Article Cite This: J. Agric. Food Chem. 2019, 67, 6466−6475
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Effect of the Solid Fat Content on Properties of Emulsion Gels and Stability of β‑Carotene Yao Lu, Like Mao,* Mengnan Cui, Fang Yuan, and Yanxiang Gao
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Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Laboratory for Food Quality and Safety, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China ABSTRACT: Whey-protein-isolate-based emulsion gels were prepared through a cold-set gelation process, and the effect of the solid fat (coconut oil) content in the oil phase on gel properties and β-carotene stability was investigated. An increase in solid fat content (0, 20, 50, 80, and 100% of the oil phase) resulted in a smaller droplet size, higher viscosity, and improved creaming stability of the emulsions. When glucono-δ-lactone was added to initiate gelation, a higher solid fat content contributed to an earlier onset of gelation and a higher storage modulus of the gels. Textural analysis indicated that the increase in the solid fat content allowed for an increase in fracture stress and Young’s modulus of the emulsion gels. Microscopic observation revealed that emulsions containing a higher solid fat content formed gels with a denser and more uniform particulate network structure. The stability of β-carotene against thermal treatment (55 °C for 12 days) and ultraviolet light exposure (8 h) was determined. The results suggested that the solidification of the oil phase can improve the stability of β-carotene, and gels with higher hardness were capable of retaining more β-carotene after the treatments. These findings indicated that emulsion gels with a solidified oil phase could be potential delivery systems for lipophilic bioactive compounds. KEYWORDS: emulsion gel, β-carotene, solid fat, rheology, stability
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the systems.13 Published studies have shown that emulsion gels were able to improve the stability of α-tocopherol,14 curcumin,15 and epigallocatechin gallate (EGCG)16 and enhance the bioaccessibility of quercetin17 and β-carotene.18 They also worked to modify the release of capsaicinoids19 and volatile compounds.20 Previous studies also concluded that properties of emulsifiers, gelling agents, oils, and environmental stresses (e.g., pH, ionic strength, and heat) played significant roles in the structures and functionalities of emulsion gels,4 of which the natures of the oil phase are of high importance for the encapsulation, delivery, and digestion of bioactive compounds.21 Oil droplets in emulsion gels are termed fillers, which can be divided into active fillers and inactive fillers, depending upon the interactions between the oil−water interface and the gel matrix.10 Active fillers can participate in the formation of gels and, thus, enhance the gel strength.22 Inactive fillers have no or minor interactions with the gel and usually weaken the gel strength.23 Literature studies have revealed that, in emulsion gels with active fillers, a higher oil content or smaller particle size was favorable for the formation of gels with a higher strength.23,24 However, the roles of the nature of the oil phase, e.g., ratio of solid fat/liquid oil, on the properties of emulsion gels were not well-understood. In fact, the solid fat content played essential roles in the properties of emulsions, e.g., droplet size, rheology, and stability,25,26 which then affected the structures and properties of emulsion gels. Liu et al.27 used a tribometer to evaluate the tribological characteristics and fat coalescence of emulsion gels and found
INTRODUCTION In the current food market, functional-food-containing bioactive compounds (e.g., carotenoids, polyphenols, and polyunsaturated fatty acids) are gaining more popularity because of their potential to reduce the risk of many chronic diseases. To better incorporate the bioactives in food, scientists have designed different delivery systems (e.g., emulsions, gels, and particles) to modify the dispersant states, improve the stability, and finally enhance the bioavailability of these bioactives.1,2 Among them, emulsions are particularly interesting as a result of their good compatibility with food systems and wide applications.2−4 However, emulsions are thermodynamically unstable systems, which are prone to flocculation, coalescence, and Ostwald ripening during manufacturing and storage, finally leading to phase separation or even emulsion breakdown.5,6 To combat these disadvantages, novel emulsions with tailor-made structures in the oil phase, water phase, and oil−water interface (e.g., multilayer emulsions and Pickering emulsions) have been developed and have presented improved performances.7−9 Emulsion gels are widely used in many foods, such as cheese and yogurt. They can be used as delivery systems to protect functional ingredients and as fat substitutes to design low-fat foods.2,5 Emulsion gels are soft solid systems formed by gelling the continuous phase of emulsions, with the oil droplets entrapped within the gel networks.10 As a special type of structured emulsions, emulsion gels can deliver functional ingredients as emulsions11 and also have good physical stability and mechanical properties as gels.12 The compact threedimensional network structures in the continuous phase of the emulsion gels can also act as physical barriers against adverse stresses (e.g., light, heat, and enzymes), offering good protection for the sensitive bioactives incorporated within © 2019 American Chemical Society
