**2.1. Materials**

) or the

) of amino acids that make up proteins can stabilize

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

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

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−

+

vegetables may not be enough to attain health benefits [7].

to inhibit the rate of their degradation [11].

162 Progress in Carotenoid Research

positively charged amino groups (–NH<sup>3</sup>

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 Extrasynthèse SA (Genay, France). Soybean lecithin Beakin LV3 was a gift from Archer Daniels Midland Co (Decatur, IL, USA). Ethanol, thimerosal, phenylmethanesulfonyl fluoride, and monobasic potassium phosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (Pittsburg, PA, USA). Deionized water was prepared by passing distilled water over a mixed bed of cationanion exchanger and was used throughout this study.

emulsion (zeta potential, ζ, mV) was measured at day 0 with a Zetasizer nano ZS (Malvern Instruments, Worcestershire, UK). Samples were diluted 100 times in 5 mM phosphate buffer

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

http://dx.doi.org/10.5772/intechopen.78601

165

The chemical stability of lutein was assessed by measuring the concentration of lutein in the lutein-enriched emulsions during storage at 5 and 15°C and analyzed right after production (0 day) and at 1, 2, 4, 6, and 7 days. The concentration of lutein was determined from absorbance measurements at 460 nm using a Beckman UV/visible model DU-530 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA). To prepare the samples for the spectrophotometric measurements, the lutein-enriched emulsions were diluted 100 times in DMSO (50 μl of emulsion was diluted into 4.95 ml of DMSO). The DMSO was used because it dissolves lutein, oil, lecithin, and protein to form transparent solutions suitable for UV/ visible analysis. The emulsion without lutein was used as a blank. A calibration curve was constructed by dissolving lutein standards in DMSO within a range from 0.5 to 5 mg/ml.

In each experiment, the results of triplicate analyses were used to test experimental variables. The data were analyzed by ANOVA using PRO GLM procedure of SAS (version 8.2, SAS Institute, Cary, NC, USA). The least significant test was used to determine significant differ-

**3.1. Relationship between casein composition and emulsion interfacial properties**

dispersion system remains stable after light exposure during storage [20].

**3.2. Physical stability of lutein emulsions prepared with bovine casein/lecithin and** 

In order to understand the effects of bovine casein or caprine casein in combination with phospholipids i.e., soybean lecithin on the chemical degradation of lutein, three sets of emulsions with corn oil were made. The three sets were stabilized by bovine casein/lecithin,

Caprine caseins have markedly higher content of β-casein than bovine casein. The specific oxidative stability found in emulsified lipids has been explained by the formation of a highly protective interface produced from β-casein as an emulsifier in the emulsions [23, 24]. Likewise, emulsions exert good protective effects on the carotenoids. Thus, it has been reported that the multilayer emulsions around the oil droplets can potentially reduce the amount of light reaching the carotenoid [19, 20]. A lutein dispersion was achieved using bovine casein or caprine caseins (caprine αs1-I-casein and caprine αs1-II-casein) as emulsifier in an emulsion beverage [19, 20]. The caprine casein emulsifier, in particular caprine αs1-II-casein, in combination with arabinogalactan, a water-soluble polysaccharide, is noteworthy because this lutein

at pH 7.0. All measurements were carried out in triplicate at 21 ± 1°C.

**2.5. Chemical stability of lutein in the emulsion**

**2.6. Statistical analysis**

ences among means at p < 0.05.

**3. Results and discussion**

**caprine casein/lecithin as emulsifiers**
