**1. Introduction**

Among the polar oxygenated xanthophylls of the carotenoids, lutein has received attention for its potent antioxidant activity [1]. Lutein may protect the DNA of photoreceptive cells in

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the retina from the harmful effects of strong light [2]. In the skin, lutein is believed to protect against UV radiation [3]. Lutein is naturally synthesized by plants, and is commercially available as a food supplement from Marigold flowers (*Tagetes erecta*) [4]. Another source of lutein, and one that is the most bioavailable of all, is the egg. A large egg yolk contains approximately 252 μg of lutein (and zeaxanthin); while there is not a tremendous amount of lutein in egg yolks, it is so bioavailable that it is taken into the bloodstream with great efficiency, giving a significant boost to the serum levels of this protective carotenoid [5]. Indeed, the inclusion of lutein in a lipophilic matrix (egg yolk phospholipids) leads to dramatic improvement in the absorption of lutein in humans [6]. However, the segment of the population that has been recommended to control dietary cholesterol intake avoids consumption of eggs. American adults have a poor supply of lutein and their intake of ~1–2 mg/day by eating fruits and vegetables may not be enough to attain health benefits [7].

droplets from aggregation by generating electrostatic repulsions. Proteins are generally relatively small molecules (about 10–50 kDa) that rapidly absorb to droplet surfaces and form thin, electrostatically charged interfacial layers. Such layers are important in the formation of

Lutein-Enriched Emulsion-Based Delivery System: Impact of Casein-Phospholipid Emulsifiers…

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Currently, caseins and whey proteins from bovine origin are the most commonly used protein-based emulsifiers in the food industry. Caseins are amphiphilic proteins with flexible structures (αs1-, αs2-, β-, and κ-caseins) whereas whey proteins are globular proteins with fairly rigid structures [β-lactoglobulin, bovine serum albumin (BSA), and immunoglobulins]. Due to their structural flexibility, caseins rapidly undergo conformational changes, with the hydrophilic groups protruding into the water phase and the hydrophobic groups into oil phase.

Phospholipids, similar to proteins, are amphiphilic molecules (loving both water and oil) with hydrophobic fatty acid tail groups and phosphoric acid esterified with glycerol and other substitutes as the hydrophilic head groups. Phospholipids are generally referred to as lecithin, and they occur in nature in the cell membranes of animals, plants, and microbial species. Phospholipids are industrially extracted from soybeans, egg yolk, milk, and sunflower kernels for use in foods. Lecithin contains a mixture of different phospholipids, with the most common being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. While lecithin ingredients are surface-active agents and facilitate the mixing of oil and water, they also are prone to coalescence because they form interfacial layers. Combining lecithin with proteins such as bovine caseins [14] can minimize this issue and help form stable emulsions.

Milks from different mammalian species present differences in their relative proportions and characteristics of caseins [15]. The degree of variability of caprine casein components from the individual milks of French-Alpine and Anglo-Nubian breeds of goats has been previously reported [16]. For both breeds the quantity of β- and κ-caseins are relatively constant while

of the ratio of caseins results in a rather distribution of structure for casein micelles (with Ca2+), which has been used for the delivery of fat-soluble bioactive compounds such as curcumin and vitamin D in aqueous solutions [17, 18]. Casein sub-micelles (without Ca2+) from bovine and caprine milks have been used for the delivery of lutein in food emulsions [19, 20].

The aim of our study was to evaluate the effects of bovine and caprine caseins (sub-micelles) in combination with phospholipids (soy lecithin) as emulsifiers on the chemical stability of lutein in corn oil-in-water emulsions at pH 7.0 during storage at 5 and 15°C. The corn oil

A commercial preparation of lutein consisting of 20% (wt/wt) lutein dissolved in corn oil was a gift from Hoffman La Roche (Pleasanton, CA). Mazola corn oil was purchased from a local supermarket. A lutein standard for chromatographic analysis was purchased from


stabilized emulsions.

the content of αs

**2.1. Materials**

allows the carotenoids to be absorbed [21].

**2. Materials and methods**

Fortification of foods and beverages with fat-soluble bioactive components such as the carotenoids, especially β-carotene, is considered important in the elimination of acute deficiency symptoms, in optimizing health, and in providing protection from many chronic ailments on a long-term basis [8, 9]. The difficulties encountered in fortifying foods with the carotenoids are primarily due to their instability (air, light) and low water solubility [10]. Since carotenoids cannot be incorporated into aqueous-based foods, an emulsion-based delivery system provides a suitable means for dispersing the lipophilic carotenoids into the aqueous environments of foods [11]. Emulsion-based delivery systems also allow for improved absorption of non-polar or fat-soluble bioactive compounds such as the carotenoids [12]. The type of emulsion used should be considered when carotenoids are added to food and beverage products to inhibit the rate of their degradation [11].