Received: Revised: Accepted: Published: 6466
February 18, 2019 April 24, 2019 May 22, 2019 May 22, 2019 DOI: 10.1021/acs.jafc.9b01156 J. Agric. Food Chem. 2019, 67, 6466−6475
Article
Journal of Agricultural and Food Chemistry
carotene. To avoid fat crystallization and clogging of the instrument, hot water was used to rinse the homogenizer before sample preparation. Characterization of the Emulsions. Droplet size, size distribution, and ζ potential of the emulsions were measured using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, U.K.) at a fixed detector angle of 90°. Refractive indexes of oil and water phases were set to 1.45 and 1.33, respectively, for the measurement. The samples were diluted 200 times with deionized water to avoid multiple scattering effects. Results were described as intensity mean diameter (nm) for droplet size, polydispersity index (PDI) for size distribution, millivolts for ζ potential. All of the measurements were performed in triplicate. Viscosity measurements were performed using a DHR-2 rheometer (TA Instruments, West Sussex, U.K.) with a parallel plate geometry (40 mm diameter and 1 mm gap). The measurement was performed at a shear rate range of 10−400 s−1 at 20 °C. Physical stability of emulsions was analyzed by measuring the transmission curve of the sample at different times using a LUMiSizer (L.U.M. GmbH, Berlin, Germany). The samples were subjected to centrifugal force, while near-infrared light illuminated the entire sample cell to measure the intensity of transmitted light as a function of time and position over the entire sample length simultaneously. The sample stability was shown as a space- and time-related transmission profile over the sample length.33 The instrumental parameters used for the measurement were as follows: sample volume, 1.8 mL; rotational speed, 4000 rpm; temperature, 15 °C; time interval, 10 s; and number of scanning times, 800. Preparation of WPI Emulsion Gels. The preparation of emulsion gels followed the study of Mao et al.,20 with some modifications. GDL (0.5%, w/w) was added to the prepared emulsions and stirred well, which were then allowed to stand at room temperature for 12 h to develop the gels. Textural Properties of WPI Emulsion Gels. Textural properties of emulsion gels were determined using a TMS-Pro texture analyzer (FTC, Rockville, MD, U.S.A.) with a cylindrical plunger (diameter of 12 mm) and a 250 N compression head. Samples for the measurement were formed in 25 mL beakers (34 mm internal diameter × 50 mm height) following the same method described above. The experiment was performed using a uniaxial compression test mode at 20 °C at a speed of 1 mm/s and a strain of 50%. The fracture stress (MPa) was the amount of force per unit area at fraction, and the fracture strain was the amount of deformation per unit length produced at fraction. Young’s modulus (MPa) was a measure of the stiffness of an isotropic elastomer, defined as the ratio between uniaxial stress and uniaxial deformation within the range applicable to Hooke’s law. All measurements were repeated 8 times and averaged. Viscoelastic Behaviors of WPI Emulsion Gels. The dynamic gelation process and rheological properties of the emulsion gels were measured using the DHR-2 rheometer (TA Instruments) coupled with a parallel plate geometry (40 mm diameter and 1 mm gap). During the measurement, emulsion samples were immediately dropped onto the plate after the addition of GDL. A thin layer of tetradecane oil was added to avoid water evaporation during gelation. The oscillation test was performed at a strain of 0.5% and a frequency of 1 Hz for 2 h, and the measurements were taken with an interval of 10 s. Storage modulus (G′), loss modulus (G″), and gelation time (Tgel, defined as the time point at which the G′−time curve and G″− time curve crossed over) were recorded. The dynamic strain sweep test was carried out to determine the linear viscoelastic range of the samples using a strain range of 0.1−100% at a constant frequency of 1 Hz. The frequency sweep test was carried out at 20 °C at a frequency range of 0.01−100 Hz at a constant strain of 0.1%. For the temperature sweep test, samples were heated from 5 to 45 °C at a heating rate of 5 °C/min with 1% strain and a 1 Hz frequency.32 Confocal Laser Scanning Microscopy (CLSM) Observation. CLSM observations of the emulsions were evaluated using a Zeiss780 inverted confocal microscope Observer.Z1 (Zeiss, Inc., Germany). Nile blue (1 mg/mL) and Nile red (1 mg/mL) were used as
