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Impact of antioxidant on the stability of β-carotene in model beverage emulsions: Role of emulsion interfacial membrane
Food Chemistry 279 (2019) 194–201
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Impact of antioxidant on the stability of β-carotene in model beverage emulsions: Role of emulsion interfacial membrane Ha Youn Songa, Tae Wha Moona,b,
⁎,1
, Seung Jun Choic,
T
⁎,1
a
Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea Center for Food and Bioconvergence, and Research Institute of Agricultural and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea c Department of Food Science and Technology, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antioxidants β-Carotene Degradation Emulsions Interfacial characteristics
The effect of the thickness and density of droplet interfacial membrane on the chemical stability of β-carotene in emulsions was investigated, and its impact on the effectiveness of oil-soluble antioxidants to retard β-carotene degradation was examined. β-Carotene was incorporated into the emulsions stabilized by PEGylated emulsifiers having various-sized hydrophilic groups. In the presence of oxidative stresses (pH, iron ions, and radicals in this study), it was observed that the interfacial thickness was relevant to the stability of β-carotene encapsulated into emulsion droplets. Particularly, iron-mediated carotene degradation was effectively retarded in the emulsions having a thin interfacial membrane than ones with a thick interfacial membrane. The interfacial denseness also affected β-carotene stability but its ability to retard β-carotene degradation was influenced by the interfacial thickness. Although β-carotene degradation rate decreased upon the addition of oil-soluble antioxidants, its antioxidant activity depended on what prooxidant promoted the degradation of β-carotene in the emulsions.
1. Introduction Carotenoids are natural pigments that are produced by plants, algae, and microorganisms. They are derivatives of tetraterpenes, consisting of 8 isoprene molecules, and are responsible for the red, orange, and yellow colors of many fruits and vegetables because their molecular structure absorbs wavelengths ranging between 400 and 550 nm (Maiani et al., 2009). More than 600 carotenoids have been identified, and they are categorized into two classes: carotenes and xanthophylls, depending on whether they contain oxygen or not, respectively (Olson & Krinsky, 1995). Regardless of the presence of oxygen in their molecular structure, since carotenoids are hydrophobic, they are present in lipid membranes or other hydrophobic regions (Britton, 1995). The antioxidant activity of carotenoids, derived from their molecular structure, bestows them with the ability to protect organic molecules and tissues from the damage caused by light and oxygen (Britton, 1995). Carotenoids, because of their antioxidant activity, have potential health benefits on the prevention of serious health disorders such as cancers and cardiovascular diseases (Krinsky, 2001). Furthermore, since some carotenoids that contain unsubstituted β-ionone rings are retinal precursors, they provide a substantial proportion of vitamin A in
the human diet (Maiani et al., 2009). Despite the significant interest of consumers for the reasons mentioned above, the incorporation of carotenoids in food products as functional ingredients is not easy because carotenoids are vulnerable to chemical and oxidative degradation and have low water solubility (Boon et al., 2008). A solution widely accepted in food industry to circumvent these drawbacks is the encapsulation of carotenoids in emulsions or nanostructured delivery systems. Emulsions have been found to be an effective way of incorporating carotenoids in food products (McClements, 2013) because the interfacial membrane isolates the oil-soluble bioactives in oil droplets from the aqueous phase containing acids, transition metals, and radicals that accelerate the chemical and/or oxidative degradation of oil-soluble bioactives. The interfacial membrane, formed by emulsifiers, plays an important role in the physicochemical stability of the emulsions containing carotenoids. It protects them from flocculation and coalescence and also prevents/ retards the interaction between the carotenoids in the emulsion droplets and the pro-oxidants in the aqueous phase by acting as a physical, or sometimes electrostatic, barrier. As described above, although the incorporation of carotenoids in emulsions is useful to reduce their degradation, the addition of
⁎
Corresponding authors at: Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea (T.W. Moon). Department of Food Science and Technology, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea (S.J. Choi). E-mail addresses: [email protected] (T.W. Moon), [email protected] (S.J. Choi). 1 Tae Wha Moon and Seung Jun Choi contributed equally to this work as corresponding authors. https://doi.org/10.1016/j.foodchem.2018.11.126 Received 26 June 2018; Received in revised form 21 November 2018; Accepted 22 November 2018 Available online 05 December 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
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mixing 5% oil phase with 95% (w/w) aqueous phase (27.94 and 100 µmol of β-carotene and TBHQ, respectively, in 1 kg of emulsion). Coarse emulsions were prepared by mixing the oil and aqueous phases together using a high-speed blender for 2 min at room temperature. To reduce the droplet size, the premixed emulsions were homogenized with five passes through a microfluidizer (MN400BF, Micronox, Seongnam, Korea) at 100 MPa. Then, the pH of every emulsion sample was adjusted to a predetermined value using 0.1 N and 1.0 N hydrochloric acid solutions. All the emulsions were stirred at the desired pH for at least 30 min under nitrogen before storing them in the dark at 25 °C to prevent any potential degradation of β-carotene by light.
antioxidants to emulsions helps in limiting the influence of storage conditions (pH, transition metals, and radicals) on the degradation of the carotenoids encapsulated in the emulsions (Berton-Carabin, Ropers, & Genot, 2014; Bou, Boon, Kweku, Hidalgo, & Decker, 2011; Qian, Decker, Xiao, & McClements, 2012b). Carotenoid degradation could be effectively retarded by adding water-soluble or oil-soluble antioxidants to emulsions. Oil-soluble antioxidants such as coenzyme Q10 and αtocopherol, and water-soluble antioxidants including ethylenediaminetetraacetic acid (EDTA), ascorbic acid, and gallyl derivatives are effective in decreasing the oxidative degradation of lycopene and β-carotene (Bou et al., 2011; Qian, Decker, Xiao, & McClements, 2012a). The combination of oil-soluble and water-soluble antioxidants can be more or less effective than the individual antioxidants (Qian et al., 2012a). The composition of the aqueous phases of the emulsions affects the ability of the antioxidants to reduce carotenoid degradation in the emulsions. The properties of emulsion droplet surfaces, particularly their charge, can also influence the effectiveness of antioxidants. The previous findings revealed that the lipid oxidation in emulsions mainly occur at the oil droplet surfaces in the presence of water-soluble prooxidants (Lee, Song, & Choi, 2018; Silvestre, Chaiyasit, Brannan, McClements, & Decker, 2000). Similar to lipid oxidation of emulsions, the decomposition of lipophilic functional compounds encapsulated in emulsion droplets could occur mainly at the oil droplet surfaces when emulsions contain the compounds what promote the degradation of lipophilic compounds. Therefore, if an emulsion contains oil-soluble antioxidants, the reaction of antioxidants with water-soluble prooxidants could occur frequently at the oil droplet surfaces. This means that the interfacial membrane plays an important role in controlling the stability of lipophilic compounds in emulsions and that its characteristics also affect the reaction rate of antioxidants with water-soluble prooxidants. However, the influence of the properties (charge, emulsifier type (synthetic or biopolymer), number of layers, thickness, density, etc.) of emulsion droplet surfaces on the ability and effectiveness of antioxidants to retard the carotenoid degradation in emulsions are not clear. Therefore, in the current study, β-carotene, a prototypical carotenoid, was encapsulated in emulsions having oil droplet interfaces of various thicknesses, and the ability of tert-butylhydroquinone (TBHQ), a widely used oil-soluble antioxidant, to postpone the oxidation of βcarotene in emulsion droplets because of the conditions of the aqueous phase was investigated. The results of this study should help in designing effective emulsion-based encapsulation systems capable of retarding β-carotene degradation during long-term storage.
