Physical and chemical stability of β-carotene-enriched nanoemulsions: Influence of pH, ionic strength, temperature, and emulsifier type
Highlights
► The influence of temperature, pH, salt, and emulsifier type on β-carotene nanoemulsion stability was examined. ► β-Carotene degradation increased with increasing temperature and decreasing pH, but was largely unaffected by salt. ► Protein-stabilised emulsions aggregated at intermediate pH, high salt, and high storage temperatures. ► β-Carotene degradation was slower in protein-stabilised than in surfactant stabilised emulsions.
Introduction
Carotenoids are a class of natural pigments mainly found in fruits and vegetables that typically have 40-carbon molecules and multiple conjugated double bonds (Failla, Huo, & Thakkar, 2007). Carotenoids are usually divided into two categories: (i) carotenes comprised entirely of carbon and hydrogen, e.g., α-carotene, β-carotene, and lycopene; and (ii) xanthophylls comprised of carbon, hydrogen, and oxygen, e.g., lutein and zeaxanthin (Failla et al., 2007). Carotenoids may be beneficial to human health when consumed at appropriate levels (Khoo, Prasad, Kong, Jiang, & Ismail, 2011). Epidemiological studies have identified a number of potential health benefits of carotenoids, e.g., an increased intake of carotenoid-rich food was correlated with a decreased risk for some cancers, cardiovascular disease, age-related macular degeneration, and cataracts (Gerster, 1993, von Lintig, 2010). Various physiological mechanisms have been proposed to account for the health benefits of carotenoids, including preventing oxidative damage, quenching singlet oxygen, altering transcriptional activity, and serving as precursors for vitamin A (Abdel-Aal and Akhtar, 2006, Failla et al., 2007, Higuera-Ciapara et al., 2006, Singh and Goyal, 2008, von Lintig, 2010). Nevertheless, their utilisation as nutraceutical ingredients within foods is currently limited because of their poor water-solubility, high melting point, chemical instability, and low bioavailability.
The relatively low bioavailability of carotenoids from natural sources has been attributed to the fact that they exist as either crystals or within protein complexes in fruit and vegetables that are not fully released during digestion within the gastrointestinal tract (Williams, Boileau, & Erdman, 1998). Carotenoids can be isolated from natural sources and used as nutraceutical ingredients, but there are a number of challenges associated with successfully incorporating them into a wide range of food and beverage products. Carotenoids have very low water-solubilities and are crystalline at ambient temperature, which usually means they have to be dissolved in oils or dispersed in other suitable matrices before they can be utilised in foods. A number of studies have shown that carotenoid bioavailability depends strongly on the composition and structure of the food matrix in which they are dispersed (Failla et al., 2008, Thakkar et al., 2007, Tyssandier et al., 2001, Tyssandier et al., 2003). Carotenoids are also strongly coloured (red/orange/yellow), which limits the types of foods that they can be incorporated into. Finally, carotenoids are highly prone to chemical degradation during food processing and storage due to the effects of chemical, mechanical, and thermal stresses (Mao et al., 2009, Nguyen and Schwartz, 1998, Tai and Chen, 2000, Xianquan et al., 2005). Attempts have therefore been made to develop effective delivery systems to improve the utilisation, bioavailability, and stability of carotenoids in foods (Mao et al., 2010, Silva et al., 2010).
Emulsion-based systems are particularly suitable for encapsulating and delivering lipophilic bioactive components (McClements, 2010, McClements et al., 2009, McClements and Li, 2010). The lipophilic components are incorporated into the oil phase prior to formation of an oil-in-water emulsion by homogenisation. The oil phase should remain liquid during the homogenisation process, and so the concentration of the lipophilic component in the oil phase should be kept below the saturation level at the homogenisation temperature. This limits the maximum amount of lipophilic materials that can be incorporated into an emulsion-based system, and sometimes means that the emulsion must be homogenised at an elevated temperature, which can promote chemical instability of labile ingredients. A number of previous studies have investigated the formation, properties, and stability of oil-in-water emulsions enriched with carotenoids. A high-pressure homogenisation method was used to prepare lycopene-enriched O/W emulsions stabilised by globular proteins or non-ionic surfactants (Ribeiro, Ax, & Schubert, 2003). High pressure homogenisation has also been investigated as a means of preparing lutein-enriched O/W emulsions stabilised by phospholipids (Losso, Khachatryan, Ogawa, Godber, & Shih, 2005) and proteins (Batista, Raymundo, Sousa, & Empis, 2006). Recently, a high pressure homogenisation method was used to prepare β-carotene enriched O/W emulsions stabilised by small molecule surfactants (Tween 20 and decaglycerol monolaurate) and biopolymers (WPI and modified starch) (Mao et al., 2009). Membrane homogenisation methods have been investigated as an alternative means of encapsulating carotenoids (astaxanthin) in O/W emulsions due to their ability to produce narrow particle size distributions, low energy requirements, and mild processing conditions (Ribeiro, Rico, Badolato, & Schubert, 2005). A number of studies have also shown that the bioavailability of carotenoids is increased when they are incorporated into O/W emulsions (Grolier et al., 1995, Parker, 1997, Ribeiro et al., 2006), which may enhance their health-promoting activities.