that a higher solid fat content in the oil phase resulted in gels with a higher level of droplet coalescence and lower friction. Geremias-Andrade et al.15 reported that the incorporation of solid lipid particles in the oil phase could enhance the strength of a heat-set protein−polysaccharide mixed gel system. When bioactives were incorporated within solidified lipid particles, they could have improved stability.28,29 Therefore, it is meaningful to elucidate the effect of the solid fat content in the oil phase on the physical properties and delivery functionality of emulsion gels. β-Carotene is widely used in the food, cosmetic, and pharmaceutical industries as a result of its coloring, antioxidant, and pro-vitamin A functions.30,31 To fulfill its benefits, different delivery systems have been developed for its incorporation in the end products.32 Among limited studies on β-caroteneloaded emulsion gels, Mun et al.18 demonstrated that βcarotene in oil-filled starch gels [with inactive fillers coated with whey protein isolate (WPI)] had higher bioaccessibility than conventional emulsions and rice starch gels. The study focused on the digestion behaviors of gels, while the stability of β-carotene was not investigated. In the current study, β-carotene was incorporated into emulsion gels with the oil phase containing different contents of solid fat and properties of emulsion gels (e.g., texture and rheology) and the stability of β-carotene against ultraviolet (UV) light and thermal treatment was investigated. It was aimed to understand the roles of the solid fat content in modifying the structures of emulsion gels and protecting βcarotene and to link the structures and delivery functionality of the gel systems. The knowledge obtained would be useful for the development of functional-food-containing bioactives.
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MATERIALS AND METHODS
Materials. WPI (BiPro) was bought from Davisco Food International (Le Sueur, MN, U.S.A.) and contained 83% protein (71%, w/w, β-lactoglobulin and 12%, w/w, α-lactabumin) and other minor ingredients (e.g., immunoglobulin and lactoferrin). Corn oil and coconut oil were purchased from a local market (Beijing, China) and used without further purification. As the supplier stated, the coconut oil contained mainly octylic, decanoic, lauric, and myristic acids. β-Carotene (>99% purity) was kindly offered by Zhejiang NHU Company, Ltd. (Zhejiang, China). Glucono-δ-lactone (GDL) and sodium azide were bought from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other chemicals used were of analytical grade. Preparation of WPI-Stabilized Emulsions. Emulsions were prepared according to a previous study,20 with slight modifications. Briefly, WPI was first dispersed in deionized water at a concentration of 5% (w/w), and 0.01% (w/w) sodium azide was added to prevent the growth of microorganisms. The mixture was stirred overnight to ensure complete dispersion and dissolution. The dispersion was heated at 85 °C for 30 min to form dispersed WPI aggregates and then cooled rapidly to room temperature with ice. Corn oil and coconut oil (the content of coconut oil in the oil phase was 0, 20, 50, 80, and 100%) were heated and stirred at 65 °C to form the mixed oil phase (20% of the emulsions and corresponding emulsion gels) and then were mixed with the WPI dispersions using a TD-5 Ultra-Turrax blender (IKA, Germany) at 10 000 rpm for 1 min to form coarse emulsions, followed by high-pressure homogenization at 50 MPa for 3 cycles using a Niro-Soavi Panda homogenizer (Parma, Italy). The final emulsions were immediately cooled to room temperature (12− 15 °C) using an ice−water mixture. To prepare emulsions containing β-carotene, β-carotene powder was added to the oil phase and heated to 140 °C with stirring to dissolve all of the β-carotene crystals, followed by mixing the oil phase and WPI dispersion for homogenization. The heating and mixing processing was completed in a short time to reduce the possible oxidation of the oil and β6467
DOI: 10.1021/acs.jafc.9b01156 J. Agric. Food Chem. 2019, 67, 6466−6475
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Journal of Agricultural and Food Chemistry fluorescent dyes for protein and oil phases. The samples were placed on a concave confocal microscope slide and covered with a glycerolcoated coverslip. The observation was carried out with a 100× magnification lens and an Ar laser (488 nm)/HeNe red laser (633 nm). Scanning Electron Microscopy (SEM) Observation. The microstructures of the emulsion gels were also observed using a SU8010 field emission electron scanning microscope (Hitachi, Inc., Japan) following the method of Wang et al.,34 with certain modifications. The samples were subjected to fixation with glutaraldehyde in cacodylate buffer, ethanol gradient dehydration, and critical point drying prior to the SEM test. The dried sample was then