2.3. Droplet size measurement The mean emulsion droplet diameters were measured using static light scattering (laser diffraction). To avoid multiple scattering effects, all emulsion samples were diluted to a droplet concentration of approximately 0.005% (w/w) using a buffer solution at the pH of the sample, and stirred continuously throughout the measurements to ensure homogeneity. The particle size distribution of the emulsions was then measured using a commercial static light scattering instrument (BT-9300ST; Bettersize Instruments, Dandong, China). The particle size data are reported as either the volume-weighted mean diameter, d43 = ∑ ni ∙di4 / ∑ ni ∙di3 , or surface-weighted mean diameter, d32 = ∑ ni ∙di3/ ∑ ni ∙di2 , where ni is the number of particles with diameter di . 2.4. Influence of transition metals and free radicals on β-carotene degradation To determine the effect of iron species on the stability of β-carotene in the emulsions, ferric chloride or ferrous sulfate stock solutions were added to the emulsion samples up to a concentration of 100 µmol/kg emulsion. Furthermore, to determine the effect of free radicals on the stability of β-carotene in emulsions, AAPH sulfate stock solutions were added to the emulsion samples up to the concentration of 100 µmol/kg emulsion. 2.5. Measurement of β-carotene concentration The β-carotene concentrations in the emulsions were determined by vortexing vigorously 2 g of emulsion with 6 g dichloromethane (SigmaAldrich) for 2 min. The mixture was then centrifuged at 3000×g for 5 min, and the solvent layer was collected. The solvent absorbance was measured at 450 nm using a UV–vis scanning spectrophotometer (Optizen Pop; Mecasys, Daegeon, Korea). The degradation rate constant (k) of β-carotene in the emulsions was calculated assuming the following first-order exponential decay model:
2. Materials and Methods 2.1. Materials β-Carotene, PEGylated emulsifiers (polyoxyethylene 10 stearyl ether (S10), polyoxyethylene 20 stearyl ether (S20), and polyoxyethylene 100 stearyl ether (S100)), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and TBHQ were purchased from Sigma-Aldrich (St. Louis. MO, USA). Polyoxyethylene alkyl ether-type emulsifiers are nonionic surfactants containing polyethyleneoxide chains as the hydrophilic groups and n-alkyl chains as the hydrophobic groups. Medium chain triglyceride (Delios S) was obtained from BASF (Ludwigshafen, Germany). All other chemicals were reagent grade and obtained from Sigma-Aldrich or Fisher Scientific (Pittsburgh, PA).
Ct = C0 ∙e−k ∙ t ,
2.2. Emulsion preparation
2.6. Statistical analysis
The oil phase of the emulsions was prepared immediately before use by dissolving β-carotene (to a final concentration of 0.3 mg/g oil) and TBHQ (to a final concentration of 2 mg/g oil) in medium chain triacylglycerol (MCT), and the aqueous phase was prepared by dissolving PEGylated emulsifiers in a 10 mM phosphate buffer (pH 7) in predetermined concentrations. Oil-in-water emulsions were prepared by
All the experiments were performed in triplicate, and the data are expressed as mean ± standard deviation. To indicate significant differences in coefficients (degradation rate constants (k) of β-carotene) between linear regressions, Chow test was performed (Chow, 1960). The statistical analyses described above were all conducted using SAS (version 9.4.; SAS Institute Inc., Cary, NC, USA).
where C0 is the initial β-carotene concentration and Ct is the β-carotene concentration remaining at time t. The value of k was determined by performing linear regression on the plot of ln(Ct / C0) versus t.
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β-carotene, the pH was adjusted to 3 and 7 and the emulsions were stored at 25 °C (Fig. 1). Regardless of the pH, the β-carotene in the emulsions gradually degraded; however, its degradation rate was extremely faster at pH 3 than at pH 7 (Fig. 1A and B). It has been reported that β-carotene degradation is faster in acidic environments (Qian et al., 2012b; Song, Moon, & Choi, 2018; Xu, Yuan, Gao, McClements, & Decker, 2013). When carotenoids are exposed to acids, carotenoid carbocations are produced, and this transformation is thought to be the first step in the degradation process of carotenoids by acids (Konovalov & Kispert, 1999). Although not as much as pH 3, the β-carotene in the emulsions at pH 7 gradually degraded during storage (Fig. 1A). Even if the pH of the emulsion is neutral, the degradation of β-carotene encapsulated in the emulsions could easily occur through its isomerization and/or oxidation when exposed to heat, UV, and oxygen (Knockaert et al., 2012). However, since all emulsions were stored in the dark at 25 °C, the β-carotene degradation at pH 7 observed in our study may be due to other factors rather temperature and/or light. One possible reason for this observation is the presence of oxygen molecules in the emulsions. Although nitrogen purging was carried out during the emulsion preparation to minimize the β-carotene degradation by oxygen as described in Section 2, it was apparent that the oxygen dissolved in the emulsion, particularly in the aqueous phase, was not completely removed. Another potential reason is isomerization and/or oxidation of the β-carotene at the elevated temperatures during the homogenization. As shown in Fig. 1A and B, the β-carotene stability seems to be inversely proportional to the interfacial thickness of the emulsion droplets. The inverse relationship between interfacial thickness and β-carotene stability at pH 3 could be explained by the adsorption of protons (H+) onto the droplet surface. The oxygen dissolved in the aqueous phase was also one of the reasons for β-carotene degradation at pH 3. Since the PEGylated emulsifiers used in this study are non-ionic, their hydrophilic groups are not charged. However,
3. Results and discussion In order to avoid any influence of the micelles formed from unadsorbed emulsifier molecules on the stability of the emulsions and the concentration of β-carotene therein (McClements, 1994; Wulff-Pérez, Torcello-Gómez, Gálvez-Ruíz, & Martín-Rodríguez, 2009), the minimum emulsifier concentration (MEC) required to prepare emulsions having the smallest oil droplet size and long-term stability was determined in a previous study (Han, Song, Moon, & Choi, 2018). The MECs of S10, S20, and S100 were 3.165, 2.926, and 0.994 mM, respectively. The initial droplet diameters of the emulsions are all fairly similar (d43 = 0.31, 0.31, and 0.33 μm and d32 = 0.26, 0.24, and 0.24 μm for S10-, S20-, and S100-stabilized emulsions, respectively) at the MECs, and did not change significantly (p > 0.05) during 21-day storage, suggesting that all the emulsions had a similar specific surface area. 3.1. Impact of pH on β-carotene stability in emulsions having various interfacial characteristics Previous studies have shown that the lipid oxidation of emulsions and the stability of the functional lipophilic compounds therein depend on the environments surrounding oil droplets (Berton-Carabin et al., 2014). Even though such functional compounds, located in the core of the emulsion droplets, are isolated from the harsh environments of the aqueous solution by the interfacial membrane, they gradually, sometimes rapidly, degrade. Since the interfacial membrane has the ability to alter the rates of the chemical reactions between oil- and aqueousphase compounds, the interfacial characteristics could be important in the lipid oxidation of emulsions and the stability of the lipophilic compounds therein. To determine the effect of the pH of the emulsions on the stability of
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Fig. 1. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ. 196
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Fig. 2. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C with ferrous irons. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ.
emulsions were calculated as 3.27, 3.02, and 1.09 nmol/m2, respectively, from droplet size and emulsifier concentration. As a result, the emulsions could have interfacial membranes with a different denseness. The value of S10-stabilized emulsion was very similar to that of S20stabilized emulsion and it was approximately 3 times higher than that of S100-stabilized emulsion. The difference in k values of β-carotene between S100- and S10-stabilized emulsions was much less than 10times. When emulsions have a similar droplet interfacial denseness (S10- and S20-stabilized emulsions in this work), it seems that those with thinner interfacial membranes have a better ability to retard βcarotene degradation. However, considering the emulsifier loading values of emulsions, it seems that there is no systematic relationship between the denseness of the interfacial membrane and the ability of the interfacial membrane to retard β-carotene degradation.
