nanoemulsions was very dependent on droplets sizes. Systems with droplets smaller than 120 nm behaved in a different way of nanoemulsions prepared by a two-step method or than conventional emulsions. Because droplets sizes were smaller than the ones obtained by

Acknowledgements

This work was supported by the National Agency for the Promotion of Science and Technology (ANPCyT) through Project PICT-2013-0897, and by the University of Buenos Aires through Project UBA-20020130100136BA. The authors wish to thank the Synchrotron Light National Laboratory (LNLS, Campinas, Brazil) for the use of the SAXS1 facilities through proposal 20150056.

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