Development of biopolymer-stabilized emulsions to enhance the stability and bioavailability of lutein

Abstract

Lutein is essential for infant visual and cognitive development, but its stability and solubility are low. This study aimed to improve the stability and bioaccessibility of lutein using biopolymer-stabilized water-oil emulsions. Commercially available octenylsuccinylated (OS) starches, including Capsule TA® (CTA), HI-CAP® 100 (HC), and Purity Gum® 2000 (PG), as well as gum arabic (GA) variants Ticaloid Acacia Max® (TAM), TICA Emulsion® 3020 (TM), and prehydrated gum arabic (PHGA), were selected as emulsifiers. By screening the effects of biopolymer concentration and oil volume fraction (Φ), emulsions stabilized with CTA, HC, or TM at concentrations of 20% and 30% and 70% Φ exhibited gel-like structures and were selected for further evaluation. After one week at 25°C, the droplet size of the emulsions stabilized with CTA and HC remained unchanged, while the droplet size of the TM emulsion increased by 1.58-fold. At 45°C, the droplet size of all emulsions increased. Lutein retention in CTA emulsions was higher than that of free lutein at both storage temperatures. All lutein emulsions exhibited higher in vitro bioaccessibility than free lutein. These findings highlight the superior stability and bioaccessibility of lutein emulsions stabilized with OS starch, making them a promising carrier for expanding lutein's application in infant and toddler food products.

Introduction

Several studies have demonstrated that lutein inhibits the progression of retinopathy of prematurity (ROP), a leading cause of blindness in premature infants, in both mouse models of ROP and human infants. Furthermore, lutein exhibits anti-inflammatory properties and is the most abundant carotenoid in the infant brain. Previous studies have demonstrated a positive correlation between serum lutein concentration and electroretinogram (ERG) response amplitude in premature infants. Lutein concentration in the infant brain has been found to correlate with levels of glutamate and gamma-aminobutyric acid, both of which are known major amino acid neurotransmitters that influence cognition. Certain populations, such as premature infants, have low serum and brain lutein levels, with MP levels being undetectable. Furthermore, formula-fed infants have lower serum lutein concentrations compared to breastfed infants. Therefore, increasing lutein intake could improve infant health, particularly in high-risk groups.

Although lutein is generally recognized as safe (GRAS) for neonates in the United States, its use as a functional ingredient in infant foods is currently limited. As a lipophilic nutrient, lutein requires the formation of micelles with lipids and bile acids in the small intestine for absorption, making it less readily absorbed than water-soluble substances. Infants fed formulas fortified with lutein exhibit a dose-dependent response, requiring four times the lutein dose of breastfed infants to achieve the same serum lutein levels. Furthermore, lutein is a sensitive bioactive compound that readily undergoes chemical degradation upon exposure to heat, light, and oxygen, limiting its use as a nutritional supplement in commercial infant foods.

The most commonly used emulsion-based delivery system for fat-soluble nutrients is the oil-in-water (O/W) emulsion, which consists of small lipid droplets dispersed in an aqueous system. Emulsion-based drug delivery systems are believed to slow the chemical degradation of carotenoids and improve their bioavailability. The use of natural emulsifiers is essential for preparing lutein emulsions in infant formula. Polysaccharides, bio-based polymers, can serve as effective emulsifiers for water-in-oil emulsions in infant formula. Their cost-effectiveness and ready availability make them suitable for commercial use. Octenylsuccinylated (OS) starch and gum arabic (GA), also known as gum acacia, are two commercially available biopolymers approved for use in infant formula at a maximum dose of 20,000 mg/kg per day. OS starch is a modified starch produced by adding octenylsuccinic anhydride groups to starch granules through esterification to enhance functionality (Figure 1A). Common natural starch sources used for this modification include various roots, tubers, cereals, and legumes. GA, derived from the African neem tree, is a natural biopolymer composed of a complex mixture of glycoproteins and polysaccharides (Figure 1B). Both OS starch and GA are used as emulsifiers for encapsulating fat-soluble nutrients such as lutein and β-carotene. However, their effectiveness in improving lutein stability and bioavailability has not been fully investigated. The goal of this study was to develop an O/W emulsion system for lutein using OS starch and GA as emulsifiers. In the current experiments, three commercial OS starches and three commercial GAs were selected as emulsifiers and tested for their emulsion stability. The selected emulsions were further evaluated for microstructure, physical and chemical stability under thermal stress, and in vitro bioavailability.