considering that the emulsifiers used in this study have the numbers of oxyethylene group in their hydrophilic groups, a negative dipole moment could be detected on the oxygen atom sites of the hydrophilic groups of PEGylated emulsifiers, indicating that their hydrophilic groups could attract protons via dipole-ion interactions (Hägerstrand et al., 2001). The pH of the interfacial region could be lower than that of the aqueous phase because the protons tend to accumulate around the oil droplet surfaces due to dipole-ion interactions. Hence, acidpromoted β-carotene degradation may be accelerated within the interfacial region. Since the number of oxyethylene groups is larger in S100 than in S10 and S20, protons could accumulate around the oil droplet surfaces of the S100-stabilized emulsion to a higher extent than around those of the S10- and S20-stabilized emulsions. This hypothesis could explain why the β-carotene stability in the emulsions having a thicker interface (stabilized by emulsifiers having a larger number of oxyethylene groups) is lower than that in emulsions having a thinner interface (stabilized by emulsifiers having a smaller number of oxyethylene groups). However, the effect of the proton accumulation around the interfacial region on β-carotene degradation did not seem to be greater than expected because the difference in the number of oxyethylene groups between S100 and S10 was 10-times but the difference in k values of β-carotene between S100- and S10-stabilized emulsions was much less than 10-times. As noted above, a similar mean droplet diameter was measured for all the emulsions but the emulsifier concentrations in emulsions were different from each other because of the different MECs between emulsifiers. If an emulsion is prepared at the MECs of S10, S20, or S100, most of the emulsifier molecules could be found at the interface between oil and water and this emulsion contains little-to-no micelle therein. Hence, emulsions should have the different emulsifier concentrations per unit droplet surface area (mass (emulsifier) loading). The emulsifier loading values for S10-, S20-, and S100-stabilized
3.2. Impact of transition metals on β-carotene stability in emulsions having various interfacial characteristics It is well known that transition metals accelerate carotenoid degradation, particularly because of their high oxidation state (Boon et al., 2008). During iron-mediated degradation of carotenoids, carotenoid radical cations are produced through the electron transfer reaction between carotenoids and Fe3+ (Gao & Kispert, 2003) that are converted into carotenoid peroxyl radical cations by interacting with oxygen (Gao & Kispert, 2003; Wei, Gao, & Kispert, 1997) or into carotenoid dications by interacting with Fe3+ (Gao, Wei, Jeevarajan, & Kispert, 1996). Then, carotenoid cation peroxyl radicals and carotenoid dications are degraded through further oxidation processes. They could also oxidize neutral carotenoids. Therefore, the rate of the abovementioned reactions could determine the rate of β-carotene degradation in emulsions. However, except the reaction of neutral carotenoids with carotenoid peroxyl radical cations and/or carotenoid dications, 197
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Fig. 3. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C with ferric irons. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ.
seems that the low solubility of iron at neutral pH could be the major factor affecting β-carotene degradation. The β-carotene degradation rate was not significantly altered by the presence of Fe3+ in the emulsions. This seems to indicate that the interfacial membrane, regardless of its thickness and/or denseness, significantly interrupted the interaction of Fe3+ with oil droplets and the formation of β-carotene radical cations at pH 7. Therefore, assuming that the interfacial membrane also interrupted the interaction of Fe2+ with oil droplets, no ironinduced β-carotene degradation mechanisms are expected to degrade the β-carotene in the emulsions containing Fe2+. Fe2+ could participate in producing some of the reactive oxygen species such as singlet oxygen (1O2) and hydroxyl radicals (HO%), strong oxidants; they could also produce superoxide radical anions (O2%−) by reacting with triplet oxygen (3O2) (Choe, 2008). O2%− could produce hydrogen peroxide (H2O2) by dismutation, and the reaction between H2O2 and O2%−
most of the processes critically affecting the carotenoid degradation rate occur at the interface between the oil and aqueous phases. This indicates that the storage stability of the β-carotene incorporated into the oil droplets could be significantly affected by their interfacial characteristics. At pH 7, Fe2+ increased the degradation rate of the β-carotene incorporated in the emulsions; however, the β-carotene degradation rate was not significantly affected by Fe3+ (Figs. 2 and 3, Table 1). A possible reason for this observation is that the interaction of iron molecules with oil droplets is reduced at this pH (Boon, McClements, Weiss, & Decker, 2009). Since the solubility of iron molecules is considerably lower at neutral pH compared with that at acidic pH, it appears that the iron molecules are less likely to interact with the oil droplets containing β-carotene. However, considering that Fe2+ accelerated β-carotene degradation at pH 7, particularly in the S100-stabilized emulsion, it
Table1 Degradation rate constant (k (day−1)) of β-carotene determined by modeling assuming a 1st-order reaction.
pH 7
pH 3
Oxidative stress
S10
S20
Without TBHQ
With TBHQ
Without TBHQ
With TBHQ
Without TBHQ
With TBHQ
No Ferrous iron Ferric iron Radicals
F
B
E
B
F
B
No Ferrous iron Ferric iron Radicals
D
bc
0.0054 0.0085b F 0.0053b BC 0.0939b E
0.0523bc 0.1007b C 0.0832b A 0.1368b B
e
S100
b
de
0.0018 0.0023d B 0.0029c B 0.0027d
D
0.0063 0.0107ab E 0.0069ab B 0.1054ab
B
B
C
0.0035d 0.0112c A 0.0126cd B 0.0027c
B
0.0029d 0.0113c A 0.0105d B 0.0022c
0.0536b 0.1273ab B 0.0944b A 0.1478ab
A
AB
0.0030 0.0032cd B 0.0034c B 0.0035cd
0.0081 0.0169a F 0.0082a C 0.1182a
B
D
A
The values with different capital-letter superscripts in a same column are significantly different (p < 0.05) by Chow test. The values with different small-letter superscripts in a same row are significantly different (p < 0.05) by Chow test. 198
a
E
0.0626a 0.1566a B 0.1408a A 0.1757a AB
0.0043cd 0.0049c B 0.0039c B 0.0046c B
B
0.0039cd 0.0123c A 0.0133c B 0.0030c
A
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Fig. 4. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C with AAPH. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ.