sputter-coated with gold using ion sputtering and observed at 15 kV. Stability of β-Carotene against UV Light. A total of 1.0 g of the emulsion gel was transferred to transparent glass bottles flushed with nitrogen and then moved to a xenon test chamber (Q-SUN, Xe-1-B, Q-Lab Corporation, Westlake, OH, U.S.A.) with an irradiance of 0.68 W/m2 at 20 °C for 8 h.33 The remaining content of β-carotene was measured at an interval of 2 h. β-Carotene extraction was performed on the whole gel sample in the bottle, and the bottle was no longer moved back to the test chamber. Stability of β-Carotene at Different Temperatures. A total of 1.0 g of the emulsion gel was added to the centrifuge tube flushed with nitrogen, and the tubes were stored in an incubator at 20 and 55 °C for 15 days in the dark. The remaining content of β-carotene was measured every 3 days. Determination of the Content of β-Carotene. A total of 1.0 g of the emulsion gel was mashed well using a glass rod with 1.0 mL of ethanol to destroy the gel structure, and β-carotene was extracted with n-hexane. The extraction process was performed in a room with very weak light. The content of β-carotene in the extract was determined by measuring the absorbance at 450 nm with an ultraviolet−visible (UV−vis) spectrophotometer (model UV 1800, Shimadzu, Japan) and referencing the standard curve of β-carotene.35 Storage stability of carotene in the samples was expressed by the retention ratio Ct/C0, where Ct was the content of β-carotene after storage for t days and C0 was the initial β-carotene content before storage.36 Statistical Analysis. Statistical analysis was performed using Origin Pro 8. All measurements were repeated 3 times, unless otherwise stated. Data were analyzed by the SPSS 20.0 software package. A one-way analysis of variance (ANOVA), followed by Tukey’s test, was applied to determine significant differences between the mean values of each test. A significance level of p < 0.05 was used throughout the study.
Table 1. Droplet Characteristics of WPI Emulsions Containing Different Solid Fat Contents in the Oil Phasea proportion of coconut oil in the oil phase (%) 0 20 50 80 100
size (nm) 349 338 335 330 316
± ± ± ± ±
2 7 3 8 4
a b b b c
ζ potential (mV)
PDI 0.31 0.24 0.24 0.26 0.24
± ± ± ± ±
0.04 0.05 0.07 0.04 0.01
a c c b c
−43.77 −45.23 −45.97 −46.10 −46.77
± ± ± ± ±
0.97 1.00 0.70 0.75 0.71
c b b a a
a Values were means of three determinations, and different lowercase letters in the same column indicated a significant difference (p < 0.05).
content of solid fat may also contribute to the smaller particle size. In terms of the distribution of the oil droplets, the system without coconut oil had the highest PDI value, indicating the presence of large particles. The content of coconut oil also presented a big influence on the rheological properties of the emulsions (Figure 1). All of the emulsions exhibited shear-thinning behaviors at the tested shear rate (0−400 s−1), which was the result of oil droplets or protein aggregation.3 In the current systems, the preheated protein had an unfolded structure with many hydrophobic sites
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RESULTS AND DISCUSSION Effect of the Solid Fat Content on the Properties of Emulsions. Many studies reported that fat solidification can result in partial coalescence of emulsion droplets, because fat crystals can pierce the interfacial films and lead to the merging of the oil phase from different droplets.37 However, some researchers argued that the susceptibility of emulsions to partial coalescence was determined by a lot of factors besides the solid fat content, especially the properties of interfacial films.38,39 In the current study, the increase of the content of coconut oil resulted in a smaller droplet size with a finer distribution (Table 1). It was probably due to the thick interfacial film covering the oil droplets, which prevented partial coalescence of the oil droplets.26 Zhu et al.40 and Liu and Tang41 revealed that protein aggregates formed through thermal treatment may behave as Pickering stabilizers to stabilize emulsions and the formed interfacial films were of high mechanical strength. Furthermore, coconut oil had a high content of medium-chain triglycerides (C8−C10), which was favored for the formation of smaller droplets.42 Third, the slight increase in the net charge of the systems with a higher
Figure 1. Viscosity of the emulsions with different solid fat contents as a function of the (A) shear rate and (B) temperature. The inset picture shows the appearance of the emulsion with a coconut oil content of 100% in the oil phase. 6468
DOI: 10.1021/acs.jafc.9b01156 J. Agric. Food Chem. 2019, 67, 6466−6475
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Journal of Agricultural and Food Chemistry
Figure 2. LUMiSizer profiles of emulsions composed of different solid fat contents. The change in the color from red to green indicated the evolution of light transmission during the measurement.