Figure 1 Structures of starch modified with octenylsuccinic anhydride (A) and gum arabic (B)

Results and Discussion

Degree of Substitution (DS) of Oxidized Starch

Oxidized starch is produced by introducing oxidized starch groups onto the hydroxyl groups on the starch granule surface via an esterification reaction. The presence of oxidized starch groups in the three commercial starches was confirmed by the presence of two peaks at 0.89 ppm and 1.23 ppm in the 1H NMR spectra (Figure 2). The DS values for CTA, HC, and PG are 0.017, 0.038, and 0.047, respectively. These peaks correspond to resonances of methyl hydrogen atoms and hydrogen atoms on the oxidized starch chains, confirming the structure of the oxidized starch, in which hydrophobic side chains are attached to the starch molecules. Chemical modification, namely esterification with OSA, introduces hydrophobic alkyl chains onto the native starch molecules. This modification alters the amphiphilic balance of the starch, increasing its hydrophobicity. The coexistence of hydrophilic and hydrophobic groups in OS starch enhances its affinity for non-polar substances (such as oils), promoting their adsorption at the oil-water interface and thereby reducing interfacial tension, a key factor in the formation and stability of emulsions. Previous studies have shown that the increased hydrophobicity of OS starches enhances their emulsifying ability compared to native starches.

Figure 2 1H NMR spectrum of octenylsuccinate starch capsules TA

Effects of crude oil volume fraction and polymer concentration on physical stability

It is well known that Φ and polymer concentration are key factors influencing emulsion formation and stability. A pilot study was conducted using a 2×5 factorial design to evaluate the effects of Φ (10%, 30%, 50%, 70%, and 90% OS starch) and biopolymer concentration (15% and 30% OS starch; 15% and 20% GA) on the physical stability of biopolymer-based emulsions after 28 days of storage (Figure 3). The EI values of emulsions stabilized by CTA, HC, PG, and TM as a function of Φ and concentration after one month of storage are shown in Figure 4. An emulsion exhibiting no phase separation after storage has an EI value of 100%. Visual inspection of samples after 28 days revealed no significant changes in the appearance of all biopolymer-stabilized emulsions and latex gels compared to fresh samples. O/W emulsions stabilized by all biopolymer types exhibited significant phase separation at Φ values of 10% and 30%. As Φ values increased to 50%, physically stable biopolymer-based emulsions were observed in some OS starch- and GA-based emulsions, including those stabilized by 30% HC, 30% PG, 15% TM, and 20% TM. Notably, the emulsion stabilized with 30% PG formed a homemade "latex" that did not foam when the bottle was inverted. When the Φ value was further increased to 70%, most biopolymer-based emulsions, including O/W emulsions stabilized with 15% CTA, 30% CTA, 15% HC, 30% HC, 15% PG, 15% TM, and 20% TM, successfully formed emulsions. Emulsions stabilized with 30% CTA, 30% HC, 15% PG, 15% TM, and 20% TM exhibited a gel-like texture, as evidenced by a lack of fluidity when the bottle was inverted. This range of gel-like emulsion formation (50%–70% Φ) is consistent with previous studies reporting that octenylsuccinylated quinoa starch granules formed gel-like emulsions within Φ values of 50%–70% and that mung bean starch formed gel-like emulsions within a range of Φ values. The increase in emulsion gel viscosity plays a crucial role in maintaining the stability and integrity of the emulsions. It prevents phase separation by reducing the rate of particle movement, delays rancidity by slowing oxidation, and mitigates the effects of various environmental factors, thereby extending storage life and making it an ideal carrier for bioactive compounds. Further increasing Φ to 90% resulted in the separation of the top oil layer and the formation of a thin aqueous phase sedimentation at the bottom, indicating that the biopolymer content was insufficient to fully stabilize the emulsion. A Φ as high as 90% may be too high to successfully prepare an O/W emulsion stabilized by OS starch and GA.