generates HO% and 1O2 (Kehrer, 2000). HO% is also produced from the decomposition of H2O2 in the presence of iron, particularly Fe2+ (Salem, El-Maazawi, & Zaki, 2000). It appears that these reactive oxygen species generated by Fe2+ may interact with oil droplets and they may initiate the degradation of β-carotene at pH 7. Thus, it was possible to speculate that the interfacial membrane of the emulsions prepared in this study well interrupted the direct interaction of iron with the emulsion droplets but it did not prevent the initiation of βcarotene degradation occurred by the attack of oxygen-containing radicals on emulsion droplets. The presence of iron, regardless of its species, caused the rapid degradation of β-carotene in emulsions during storage at acidic pH (Figs. 2B and 3B, and Table 1). Considering the oxidation mechanism of β-carotene described above, it is not difficult to explain this observation. Although the interfacial membrane significantly interrupts the interaction of iron with oil droplets, iron could interact with the droplets more frequently at pH 3 than at pH 7 because of the higher solubility of iron at acidic pH. β-Carotene was gradually degraded during its storage in the presence of Fe3+; however, the level of β-carotene dropped sharply at the initial stage of its storage in the presence of Fe2+. It may be not difficult to identify the reason for this sharp drop of the β-carotene concentration in the emulsions containing Fe2+ at the beginning of the storage. In emulsions containing Fe3+ at pH 3, βcarotene degradation could be initiated by protons rather than Fe3+. The decomposition of the β-carotene carbocations by Fe3+ could occur at the emulsion droplet surfaces or they could also react with neutral βcarotene molecules in the oil droplets. In the emulsions containing Fe2+ at pH 3, β-carotene oxidation was initiated at the oil droplet surfaces by protons and oxygen-containing radicals. Then, the products of those reactions were decomposed at the oil droplet surfaces by Fe2+, or they reacted with the neutral β-carotene molecules. As described above, since the degradation mechanism of β-carotene by Fe2+ in emulsions is
more varied than that involving Fe3+, the β-carotene in emulsions could be degraded faster by Fe2+ than Fe3+. Considering that the rate of decomposition of lipid hydroperoxides by Fe2+ is higher than that by Fe3+ (Mei, McClements, Wu, & Decker, 1998), the rate of decomposition of β-carotene peroxyl radicals, intermediates of the oxidative degradation of β-carotene, by Fe2+ could be higher than that by Fe3+. As shown in Table 1, the S100-stabilized emulsion, the one having the thickest interfacial membrane among the emulsions investigated in this study, showed the highest k value between emulsions stored at the same condition. This observation could be explained through the hypothesis presented above to explain why the acid-mediated degradation of β-carotene was faster in the emulsions having a thicker interface (stabilized by emulsifiers having a larger number of oxyethylene groups) than in the ones having a thinner interface (stabilized by emulsifiers having a smaller number of oxyethylene groups). A negative dipole moment on oxygen atoms of the hydrophilic groups of emulsifiers could attract iron molecules, regardless of their oxidative state and one molecule of S100 could accumulate iron molecules to a higher amount than S10 and/or S20. Hence, a higher amount of iron molecules could accumulate around the oil droplet surfaces of the S100-stabilized emulsion than around those of the S10- and S20-stabilized ones, leading to the faster degradation of β-carotene in S100-stabilized emulsion than in S10- and S20-stabilized ones. Therefore, the stability of β-carotene in emulsions in the presence of iron molecules seems to be inversely related to the interfacial thickness of the oil droplets. However, as shown in Table 1, the interfacial denseness plays a minor role in β-carotene degradation. 3.3. Impact of free radicals on β-carotene stability in emulsions having various interfacial characteristics Lipid containing foods may contain free radicals, generated through 199
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the TBHQ in the core of the emulsion droplets may convert the β-carotene radicals back to β-carotene. At pH 7, it was apparent that the effectiveness of TBHQ to inhibit the degradation of the β-carotene incorporated in the emulsions was affected by the interfacial thickness rather than by its denseness. It is not clear how interfacial thickness affected the effectiveness of TBHQ at pH 7. However, at pH 3, the correlation between interfacial thickness and/or denseness and the ability of TBHQ to inhibit β-carotene degradation was not clear. Interestingly, although TBHQ effectively degraded the β-carotene in emulsions at pH 3, it was considerably less effective in the emulsions containing Fe2+ and Fe3+ than in the emulsions containing other prooxidants (Table 1). This could be due to the iron-reducing activity of TBHQ. Phenolic compounds are able to reduce Fe3+ to Fe2+, and their reducing ability is larger at lower pH values (Dunford, 1987). TBHQ, a phenolic antioxidant, reduced Fe3+ to Fe2+, and the prooxidant activity of iron in the emulsions increased because the reactivity of Fe2+ is higher than that of Fe3+. Therefore, in emulsions containing iron molecules at pH 3, TBHQ blocked the interaction of the prooxidants present in the aqueous phase with the oil droplets, and neutral β-carotene was generated from β-carotene radicals; however, the more reactive Fe2+ was accumulated in the aqueous phase. The metal reducing activity of TBHQ could explain why it was less effective in inhibiting β-carotene degradation at low pH in emulsions containing iron.
lipid oxidation during food processing, up to a certain level (Choe & Min, 2006). These free radicals could accelerate the oxidation of neutral lipids, and could also initiate the degradation of the functional compounds in food media by reacting with them. If these free radicals are oil-soluble and located in the interior of oil droplets, they could attack neutral lipids and/or oil-soluble functional compounds in emulsion systems without any physical interference. However, if the free radicals are water-soluble, since they have to interact with oil droplets to attack the oil-soluble functional compounds therein, the degradation of the oil-soluble functional compounds in emulsions could be greatly influenced by the nature of the interface (Miyashita, 2008). Therefore, using AAPH, a water-soluble free radical generator, the effect of free radicals on β-carotene stability in emulsions having different thicknesses was evaluated (Fig. 4). Independent of the interfacial thickness and pH, over 90% of βcarotene was degraded during 21-day storage. The interaction of free radicals with β-carotene in oil droplets was the main driving force for βcarotene degradation at pH 7, whereas, in addition to the interaction mentioned above, it seems that β-carotene was also degraded by the interaction of protons with β-carotene at pH 3. The higher k values of βcarotene in emulsions at pH 3 are a strong evidence for this hypothesis (Table 1). This suggests that the interfacial membrane of the emulsions investigated did not have the ability to interrupt the entry of free radicals in the oil droplets. As described above, the degradation of βcarotene in emulsions by oxygen-containing radicals is not well impeded by the interfacial membrane. Therefore, it appears that the attack of AAPH radicals on the β-carotene at the emulsion droplet surfaces is not well blocked by the interfacial membrane either. As shown in Fig. 2A and B, when the oxygen-containing radicals act as prooxidants, the effect of interfacial denseness is unclear; however, it seems that interfacial thickness has the tendency to affect storage stability of βcarotene. However, when AAPH radicals were present in the emulsions, although it appeared that the ability of the interfacial membrane to retard β-carotene degradation was inversely proportional to the interfacial thickness, β-carotene concentration rapidly dropped regardless of the interfacial thickness. A possible explanation for this observation is the high concentration of the AAPH radicals existent in the emulsions. Compared with the concentration of the oxygen-containing radicals in iron-containing emulsions, the concentration of the AAPH radicals is enormous. When radicals are present in food media, several reactions of the radicals with carotenoids, including electron transfer (Young & Lowe, 2001), hydrogen abstraction (Woodall, Lee, Weesie, Jackson, & Britton, 1997), and the addition of radical species to form carotenoidradical adducts (Mortensen, 2002) are possible. Therefore, vigorous attacks of the AAPH radicals on the oil droplets through the reactions mentioned above were observed despite the thickness-dependent defensive ability of the interfacial membrane. It was also apparent that βcarotene stability was not influenced by interfacial denseness.
4. Conclusion The results show that the interfacial characteristics, particularly thickness, highly affect the stability of β-carotene encapsulated within emulsions. It is apparent that the ability of the interfacial membrane to retard β-carotene degradation in emulsions is inversely related to its thickness. The interaction of the water-soluble compounds (transition metals and free radicals in this work), which are able to contribute to βcarotene degradation, with the emulsion droplets containing β-carotene is more effectively retarded in the emulsions having a thinner interfacial membrane than emulsions having a thicker one. TBHQ, the oilsoluble phenolic antioxidant used in the current study, was found to increase the stability of β-carotene in emulsions against the chemical degradation promoted by the compounds in the aqueous phase; however, its ability to increase the β-carotene stability varied with what oxidative stress promoted the degradation of β-carotene in the emulsions. Independent on emulsion pH, TBHQ effectively prevent β-carotene in emulsions from radical-mediated degradation. TBHQ was still effective to inhibit iron-mediated degradation of β-carotene at neutral pH but its ability to increase the stability of β-carotene against prooxidant metals at acidic pH. Therefore, our results have important applications for the proper selection of emulsifiers capable of producing emulsions that can encapsulate and protect β-carotene in commercial food products and for the rational selection of antioxidants to achieve the objective mentioned above.
