Protection of β-carotene from chemical degradation in emulsion-based delivery systems using antioxidant interfacial complexes: Catechin-egg white protein conjugates
Graphic abstract
Introduction
β-carotene (BC) is an important carotenoid present in various fruits and vegetables, including sweet potato, carrot, spinach, papaya, mango, etc. (Berman et al., 2015, Rao and Rao, 2007). It is widely utilized as a natural colorant and antioxidant in foods (Coronel-Aguilera and San Martín-González, 2015, Esther et al., 2012). Evidence from numerous cohort-based studies suggest that consumption of foods rich in BC may be beneficial to human health by decreasing the risk of cancer, cardiovascular disease, and cataracts (Hou, Liu, Lei, & Gao, 2014). Nevertheless, the poor water-solubility of BC currently limits its application in many aqueous-based foods and beverages (Akinosho and Wicker, 2015, McClements and Xiao, 2012). Moreover, BC is highly susceptible to chemical degradation during storage because it is a polyunsaturated molecule that contains 11 double bonds (Qian, Decker, Xiao, & McClements, 2012). Indeed, BC degradation occurs rapidly when it is exposed to certain environmental conditions, such as heat, light, oxygen, and transition metals (Boon et al., 2010, Tan et al., 2016). Challenges associated with the poor water-solubility and chemical instability of BC can often be overcome by encapsulating it within emulsion-based delivery systems (Mao and Miao, 2015, McClements and Li, 2010, Wang et al., 2015). These oil-in-water emulsions consist of BC dissolved in small lipid droplets that are dispersed in water. Due to increasing consumer demand for “clean-label” food products there has been considerable interest in formulating emulsions using natural emulsifiers, such as proteins, polysaccharides, phospholipids, and biosurfactants (McClements & Gumus, 2016). In particular, proteins have been widely used as emulsifiers because of their ready availability, strong surface activity, and good stabilizing properties (Lam & Nickerson, 2013). Another important role that protein-based emulsifiers may play at an oil-water interface is to act as interfacial antioxidants that inhibit the oxidation of encapsulated lipids (Berton-Carabin et al., 2014, Genot et al., 2013). Antioxidants are believed to be more effective in emulsions when they are located at the droplet surfaces because this is the site where the lipid oxidation reaction occurs (Jacobsen, 2015, Waraho et al., 2011). A major objective of the current study was therefore to develop protein-based antioxidant emulsifiers that could be used to form and stabilize oil-in-water emulsions, as well as to protect encapsulated BC from oxidation.
Previous studies have demonstrated the potential of using polyphenol-protein conjugates to encapsulate and protect lipophilic bioactive compounds (Almajano et al., 2007, Wei et al., 2015). For instance, emulsions stabilized by polyphenol-protein conjugates were reported to have better physical and chemical stability than those stabilized by proteins alone (Liu et al., 2016, Yi et al., 2015b). The amphiphilic protein molecules anchor the conjugates to the oil–water interfaces where they form a coating around the oil droplets that helps protect them from aggregation. On the other hand, the polyphenol part of the conjugates provides antioxidant activity that can protect polyunsaturated lipids from oxidation. Various chemical and enzymatic approaches are available to fabricate polyphenol-protein conjugates (Liu, Ma, Gao, & McClements, 2017). For example, Rawel and co-workers used the alkaline method to prepare chlorogenic acid-soy protein, caffeic acid-soy protein, gallic acid-soy protein, and gallic acid-lysozyme conjugates (Rawel et al., 2002, Rawel et al., 2000). Conjugation of the polyphenols and proteins was reported to influence the structural characteristics, physicochemical properties, and in-vitro digestibility of the proteins used. Chung and co-workers synthesized catechin-gelatin conjugates using an enzymatic method that involved laccase-catalyzed oxidation (Chung, Kurisawa, Uyama, & Kobayashi, 2003). The conjugates formed had strong free radical scavenging activity and protective effects against the oxidation of human low-density lipoprotein. Recently, a free radical grafting approach was reported to improve the physical and chemical stability of biopolymer-based emulsions (Wei and Gao, 2016, Yi et al., 2016). The conjugates prepared by this method exhibited higher antioxidant activity than those prepared using alkaline treatment (You, Luo, & Wu, 2014). In contrast to the conventional free radical method, a hydrogen peroxide-ascorbic acid pair was employed as a radical initiator system (Lei, Liu, Yuan, & Gao, 2014). The hydroxyl radicals generated by this reaction attack labile residues on the proteins, such as H-atoms in R-methylene groups (− CH2) or in amino-groups (− NH), thereby leading to the formation of protein radicals. These radicals then react with the polyphenols promoting the formation of protein–polyphenol conjugates. The advantage of this approach is that the reaction can be performed at room temperature and does not require the presence of metal irons or organic solvents, and thus the synthesized materials can easily be collected and purified (Spizzirri et al., 2009).