Figure 3: Emulsion Image

Figure 4: Radar Plot

Effect of Biopolymer Concentration on the Physical Stability of Lutein Emulsions

Figure 5 shows the visual appearance of lutein emulsions stabilized with CTA, HC, PG, and TM at different biopolymer concentrations (15%, 20%, or 30%) and an oil phase volume fraction Φ of 70% immediately after preparation and after 28 days of storage. The macrostructure of the biopolymer-stabilized lutein emulsions is similar to that of the corresponding biopolymer-fluid-stabilized emulsions, indicating that the addition of lutein did not significantly affect the stability of the emulsion system. This demonstrates that biopolymer-stabilized lutein emulsions can effectively serve as carriers for encapsulating and delivering lutein.

Figure 5 Optical Appearance

Microstructure

The microstructure of lutein emulsions stabilized with 30% CTA, 30% HC, and 20% TM was examined using CLSM (Figure 6). Under 488 nm excitation, the lutein-containing oil droplets exhibited natural fluorescence, appearing green, while the aqueous phase, stained with Nile blue, appeared purple under 633 nm excitation. As shown in Figures 6A-C, all three biopolymer-based lutein emulsions exhibited tightly packed and uniform oil droplets, indicating the formation of a compact emulsion network and the presence of an emulsion gel, consistent with visual observations. The proximity of the oil droplets results in strong steric repulsive interactions between them, contributing to the good physical stability of the emulsions. Similar microstructures were observed in CLSM images of emulsion gels stabilized with OS starch and β-conglycinin. A study of emulsion gels stabilized with OS quinoa starch granules found that the spacing between oil droplets was smaller than that between oil droplets in emulsions that did not form a gel. The aqueous phase surrounded the droplet surface, effectively encapsulating the oil droplet, indicating the formation of an O/W emulsion (Figures 6D-E). The formed thin film adequately covered the droplet, indicating an appropriate biopolymer concentration in the emulsion system. Adequate biopolymer concentration is crucial for emulsion stability. For example, previous studies have shown that emulsions containing emulsifiers that form relatively thick films are less susceptible to partial coalescence (i.e., droplet merging) than emulsions stabilized with emulsifiers that produce thinner layers. Overlay images (Figures 6G-I) further confirmed the formation of consistent O/W emulsions stabilized by the biopolymers CTA, HC, and TM. In the TM-stabilized lutein emulsion, the addition of the dye solution altered the close-packed structure and droplet size. Therefore, as shown in the CLSM image of the unstained lutein emulsion, the droplet size and distribution (Figure 7) were determined from the image of the unstained lutein emulsion.

Figure 6 Lutein emulsion images in green, purple, and overlapping fluorescence fields

Figure 7 Lutein emulsion droplet distribution

Stability of lutein emulsions stabilized with biopolymers

The physical stability (i.e., change in droplet size) and chemical stability (i.e., lutein retention) of lutein emulsions stabilized with 30% CTA, 30% HC, and 20% TM were quantified over a week of storage at 25 and 45°C, as shown in Figure 8. After one week of storage at 25°C, the droplet size of the lutein emulsions stabilized with OS starch, CTA, and HC did not change significantly, while the droplet size of the emulsion stabilized with TM increased by 1.58-fold compared to the fresh sample (P < 0.05) (Figure 8A). This increase in droplet size indicates flocculation and coalescence, indicating emulsion instability. Therefore, the lutein emulsion stabilized with OS starch exhibited greater emulsion stability than the lutein emulsion stabilized with GA.