3.4. Impact of antioxidants on β-carotene stability in emulsions having different interfacial characteristics To understand how antioxidants inhibit the chemical degradation of the β-carotene encapsulated in emulsions of various interfacial thicknesses, emulsions containing TBHQ (an oil-soluble antioxidant) were prepared. The addition of TBHQ effectively inhibited the chemical degradation of β-carotene (Figs. 1–4, and Table 1) due to its ability to scavenge the reactive free radicals. When β-carotene and TBHQ coexist in the emulsion droplets, although β-carotene and TBHQ are both oilsoluble molecules, TBHQ preferentially accumulates near the emulsion droplet surfaces because its log P (2.94) is lower than that of β-carotene (17.62). Therefore, when the prooxidants in the aqueous phase of the emulsions interact with the oil droplets, they frequently react with TBHQ rather than with β-carotene (Silvestre et al., 2000). In conclusion, the TBHQ molecules located near the emulsion droplet surfaces directly blocked the attack of the prooxidants in the aqueous phase, and
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03930215), Republic of Korea. 200
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Impact of antioxidant on the stability of β-carotene in model beverage emulsions: Role of emulsion interfacial membrane Ha Youn Songa, Tae Wha Moona,b,
⁎,1
, Seung Jun Choic,
T
⁎,1
a
Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea Center for Food and Bioconvergence, and Research Institute of Agricultural and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea c Department of Food Science and Technology, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Antioxidants β-Carotene Degradation Emulsions Interfacial characteristics
The effect of the thickness and density of droplet interfacial membrane on the chemical stability of β-carotene in emulsions was investigated, and its impact on the effectiveness of oil-soluble antioxidants to retard β-carotene degradation was examined. β-Carotene was incorporated into the emulsions stabilized by PEGylated emulsifiers having various-sized hydrophilic groups. In the presence of oxidative stresses (pH, iron ions, and radicals in this study), it was observed that the interfacial thickness was relevant to the stability of β-carotene encapsulated into emulsion droplets. Particularly, iron-mediated carotene degradation was effectively retarded in the emulsions having a thin interfacial membrane than ones with a thick interfacial membrane. The interfacial denseness also affected β-carotene stability but its ability to retard β-carotene degradation was influenced by the interfacial thickness. Although β-carotene degradation rate decreased upon the addition of oil-soluble antioxidants, its antioxidant activity depended on what prooxidant promoted the degradation of β-carotene in the emulsions.
1. Introduction Carotenoids are natural pigments that are produced by plants, algae, and microorganisms. They are derivatives of tetraterpenes, consisting of 8 isoprene molecules, and are responsible for the red, orange, and yellow colors of many fruits and vegetables because their molecular structure absorbs wavelengths ranging between 400 and 550 nm (Maiani et al., 2009). More than 600 carotenoids have been identified, and they are categorized into two classes: carotenes and xanthophylls, depending on whether they contain oxygen or not, respectively (Olson & Krinsky, 1995). Regardless of the presence of oxygen in their molecular structure, since carotenoids are hydrophobic, they are present in lipid membranes or other hydrophobic regions (Britton, 1995). The antioxidant activity of carotenoids, derived from their molecular structure, bestows them with the ability to protect organic molecules and tissues from the damage caused by light and oxygen (Britton, 1995). Carotenoids, because of their antioxidant activity, have potential health benefits on the prevention of serious health disorders such as cancers and cardiovascular diseases (Krinsky, 2001). Furthermore, since some carotenoids that contain unsubstituted β-ionone rings are retinal precursors, they provide a substantial proportion of vitamin A in
the human diet (Maiani et al., 2009). Despite the significant interest of consumers for the reasons mentioned above, the incorporation of carotenoids in food products as functional ingredients is not easy because carotenoids are vulnerable to chemical and oxidative degradation and have low water solubility (Boon et al., 2008). A solution widely accepted in food industry to circumvent these drawbacks is the encapsulation of carotenoids in emulsions or nanostructured delivery systems. Emulsions have been found to be an effective way of incorporating carotenoids in food products (McClements, 2013) because the interfacial membrane isolates the oil-soluble bioactives in oil droplets from the aqueous phase containing acids, transition metals, and radicals that accelerate the chemical and/or oxidative degradation of oil-soluble bioactives. The interfacial membrane, formed by emulsifiers, plays an important role in the physicochemical stability of the emulsions containing carotenoids. It protects them from flocculation and coalescence and also prevents/ retards the interaction between the carotenoids in the emulsion droplets and the pro-oxidants in the aqueous phase by acting as a physical, or sometimes electrostatic, barrier. As described above, although the incorporation of carotenoids in emulsions is useful to reduce their degradation, the addition of
⁎
Corresponding authors at: Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea (T.W. Moon). Department of Food Science and Technology, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea (S.J. Choi). E-mail addresses: [email protected] (T.W. Moon), [email protected] (S.J. Choi). 1 Tae Wha Moon and Seung Jun Choi contributed equally to this work as corresponding authors. https://doi.org/10.1016/j.foodchem.2018.11.126 Received 26 June 2018; Received in revised form 21 November 2018; Accepted 22 November 2018 Available online 05 December 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
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mixing 5% oil phase with 95% (w/w) aqueous phase (27.94 and 100 µmol of β-carotene and TBHQ, respectively, in 1 kg of emulsion). Coarse emulsions were prepared by mixing the oil and aqueous phases together using a high-speed blender for 2 min at room temperature. To reduce the droplet size, the premixed emulsions were homogenized with five passes through a microfluidizer (MN400BF, Micronox, Seongnam, Korea) at 100 MPa. Then, the pH of every emulsion sample was adjusted to a predetermined value using 0.1 N and 1.0 N hydrochloric acid solutions. All the emulsions were stirred at the desired pH for at least 30 min under nitrogen before storing them in the dark at 25 °C to prevent any potential degradation of β-carotene by light.
antioxidants to emulsions helps in limiting the influence of storage conditions (pH, transition metals, and radicals) on the degradation of the carotenoids encapsulated in the emulsions (Berton-Carabin, Ropers, & Genot, 2014; Bou, Boon, Kweku, Hidalgo, & Decker, 2011; Qian, Decker, Xiao, & McClements, 2012b). Carotenoid degradation could be effectively retarded by adding water-soluble or oil-soluble antioxidants to emulsions. Oil-soluble antioxidants such as coenzyme Q10 and αtocopherol, and water-soluble antioxidants including ethylenediaminetetraacetic acid (EDTA), ascorbic acid, and gallyl derivatives are effective in decreasing the oxidative degradation of lycopene and β-carotene (Bou et al., 2011; Qian, Decker, Xiao, & McClements, 2012a). The combination of oil-soluble and water-soluble antioxidants can be more or less effective than the individual antioxidants (Qian et al., 2012a). The composition of the aqueous phases of the emulsions affects the ability of the antioxidants to reduce carotenoid degradation in the emulsions. The properties of emulsion droplet surfaces, particularly their charge, can also influence the effectiveness of antioxidants. The previous findings revealed that the lipid oxidation in emulsions mainly occur at the oil droplet surfaces in the presence of water-soluble prooxidants (Lee, Song, & Choi, 2018; Silvestre, Chaiyasit, Brannan, McClements, & Decker, 2000). Similar to lipid oxidation of emulsions, the decomposition of lipophilic functional compounds encapsulated in emulsion droplets could occur mainly at the oil droplet surfaces when emulsions contain the compounds what promote the degradation of lipophilic compounds. Therefore, if an emulsion contains oil-soluble antioxidants, the reaction of antioxidants with water-soluble prooxidants could occur frequently at the oil droplet surfaces. This means that the interfacial membrane plays an important role in controlling the stability of lipophilic compounds in emulsions and that its characteristics also affect the reaction rate of antioxidants with water-soluble prooxidants. However, the influence of the properties (charge, emulsifier type (synthetic or biopolymer), number of layers, thickness, density, etc.) of emulsion droplet surfaces on the ability and effectiveness of antioxidants to retard the carotenoid degradation in emulsions are not clear. Therefore, in the current study, β-carotene, a prototypical carotenoid, was encapsulated in emulsions having oil droplet interfaces of various thicknesses, and the ability of tert-butylhydroquinone (TBHQ), a widely used oil-soluble antioxidant, to postpone the oxidation of βcarotene in emulsion droplets because of the conditions of the aqueous phase was investigated. The results of this study should help in designing effective emulsion-based encapsulation systems capable of retarding β-carotene degradation during long-term storage.