Food emulsions are usually one of the simplest forms of oil-in-water emulsions, consisting of small oil droplets dispersed within an aqueous medium, with the oil droplets having mean diameters ranging from 10 to 100 nm (nanoemulsions) or 100 nm to 100 μm (conventional emulsions). The formation of successful emulsion-based food and beverage products requires emulsifiers [13]. Emulsifiers are surface-active (surfactants) substances that play a crucial role in the mixing of two immiscible liquids. These surface active substances normally have a polar (hydrophilic) and a non-polar (hydrophobic) ends that break the surface tension between different liquids thereby, facilitating the formation of the mix and maintaining the stability of the mix. Food manufacturers have traditionally used both synthetic and natural emulsifiers in food formulations; however, the clean label movement is creating a new trend toward the use of natural emulsifiers. In this context, the interest of the general public and the food industry professionals are toward identifying, characterizing, and utilizing naturally occurring substances such as proteins, polysaccharides, phospholipids, and saponins as emulsifiers in formulated foods. The desire is to find which naturally occurring compounds have the appropriate properties to efficiently form stabilized emulsions in foods with possible commercial applications.

Proteins contain a mixture of hydrophilic and hydrophobic amino acids along their polypeptide chains that render them naturally surface active agents. This characteristic enables most proteins to quickly absorb to oil-in-water interfaces and coat the oil droplets that are formed during mixing and homogenization. The negatively charged carboxylic groups (–COO− ) or the positively charged amino groups (–NH<sup>3</sup> + ) of amino acids that make up proteins can stabilize droplets from aggregation by generating electrostatic repulsions. Proteins are generally relatively small molecules (about 10–50 kDa) that rapidly absorb to droplet surfaces and form thin, electrostatically charged interfacial layers. Such layers are important in the formation of stabilized emulsions.

Currently, caseins and whey proteins from bovine origin are the most commonly used protein-based emulsifiers in the food industry. Caseins are amphiphilic proteins with flexible structures (αs1-, αs2-, β-, and κ-caseins) whereas whey proteins are globular proteins with fairly rigid structures [β-lactoglobulin, bovine serum albumin (BSA), and immunoglobulins]. Due to their structural flexibility, caseins rapidly undergo conformational changes, with the hydrophilic groups protruding into the water phase and the hydrophobic groups into oil phase.

Phospholipids, similar to proteins, are amphiphilic molecules (loving both water and oil) with hydrophobic fatty acid tail groups and phosphoric acid esterified with glycerol and other substitutes as the hydrophilic head groups. Phospholipids are generally referred to as lecithin, and they occur in nature in the cell membranes of animals, plants, and microbial species. Phospholipids are industrially extracted from soybeans, egg yolk, milk, and sunflower kernels for use in foods. Lecithin contains a mixture of different phospholipids, with the most common being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. While lecithin ingredients are surface-active agents and facilitate the mixing of oil and water, they also are prone to coalescence because they form interfacial layers. Combining lecithin with proteins such as bovine caseins [14] can minimize this issue and help form stable emulsions.

Milks from different mammalian species present differences in their relative proportions and characteristics of caseins [15]. The degree of variability of caprine casein components from the individual milks of French-Alpine and Anglo-Nubian breeds of goats has been previously reported [16]. For both breeds the quantity of β- and κ-caseins are relatively constant while the content of αs -caseins of these breeds vary significantly [16]. In bovine milk the constancy of the ratio of caseins results in a rather distribution of structure for casein micelles (with Ca2+), which has been used for the delivery of fat-soluble bioactive compounds such as curcumin and vitamin D in aqueous solutions [17, 18]. Casein sub-micelles (without Ca2+) from bovine and caprine milks have been used for the delivery of lutein in food emulsions [19, 20].

The aim of our study was to evaluate the effects of bovine and caprine caseins (sub-micelles) in combination with phospholipids (soy lecithin) as emulsifiers on the chemical stability of lutein in corn oil-in-water emulsions at pH 7.0 during storage at 5 and 15°C. The corn oil allows the carotenoids to be absorbed [21].