exposed, which facilitated interdroplet association and protein aggregation. Similar findings were well-addressed in literature studies.43 The higher viscosity of the emulsions with a higher solid fat content suggested the lower mobility of the oil droplets with solid fat as a result of their higher density.44 Second, emulsion droplets with a smaller size could also contribute to higher resistance of the systems against shearing, leading to increased viscosity.45 On the other hand, more oil droplets could be present in the emulsions with a higher solid fat content, because they had a smaller particle size. The denser packed oil droplets contributed to higher viscosity of the systems. It was well-expected that the viscosity of the emulsions was decreased with the increase in the temperature. However, a sudden increase followed by fast decrease in viscosity was observed at the temperature between 20 and 25 °C in the systems with a higher solid fat content (80 and 100%) (Figure 1B). This temperature range was close to the melting point of coconut oil (25−27 °C), and the melting of coconut oil resulted in the fluctuation in emulsion viscosity. Such fluctuation was not observed in the systems with a lower fraction of solid fat. No significant difference in emulsion stability was observed through the centrifugal test, because all of the emulsions showed similar evolution of the intensity of transmitted light (Figure 2). It was probably due to the small difference in the droplet size of the emulsions and relatively high viscosity (discussed later). During the test, the transmission intensity was increased at the bottom of the sample cuvettes, indicating the reduction in number of oil droplets, which were migrated to the top of the samples. It should be kept in mind that the
current study was an accelerated test conducted in a centrifugal field and all of the emulsions showed good stability when stored at quiescent conditions, which was attributed to the higher content of protein in the systems.3 Effect of the Solid Fat Content on the Rheological Properties of Emulsion Gels. The current study applied a cold-set gelation process (with GDL) to obtain emulsion gels. Because the oil droplets were coated with protein, they worked as active fillers, which could play significant roles in the structures of the emulsion gels. The change in the solid fat content had a great influence on the gelation kinetics and the rheological properties of the resulting gels. Figure 3 shows the storage modulus (G′) of emulsion gels as affected by oscillation frequency (0.01−100 Hz) and measuring temperature (5−45 °C). G′ referred to the energy stored by the material, reflecting the elastic property of the materials.10 It was observed that the gels with a higher solid fat content had higher G′, indicating higher gel strength.20,46 Figure 3A suggests that G′ of the emulsion gel without coconut oil was frequency-dependent and G′ of the gel was increased with oscillation frequency. The dependence was the characteristic of weak gels, which were more liable to deformation against mechanical forces.28 For a typical strong gel system, both storage and loss moduli were essentially not affected by frequency.47 In the current study, G′ of the gels containing solid fat did not show significant frequency dependence. This difference in frequency dependence of the gels reflected the difference in the interaction forces that maintain the gel network.41 It was thus inferred that the incorporation of the solid fat content in the oil phase can promote the transition of 6469
DOI: 10.1021/acs.jafc.9b01156 J. Agric. Food Chem. 2019, 67, 6466−6475
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Journal of Agricultural and Food Chemistry
mainly formed through electrostatic interactions, hydrogen bonding, disulfide bonding, and hydrophobic interactions.23 Among them, the hydrogen bonds and the electrostatic interactions were greatly affected by the temperature and decreased with the increase in the temperature.48 The gelation time (Tgel) was defined as the time point at which the G′−time curve intersected the G″−time curve.20 Figure 4 reveals that Tgel of the emulsion gels was decreased
Figure 4. Gelation time of the emulsion gels with different solid fat contents.
significantly with the increase in the coconut oil content in the oil phase. For example, gelation of the system free of solid oil occurred at 84.53 min, while that of the system with 80% solid fat initiated at 51.83 min. The earlier onset of gelation in gels with a higher solid fat content was the result of the larger interfacial area covering the droplets, which provided more interaction sites between the oil droplets and the matrix. Besides, the emulsions (before gelation) with solid fat had higher G′ than that free of solid fat (results not shown). Effect of the Solid Fat Content on the Textural Properties of Emulsion Gels. Table 2 summarizes the
Figure 3. Viscoelastical properties of the emulsion gels with different solid fat contents as a function of the (A) frequency and (B) temperature.