2.3. Droplet size measurement The mean emulsion droplet diameters were measured using static light scattering (laser diffraction). To avoid multiple scattering effects, all emulsion samples were diluted to a droplet concentration of approximately 0.005% (w/w) using a buffer solution at the pH of the sample, and stirred continuously throughout the measurements to ensure homogeneity. The particle size distribution of the emulsions was then measured using a commercial static light scattering instrument (BT-9300ST; Bettersize Instruments, Dandong, China). The particle size data are reported as either the volume-weighted mean diameter, d43 = ∑ ni ∙di4 / ∑ ni ∙di3 , or surface-weighted mean diameter, d32 = ∑ ni ∙di3/ ∑ ni ∙di2 , where ni is the number of particles with diameter di . 2.4. Influence of transition metals and free radicals on β-carotene degradation To determine the effect of iron species on the stability of β-carotene in the emulsions, ferric chloride or ferrous sulfate stock solutions were added to the emulsion samples up to a concentration of 100 µmol/kg emulsion. Furthermore, to determine the effect of free radicals on the stability of β-carotene in emulsions, AAPH sulfate stock solutions were added to the emulsion samples up to the concentration of 100 µmol/kg emulsion. 2.5. Measurement of β-carotene concentration The β-carotene concentrations in the emulsions were determined by vortexing vigorously 2 g of emulsion with 6 g dichloromethane (SigmaAldrich) for 2 min. The mixture was then centrifuged at 3000×g for 5 min, and the solvent layer was collected. The solvent absorbance was measured at 450 nm using a UV–vis scanning spectrophotometer (Optizen Pop; Mecasys, Daegeon, Korea). The degradation rate constant (k) of β-carotene in the emulsions was calculated assuming the following first-order exponential decay model:
2. Materials and Methods 2.1. Materials β-Carotene, PEGylated emulsifiers (polyoxyethylene 10 stearyl ether (S10), polyoxyethylene 20 stearyl ether (S20), and polyoxyethylene 100 stearyl ether (S100)), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and TBHQ were purchased from Sigma-Aldrich (St. Louis. MO, USA). Polyoxyethylene alkyl ether-type emulsifiers are nonionic surfactants containing polyethyleneoxide chains as the hydrophilic groups and n-alkyl chains as the hydrophobic groups. Medium chain triglyceride (Delios S) was obtained from BASF (Ludwigshafen, Germany). All other chemicals were reagent grade and obtained from Sigma-Aldrich or Fisher Scientific (Pittsburgh, PA).
Ct = C0 ∙e−k ∙ t ,
2.2. Emulsion preparation
2.6. Statistical analysis
The oil phase of the emulsions was prepared immediately before use by dissolving β-carotene (to a final concentration of 0.3 mg/g oil) and TBHQ (to a final concentration of 2 mg/g oil) in medium chain triacylglycerol (MCT), and the aqueous phase was prepared by dissolving PEGylated emulsifiers in a 10 mM phosphate buffer (pH 7) in predetermined concentrations. Oil-in-water emulsions were prepared by
All the experiments were performed in triplicate, and the data are expressed as mean ± standard deviation. To indicate significant differences in coefficients (degradation rate constants (k) of β-carotene) between linear regressions, Chow test was performed (Chow, 1960). The statistical analyses described above were all conducted using SAS (version 9.4.; SAS Institute Inc., Cary, NC, USA).
where C0 is the initial β-carotene concentration and Ct is the β-carotene concentration remaining at time t. The value of k was determined by performing linear regression on the plot of ln(Ct / C0) versus t.
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β-carotene, the pH was adjusted to 3 and 7 and the emulsions were stored at 25 °C (Fig. 1). Regardless of the pH, the β-carotene in the emulsions gradually degraded; however, its degradation rate was extremely faster at pH 3 than at pH 7 (Fig. 1A and B). It has been reported that β-carotene degradation is faster in acidic environments (Qian et al., 2012b; Song, Moon, & Choi, 2018; Xu, Yuan, Gao, McClements, & Decker, 2013). When carotenoids are exposed to acids, carotenoid carbocations are produced, and this transformation is thought to be the first step in the degradation process of carotenoids by acids (Konovalov & Kispert, 1999). Although not as much as pH 3, the β-carotene in the emulsions at pH 7 gradually degraded during storage (Fig. 1A). Even if the pH of the emulsion is neutral, the degradation of β-carotene encapsulated in the emulsions could easily occur through its isomerization and/or oxidation when exposed to heat, UV, and oxygen (Knockaert et al., 2012). However, since all emulsions were stored in the dark at 25 °C, the β-carotene degradation at pH 7 observed in our study may be due to other factors rather temperature and/or light. One possible reason for this observation is the presence of oxygen molecules in the emulsions. Although nitrogen purging was carried out during the emulsion preparation to minimize the β-carotene degradation by oxygen as described in Section 2, it was apparent that the oxygen dissolved in the emulsion, particularly in the aqueous phase, was not completely removed. Another potential reason is isomerization and/or oxidation of the β-carotene at the elevated temperatures during the homogenization. As shown in Fig. 1A and B, the β-carotene stability seems to be inversely proportional to the interfacial thickness of the emulsion droplets. The inverse relationship between interfacial thickness and β-carotene stability at pH 3 could be explained by the adsorption of protons (H+) onto the droplet surface. The oxygen dissolved in the aqueous phase was also one of the reasons for β-carotene degradation at pH 3. Since the PEGylated emulsifiers used in this study are non-ionic, their hydrophilic groups are not charged. However,
3. Results and discussion In order to avoid any influence of the micelles formed from unadsorbed emulsifier molecules on the stability of the emulsions and the concentration of β-carotene therein (McClements, 1994; Wulff-Pérez, Torcello-Gómez, Gálvez-Ruíz, & Martín-Rodríguez, 2009), the minimum emulsifier concentration (MEC) required to prepare emulsions having the smallest oil droplet size and long-term stability was determined in a previous study (Han, Song, Moon, & Choi, 2018). The MECs of S10, S20, and S100 were 3.165, 2.926, and 0.994 mM, respectively. The initial droplet diameters of the emulsions are all fairly similar (d43 = 0.31, 0.31, and 0.33 μm and d32 = 0.26, 0.24, and 0.24 μm for S10-, S20-, and S100-stabilized emulsions, respectively) at the MECs, and did not change significantly (p > 0.05) during 21-day storage, suggesting that all the emulsions had a similar specific surface area. 3.1. Impact of pH on β-carotene stability in emulsions having various interfacial characteristics Previous studies have shown that the lipid oxidation of emulsions and the stability of the functional lipophilic compounds therein depend on the environments surrounding oil droplets (Berton-Carabin et al., 2014). Even though such functional compounds, located in the core of the emulsion droplets, are isolated from the harsh environments of the aqueous solution by the interfacial membrane, they gradually, sometimes rapidly, degrade. Since the interfacial membrane has the ability to alter the rates of the chemical reactions between oil- and aqueousphase compounds, the interfacial characteristics could be important in the lipid oxidation of emulsions and the stability of the lipophilic compounds therein. To determine the effect of the pH of the emulsions on the stability of
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Fig. 1. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ. 196
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Fig. 2. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C with ferrous irons. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ.
emulsions were calculated as 3.27, 3.02, and 1.09 nmol/m2, respectively, from droplet size and emulsifier concentration. As a result, the emulsions could have interfacial membranes with a different denseness. The value of S10-stabilized emulsion was very similar to that of S20stabilized emulsion and it was approximately 3 times higher than that of S100-stabilized emulsion. The difference in k values of β-carotene between S100- and S10-stabilized emulsions was much less than 10times. When emulsions have a similar droplet interfacial denseness (S10- and S20-stabilized emulsions in this work), it seems that those with thinner interfacial membranes have a better ability to retard βcarotene degradation. However, considering the emulsifier loading values of emulsions, it seems that there is no systematic relationship between the denseness of the interfacial membrane and the ability of the interfacial membrane to retard β-carotene degradation.