Figure 8. Changes in Droplet Size and Lutein Retention in Lutein Emulsions

To assess chemical stability, the initial lutein content was set at 100%, and lutein retention was monitored. On day 7, lutein retention in the control and emulsions stabilized with CTA, HC, and TM was 79%, 88%, 89%, and 86%, respectively (Figure 8B). These results are consistent with those of other studies, which have reported degradation rates of approximately 10% to 14% in biopolymer-stabilized O/W emulsions after 7 days of storage at 20°C. Compared with CTA- and HC-stabilized emulsions, TM-stabilized lutein emulsions exhibited significantly lower lutein retention. The reduced lutein retention in TM-stabilized emulsions may be attributed to changes in droplet structure. Droplet coalescence in TM-stabilized emulsions after one week of storage (data not shown) is likely due to reduced droplet coverage by TM, resulting in greater lutein degradation upon exposure to oxidation.

After storage at 45°C for one week, the particle sizes of lutein emulsions stabilized with CTA, HC, and TM were 1.34-, 2.38-, and 1.55-fold larger than those of the fresh sample, respectively (P < 0.05) (Figure 8C). Lutein retention in free lutein and emulsions stabilized with CTA, HC, and TM was 78%, 86%, 46%, and 63%, respectively (Figure 8D). Previous studies have shown that higher storage temperatures accelerate the degradation of carotenoids in emulsion systems. With the exception of the CTA-stabilized emulsion, lutein emulsions stabilized with HC and TM were physically and chemically unstable at higher temperatures. On the one hand, the homogenization step may have increased the surface area of the lutein droplets in the emulsions, exposing them to more air and promoting their degradation at elevated temperatures. On the other hand, the control group, using lutein esters, showed superior stability compared to free lutein under the same light and heat stress conditions. However, the CTA-stabilized lutein emulsion effectively inhibited lutein degradation, as evidenced by relatively stable droplet size and significantly improved color retention compared to the other two emulsions.

In Vitro Bioaccessibility

Bioaccessibility refers to the amount of a compound released from a food matrix into the gastrointestinal tract and available for human absorption.In vitro digestion models are commonly used to assess the bioaccessibility of bioactive compounds in the gastrointestinal system. In this study, a static in vitro model was used to maintain a constant ratio of enzymes, salts, and bile acids throughout the digestion process.

The in vitro bioaccessibility of lutein in three lutein emulsions is shown in Figure 9. Lutein in olive oil was used as a control. Compared to lutein oil, the lutein emulsions showed significantly improved in vitro bioaccessibility. The order of lutein bioaccessibility was: TM (70%) > CTA (68%) > HC (38%) > lutein in olive oil (30%). Lutein bioaccessibility was 2.26-, 1.26-, and 2.33-fold higher in CTA-, HC-, and TM-stabilized lutein emulsions compared to free lutein, respectively.

Figure 9 In vitro bioaccessibility of lutein emulsions stabilized by encapsulated TA

Conclusion

In this study, we successfully developed biopolymer-based lutein emulsions and examined their microstructure, physical and chemical stability, and in vitro bioaccessibility under heat stress. In various OS starch and GA tested, CTA, HC, PG, and TM proved to be effective emulsifiers, allowing the formation of stable biopolymer-based emulsions at concentrations ranging from 15% to 30% and an oil phase volume fraction (Φ) of 70%. Emulsions with higher concentrations of CTA, HC, and TM exhibited a gel-like structure, indicating enhanced storage stability. Therefore, lutein emulsions stabilized with 30% CTA, 30% HC, and 20% TM were selected for further evaluation. These biopolymer-based lutein emulsions exhibited small and uniform particle sizes ranging from 1.18 μm to 1.45 μm. Thermal stability testing demonstrated that the CTA-stabilized lutein emulsions exhibited minimal changes in droplet size and effectively prevented lutein degradation at elevated temperatures. In vitro digestion experiments demonstrated improved lutein bioaccessibility in CTA- and TM-stabilized emulsions. Overall, the lutein emulsion stabilized by OS starch CTA exhibited the best stability in terms of droplet aggregation, color retention, and in vitro release. These findings open a promising avenue for developing biopolymer-based emulsions as promising carriers of lutein in infant foods.