Recently, there has been great interest in utilising nanoemulsions to encapsulate bioactive components for applications in food and beverage products (McClements, 2011b, McClements and Rao, 2011). Oil-in-water nanoemulsions consist of small lipid droplets (r < 100 nm) dispersed within an aqueous continuous phase. Similar to conventional emulsions, nanoemulsions are thermodynamically unstable systems that tend to breakdown over time. Nevertheless, they do have some potential advantages over conventional emulsions: they can greatly increase the bioavailability of lipophilic substances; they scatter light weakly and so can be incorporated into optically transparent products; and they have a high stability to particle aggregation and gravitational separation (Acosta, 2009, McClements, 2011a). Nanoemulsions containing carotenoids have previously been prepared using high pressure homogenisation (Mao et al., 2009, Mao et al., 2010) and combined homogenisation/solvent displacement (Silva et al., 2010, Tan and Nakajima, 2005a, Tan and Nakajima, 2005b) methods. Commercially, colloidal dispersions containing β-carotene are typically stabilised against chemical degradation by adding antioxidants, reducing oxygen levels, and minimising exposure to light and pro-oxidants. However, once a sealed product is opened and exposed to the atmosphere some of these protective measures may be lost, and so it is important to understand the major factors that influence β-carotene stability.
In the present study, a high pressure homogenisation method was used to prepare nanoemulsions containing β-carotene, and then test their stability to environmental stresses that might be encountered in typical food and beverage applications (pH, ionic strength, and temperature). A primary goal of this study is to formulate nanoemulsions entirely from food-grade ingredients that are perceived to be safe and label friendly. We therefore utilised orange oil as the carrier oil phase since this is widely used for this purpose in commercial beverage emulsions, and utilised a globular protein (β-lactoglobulin) as the emulsifier since whey proteins are already widely used for this purpose in food and beverage products (McClements, 2005). In addition, previous studies have shown that certain types of food protein are effective at reducing the oxidation rate of emulsified lipids, such as polyunsaturated oils (Berton et al., 2011, Hu et al., 2003, McClements and Decker, 2000, Waraho et al., 2011). It was therefore hypothesised that coating the lipid droplets with a protein layer may improve the chemical stability of the encapsulated β-carotene. For this reason, the rate of chemical degradation of β-carotene in protein-stabilised and surfactant-stabilised nanoemulsions was compared. The results of this study will be useful for designing effective delivery systems to encapsulate and stabilise β-carotene for application within food, beverage, and pharmaceutical products.
Section snippets
Materials
Orange oil was supplied by a food ingredient manufacturer (Givaudan Flavors Corporation, Cincinnati, OH). Food grade β-lactoglobulin was obtained from Davisco Foods International Inc. (Le Sueur, MN). Beta-carotene (Type I, C9750) and Tween 20 were purchased from the Sigma Chemical Company (St. Louis, MO). All other chemicals used were of analytical grade. Double-distiled water was used to prepare all solutions and emulsions.
Preparation of the β-carotene O/W emulsions
An oil phase was prepared by dispersing 0.25% (w/w) of crystalline
Nanoemulsion formation and physical stability
Nanoemulsions were prepared by homogenising 10% oil phase (0.25% β-carotene in orange oil) with 90% aqueous phase (2% β-Lg in buffer solution). The mean particle radius obtained was 78 nm, confirming that nanoemulsions were formed (i.e., r < 100 nm). A monomodal particle size distribution was obtained immediately after homogenisation, with the majority of particles being <100 nm in radius (Fig. 1). The mean particle radius (r = 79 nm) and particle size distribution (Fig. 1) did not change appreciably
Conclusions
This study has shown that β-carotene can be effectively encapsulated within food-grade nanoemulsions stabilised by globular proteins or non-ionic surfactants. A number of important factors that influence the chemical and physical stability of these nanoemulsions were identified. During storage, encapsulated β-carotene had a tendency to chemically degrade, which led to colour fading over time. The rate of colour fading increased with increasing storage temperature, was fastest at the most acidic
Acknowledgements
This material is based upon work supported by the Cooperative State Research, Extension, Education Service, United State Department of Agriculture, Massachusetts Agricultural Experiment Station and a United States Department of Agriculture, CREES, NRI and AFRI Grants. We greatly thank Davisco Foods International for donating the β-lactoglobulin.
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