considering that the emulsifiers used in this study have the numbers of oxyethylene group in their hydrophilic groups, a negative dipole moment could be detected on the oxygen atom sites of the hydrophilic groups of PEGylated emulsifiers, indicating that their hydrophilic groups could attract protons via dipole-ion interactions (Hägerstrand et al., 2001). The pH of the interfacial region could be lower than that of the aqueous phase because the protons tend to accumulate around the oil droplet surfaces due to dipole-ion interactions. Hence, acidpromoted β-carotene degradation may be accelerated within the interfacial region. Since the number of oxyethylene groups is larger in S100 than in S10 and S20, protons could accumulate around the oil droplet surfaces of the S100-stabilized emulsion to a higher extent than around those of the S10- and S20-stabilized emulsions. This hypothesis could explain why the β-carotene stability in the emulsions having a thicker interface (stabilized by emulsifiers having a larger number of oxyethylene groups) is lower than that in emulsions having a thinner interface (stabilized by emulsifiers having a smaller number of oxyethylene groups). However, the effect of the proton accumulation around the interfacial region on β-carotene degradation did not seem to be greater than expected because the difference in the number of oxyethylene groups between S100 and S10 was 10-times but the difference in k values of β-carotene between S100- and S10-stabilized emulsions was much less than 10-times. As noted above, a similar mean droplet diameter was measured for all the emulsions but the emulsifier concentrations in emulsions were different from each other because of the different MECs between emulsifiers. If an emulsion is prepared at the MECs of S10, S20, or S100, most of the emulsifier molecules could be found at the interface between oil and water and this emulsion contains little-to-no micelle therein. Hence, emulsions should have the different emulsifier concentrations per unit droplet surface area (mass (emulsifier) loading). The emulsifier loading values for S10-, S20-, and S100-stabilized
3.2. Impact of transition metals on β-carotene stability in emulsions having various interfacial characteristics It is well known that transition metals accelerate carotenoid degradation, particularly because of their high oxidation state (Boon et al., 2008). During iron-mediated degradation of carotenoids, carotenoid radical cations are produced through the electron transfer reaction between carotenoids and Fe3+ (Gao & Kispert, 2003) that are converted into carotenoid peroxyl radical cations by interacting with oxygen (Gao & Kispert, 2003; Wei, Gao, & Kispert, 1997) or into carotenoid dications by interacting with Fe3+ (Gao, Wei, Jeevarajan, & Kispert, 1996). Then, carotenoid cation peroxyl radicals and carotenoid dications are degraded through further oxidation processes. They could also oxidize neutral carotenoids. Therefore, the rate of the abovementioned reactions could determine the rate of β-carotene degradation in emulsions. However, except the reaction of neutral carotenoids with carotenoid peroxyl radical cations and/or carotenoid dications, 197
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Fig. 3. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C with ferric irons. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ.
seems that the low solubility of iron at neutral pH could be the major factor affecting β-carotene degradation. The β-carotene degradation rate was not significantly altered by the presence of Fe3+ in the emulsions. This seems to indicate that the interfacial membrane, regardless of its thickness and/or denseness, significantly interrupted the interaction of Fe3+ with oil droplets and the formation of β-carotene radical cations at pH 7. Therefore, assuming that the interfacial membrane also interrupted the interaction of Fe2+ with oil droplets, no ironinduced β-carotene degradation mechanisms are expected to degrade the β-carotene in the emulsions containing Fe2+. Fe2+ could participate in producing some of the reactive oxygen species such as singlet oxygen (1O2) and hydroxyl radicals (HO%), strong oxidants; they could also produce superoxide radical anions (O2%−) by reacting with triplet oxygen (3O2) (Choe, 2008). O2%− could produce hydrogen peroxide (H2O2) by dismutation, and the reaction between H2O2 and O2%−
most of the processes critically affecting the carotenoid degradation rate occur at the interface between the oil and aqueous phases. This indicates that the storage stability of the β-carotene incorporated into the oil droplets could be significantly affected by their interfacial characteristics. At pH 7, Fe2+ increased the degradation rate of the β-carotene incorporated in the emulsions; however, the β-carotene degradation rate was not significantly affected by Fe3+ (Figs. 2 and 3, Table 1). A possible reason for this observation is that the interaction of iron molecules with oil droplets is reduced at this pH (Boon, McClements, Weiss, & Decker, 2009). Since the solubility of iron molecules is considerably lower at neutral pH compared with that at acidic pH, it appears that the iron molecules are less likely to interact with the oil droplets containing β-carotene. However, considering that Fe2+ accelerated β-carotene degradation at pH 7, particularly in the S100-stabilized emulsion, it
Table1 Degradation rate constant (k (day−1)) of β-carotene determined by modeling assuming a 1st-order reaction.
pH 7
pH 3
Oxidative stress
S10
S20
Without TBHQ
With TBHQ
Without TBHQ
With TBHQ
Without TBHQ
With TBHQ
No Ferrous iron Ferric iron Radicals
F
B
E
B
F
B
No Ferrous iron Ferric iron Radicals
D
bc
0.0054 0.0085b F 0.0053b BC 0.0939b E
0.0523bc 0.1007b C 0.0832b A 0.1368b B
e
S100
b
de
0.0018 0.0023d B 0.0029c B 0.0027d
D
0.0063 0.0107ab E 0.0069ab B 0.1054ab
B
B
C
0.0035d 0.0112c A 0.0126cd B 0.0027c
B
0.0029d 0.0113c A 0.0105d B 0.0022c
0.0536b 0.1273ab B 0.0944b A 0.1478ab
A
AB
0.0030 0.0032cd B 0.0034c B 0.0035cd
0.0081 0.0169a F 0.0082a C 0.1182a
B
D
A
The values with different capital-letter superscripts in a same column are significantly different (p < 0.05) by Chow test. The values with different small-letter superscripts in a same row are significantly different (p < 0.05) by Chow test. 198
a
E
0.0626a 0.1566a B 0.1408a A 0.1757a AB
0.0043cd 0.0049c B 0.0039c B 0.0046c B
B
0.0039cd 0.0123c A 0.0133c B 0.0030c
A
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Fig. 4. Effect of TBHQ on the stability of β-carotene in emulsions stored at 25 °C with AAPH. A: pH 7 with no TBHQ, B: pH 3 with no TBHQ, C: pH 7 with TBHQ, D: pH 3 with TBHQ.