In the present study, egg white protein (EWP) and catechin (CT) were used to prepare conjugates suitable for utilization as emulsifiers. EWPs are widely used as food ingredients due to their high nutritional value and desirable functional attributes, such as gelation, emulsification, and foaming (Foegeding and Davis, 2011, Lam and Nickerson, 2013). EWPs consist of a mixture of globular proteins that have both hydrophilic and hydrophobic regions on their surfaces (Day et al., 2014, Yu et al., 2014). Consequently, EWPs are surface active molecules that can adsorb to oil droplet surfaces and form an interfacial coating that protects them from aggregation through a combination of electrostatic and steric repulsion (Chang et al., 2016, Lam and Nickerson, 2013). Catechin, which belongs to the flavonoid class, is present at a significant level in green tea and wine (Higdon and Frei, 2003, Kurisawa et al., 2003, Vuong et al., 2011). As shown in Fig. 1, catechin possesses two benzene rings (A- and B-rings), each bearing two phenolic hydroxyl groups connected to a dihydropyran heterocycle (C-ring) with a hydroxyl group at position 3. These hydroxyl groups are reported to be responsible for the pharmacological activities of catechin, including its anti-inflammation, anti-cancer, and immune modulation effects (Adhami et al., 2004, Butt and Sultan, 2009). These potentially beneficial effects have been partly attributed to the antioxidant activity of catechin, which is usually ascribed to its radical scavenging capacity and metal chelating activity (Intra & Kuo, 2007). The conjugation of catechin to EWP may therefore lead to the formation of protein-based antioxidant emulsifiers that can improve the physical and chemical stability of emulsion-based delivery systems.
The objective of this study was therefore to synthesize CT-EWP conjugates using a free radical method, characterize their molecular characteristics, and then establish their ability to form and stabilize emulsion-based delivery systems for BC.
Section snippets
Materials
Hen eggs were provided by Kangde Co., Ltd. (Jiangsu Province, China). Sunflower oil was obtained from a local supermarket. (+)-Catechin hydrate (CT) (≥ 98% HPLC), 2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-anilino-8-naphthalensulfonate (ANS), β-carotene (BC) (≥ 97% UV), and dialysis bag (MWCO 12,000–14,000 Da) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical
SDS-PAGE
Initially, the molecular weights of the major components in EWP, CT + EWP mixtures, and CT-EWP conjugates were determined by SDS-PAGE (Fig. 2). Three main bands were observed in the EWP with molecular weights around 14, 45, and 77 kDa, which correspond to lysozyme, ovalbumin, and ovotransferrin, respectively (Mine, 1995). The SDS-PAGE profile of the CT + EWP mixture was similar to that of EWP alone. Due to the weak non-covalent (physical) interactions between catechin and egg white proteins, some
Conclusion
In summary, a protein-based emulsifier with strong antioxidant activity was synthesized by the conjugation of catechin to egg white proteins using a hydrogen peroxide-ascorbic acid pair as a radical initiator. The covalent attachment of catechin to EWP was confirmed by electrophoresis and chromatography. Emulsions prepared using CT-EWP conjugates exhibited better physical stability at high temperatures and high ionic strengths those prepared using protein alone, which may have been due to an
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