generates HO% and 1O2 (Kehrer, 2000). HO% is also produced from the decomposition of H2O2 in the presence of iron, particularly Fe2+ (Salem, El-Maazawi, & Zaki, 2000). It appears that these reactive oxygen species generated by Fe2+ may interact with oil droplets and they may initiate the degradation of β-carotene at pH 7. Thus, it was possible to speculate that the interfacial membrane of the emulsions prepared in this study well interrupted the direct interaction of iron with the emulsion droplets but it did not prevent the initiation of βcarotene degradation occurred by the attack of oxygen-containing radicals on emulsion droplets. The presence of iron, regardless of its species, caused the rapid degradation of β-carotene in emulsions during storage at acidic pH (Figs. 2B and 3B, and Table 1). Considering the oxidation mechanism of β-carotene described above, it is not difficult to explain this observation. Although the interfacial membrane significantly interrupts the interaction of iron with oil droplets, iron could interact with the droplets more frequently at pH 3 than at pH 7 because of the higher solubility of iron at acidic pH. β-Carotene was gradually degraded during its storage in the presence of Fe3+; however, the level of β-carotene dropped sharply at the initial stage of its storage in the presence of Fe2+. It may be not difficult to identify the reason for this sharp drop of the β-carotene concentration in the emulsions containing Fe2+ at the beginning of the storage. In emulsions containing Fe3+ at pH 3, βcarotene degradation could be initiated by protons rather than Fe3+. The decomposition of the β-carotene carbocations by Fe3+ could occur at the emulsion droplet surfaces or they could also react with neutral βcarotene molecules in the oil droplets. In the emulsions containing Fe2+ at pH 3, β-carotene oxidation was initiated at the oil droplet surfaces by protons and oxygen-containing radicals. Then, the products of those reactions were decomposed at the oil droplet surfaces by Fe2+, or they reacted with the neutral β-carotene molecules. As described above, since the degradation mechanism of β-carotene by Fe2+ in emulsions is
more varied than that involving Fe3+, the β-carotene in emulsions could be degraded faster by Fe2+ than Fe3+. Considering that the rate of decomposition of lipid hydroperoxides by Fe2+ is higher than that by Fe3+ (Mei, McClements, Wu, & Decker, 1998), the rate of decomposition of β-carotene peroxyl radicals, intermediates of the oxidative degradation of β-carotene, by Fe2+ could be higher than that by Fe3+. As shown in Table 1, the S100-stabilized emulsion, the one having the thickest interfacial membrane among the emulsions investigated in this study, showed the highest k value between emulsions stored at the same condition. This observation could be explained through the hypothesis presented above to explain why the acid-mediated degradation of β-carotene was faster in the emulsions having a thicker interface (stabilized by emulsifiers having a larger number of oxyethylene groups) than in the ones having a thinner interface (stabilized by emulsifiers having a smaller number of oxyethylene groups). A negative dipole moment on oxygen atoms of the hydrophilic groups of emulsifiers could attract iron molecules, regardless of their oxidative state and one molecule of S100 could accumulate iron molecules to a higher amount than S10 and/or S20. Hence, a higher amount of iron molecules could accumulate around the oil droplet surfaces of the S100-stabilized emulsion than around those of the S10- and S20-stabilized ones, leading to the faster degradation of β-carotene in S100-stabilized emulsion than in S10- and S20-stabilized ones. Therefore, the stability of β-carotene in emulsions in the presence of iron molecules seems to be inversely related to the interfacial thickness of the oil droplets. However, as shown in Table 1, the interfacial denseness plays a minor role in β-carotene degradation. 3.3. Impact of free radicals on β-carotene stability in emulsions having various interfacial characteristics Lipid containing foods may contain free radicals, generated through 199
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the TBHQ in the core of the emulsion droplets may convert the β-carotene radicals back to β-carotene. At pH 7, it was apparent that the effectiveness of TBHQ to inhibit the degradation of the β-carotene incorporated in the emulsions was affected by the interfacial thickness rather than by its denseness. It is not clear how interfacial thickness affected the effectiveness of TBHQ at pH 7. However, at pH 3, the correlation between interfacial thickness and/or denseness and the ability of TBHQ to inhibit β-carotene degradation was not clear. Interestingly, although TBHQ effectively degraded the β-carotene in emulsions at pH 3, it was considerably less effective in the emulsions containing Fe2+ and Fe3+ than in the emulsions containing other prooxidants (Table 1). This could be due to the iron-reducing activity of TBHQ. Phenolic compounds are able to reduce Fe3+ to Fe2+, and their reducing ability is larger at lower pH values (Dunford, 1987). TBHQ, a phenolic antioxidant, reduced Fe3+ to Fe2+, and the prooxidant activity of iron in the emulsions increased because the reactivity of Fe2+ is higher than that of Fe3+. Therefore, in emulsions containing iron molecules at pH 3, TBHQ blocked the interaction of the prooxidants present in the aqueous phase with the oil droplets, and neutral β-carotene was generated from β-carotene radicals; however, the more reactive Fe2+ was accumulated in the aqueous phase. The metal reducing activity of TBHQ could explain why it was less effective in inhibiting β-carotene degradation at low pH in emulsions containing iron.
lipid oxidation during food processing, up to a certain level (Choe & Min, 2006). These free radicals could accelerate the oxidation of neutral lipids, and could also initiate the degradation of the functional compounds in food media by reacting with them. If these free radicals are oil-soluble and located in the interior of oil droplets, they could attack neutral lipids and/or oil-soluble functional compounds in emulsion systems without any physical interference. However, if the free radicals are water-soluble, since they have to interact with oil droplets to attack the oil-soluble functional compounds therein, the degradation of the oil-soluble functional compounds in emulsions could be greatly influenced by the nature of the interface (Miyashita, 2008). Therefore, using AAPH, a water-soluble free radical generator, the effect of free radicals on β-carotene stability in emulsions having different thicknesses was evaluated (Fig. 4). Independent of the interfacial thickness and pH, over 90% of βcarotene was degraded during 21-day storage. The interaction of free radicals with β-carotene in oil droplets was the main driving force for βcarotene degradation at pH 7, whereas, in addition to the interaction mentioned above, it seems that β-carotene was also degraded by the interaction of protons with β-carotene at pH 3. The higher k values of βcarotene in emulsions at pH 3 are a strong evidence for this hypothesis (Table 1). This suggests that the interfacial membrane of the emulsions investigated did not have the ability to interrupt the entry of free radicals in the oil droplets. As described above, the degradation of βcarotene in emulsions by oxygen-containing radicals is not well impeded by the interfacial membrane. Therefore, it appears that the attack of AAPH radicals on the β-carotene at the emulsion droplet surfaces is not well blocked by the interfacial membrane either. As shown in Fig. 2A and B, when the oxygen-containing radicals act as prooxidants, the effect of interfacial denseness is unclear; however, it seems that interfacial thickness has the tendency to affect storage stability of βcarotene. However, when AAPH radicals were present in the emulsions, although it appeared that the ability of the interfacial membrane to retard β-carotene degradation was inversely proportional to the interfacial thickness, β-carotene concentration rapidly dropped regardless of the interfacial thickness. A possible explanation for this observation is the high concentration of the AAPH radicals existent in the emulsions. Compared with the concentration of the oxygen-containing radicals in iron-containing emulsions, the concentration of the AAPH radicals is enormous. When radicals are present in food media, several reactions of the radicals with carotenoids, including electron transfer (Young & Lowe, 2001), hydrogen abstraction (Woodall, Lee, Weesie, Jackson, & Britton, 1997), and the addition of radical species to form carotenoidradical adducts (Mortensen, 2002) are possible. Therefore, vigorous attacks of the AAPH radicals on the oil droplets through the reactions mentioned above were observed despite the thickness-dependent defensive ability of the interfacial membrane. It was also apparent that βcarotene stability was not influenced by interfacial denseness.
4. Conclusion The results show that the interfacial characteristics, particularly thickness, highly affect the stability of β-carotene encapsulated within emulsions. It is apparent that the ability of the interfacial membrane to retard β-carotene degradation in emulsions is inversely related to its thickness. The interaction of the water-soluble compounds (transition metals and free radicals in this work), which are able to contribute to βcarotene degradation, with the emulsion droplets containing β-carotene is more effectively retarded in the emulsions having a thinner interfacial membrane than emulsions having a thicker one. TBHQ, the oilsoluble phenolic antioxidant used in the current study, was found to increase the stability of β-carotene in emulsions against the chemical degradation promoted by the compounds in the aqueous phase; however, its ability to increase the β-carotene stability varied with what oxidative stress promoted the degradation of β-carotene in the emulsions. Independent on emulsion pH, TBHQ effectively prevent β-carotene in emulsions from radical-mediated degradation. TBHQ was still effective to inhibit iron-mediated degradation of β-carotene at neutral pH but its ability to increase the stability of β-carotene against prooxidant metals at acidic pH. Therefore, our results have important applications for the proper selection of emulsifiers capable of producing emulsions that can encapsulate and protect β-carotene in commercial food products and for the rational selection of antioxidants to achieve the objective mentioned above.
3.4. Impact of antioxidants on β-carotene stability in emulsions having different interfacial characteristics To understand how antioxidants inhibit the chemical degradation of the β-carotene encapsulated in emulsions of various interfacial thicknesses, emulsions containing TBHQ (an oil-soluble antioxidant) were prepared. The addition of TBHQ effectively inhibited the chemical degradation of β-carotene (Figs. 1–4, and Table 1) due to its ability to scavenge the reactive free radicals. When β-carotene and TBHQ coexist in the emulsion droplets, although β-carotene and TBHQ are both oilsoluble molecules, TBHQ preferentially accumulates near the emulsion droplet surfaces because its log P (2.94) is lower than that of β-carotene (17.62). Therefore, when the prooxidants in the aqueous phase of the emulsions interact with the oil droplets, they frequently react with TBHQ rather than with β-carotene (Silvestre et al., 2000). In conclusion, the TBHQ molecules located near the emulsion droplet surfaces directly blocked the attack of the prooxidants in the aqueous phase, and
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03930215), Republic of Korea. 200
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