the weak gels to strong gels, because the roles of the active fillers were strengthened when the oil droplets were solidified. The solidified oil droplets had enhanced mechanical strength, which could provide more support for the protein gel network.25 When the solid fat turned to liquid at temperature above the melting point of coconut oil, G′ of the gels was decreased with the temperature (Figure 3B). For example, with the increase of the temperature from 5 to 45 °C, G′ of the gel with 100% coconut oil was decreased from 11 to 8.4 kPa and that of the gel with 50% coconut oil was decreased from 7.8 to 5.7 kPa. However, the coconut oil content did not have a significant effect on the slope of the curves, indicating that the melting of the solid oil phase did not result in a rapid decrease in the gel storage modulus or most coconut oil remained in the solid state during the short measurement. This might be attributed to the fact that the gel with a higher coconut oil content formed a denser and stronger network structure, which held the oil droplets tightly in the network. Moreover, the solid fat worked in the building up of a strong protein gel network, and the melting of solid fat might not be big enough to affect the strength of the gel structure. In comparison to the gel with 100% corn oil, the gels containing coconut oil had a higher storage modulus, even when the solid fat was melted. In fact, the gel without solid fat also showed a decrease in G′ when the temperature was increased, indicating weakened interactions in the gel matrix. In acid-induced gels, the gel networks were
Table 2. Texture Properties of the Emulsion Gels with Different Solid Fat Contentsa proportion of coconut oil in the oil phase (%) 0 20 50 80 100
fracture stress (MPa) 2.98 3.27 3.79 4.06 4.72
± ± ± ± ±
0.07 0.15 0.22 0.24 0.17
c c b b a
fracture strain 0.46 0.47 0.48 0.49 0.51
± ± ± ± ±
0.02 0.04 0.04 0.06 0.04
c b b b a
Young’s modulus (MPa) 6.36 6.94 7.96 8.24 9.17
± ± ± ± ±
0.41 0.20 0.25 0.31 0.31
a b c c d
a Values were means of eight determinations, and different lowercase letters in the same column indicated a significant difference (p < 0.05).
textural properties of emulsion gels, including fracture stress, fracture strain, and Young’s modulus. It could be observed that the increase in the solid fat content enhanced the fracture stress of the emulsion gels. In the system free of coconut oil, fracture stress of the gel was 2.98 ± 0.07 MPa; at the coconut oil content of 100%, fracture stress was increased to 4.72 ± 6470
DOI: 10.1021/acs.jafc.9b01156 J. Agric. Food Chem. 2019, 67, 6466−6475
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Journal of Agricultural and Food Chemistry 0.17 MPa. The findings supported the result of rheological analysis, because solidified oil droplets could strengthen the gel network to a higher level than liquid oil droplets.49 Second, gels with a higher solid fat content had a smaller droplet size (Table 1), which created a bigger surface area for the interaction with the gel matrix.24,50 Third, the relatively larger number of oil droplets allowed stronger filler effects. More importantly, it was observed that Young’s modulus of the emulsion gel increased with the increase of the solid fat content, which indicated that oil droplets participated in building up the matrix structure through the interaction between the protein in the continuous phase and at the interface. van der Poel’s theory believed that the modulus of the gel increased as the modulus of the filler increased,18 which supported our findings. In fact, these findings were in agreement with many previous reports.22,27,49 Microstructures of Emulsion Gel. CLSM and SEM were used to observe the microstructures of the emulsions and corresponding gels (Figure 5). It was found that the emulsions with a lower solid fat had many bigger droplets (in green), and the emulsions with a high solid fat content were more evenly distributed with smaller particles. The results were in agreement with particle size analysis from zetasizer. The SEM images presented the large differences in the microstructures of the gels with different solid fat contents. All of the gels had a typical particulate gel network, and oil droplets of different sizes were trapped. Literature studies indicated that whey protein can form particulate gels or filamentous gels, depending upon the balance between attractive and repulsive forces among denatured protein molecules during aggregation. A higher electrostatic repulsion force is favored for the development of filamentous gels, and a lower force is favored for particulate gels.51,52 Gels in the current study were formed with the assistance of GDL, which lowered the pH of the systems to the isoelectrical point of the protein and allowed for the association of protein particles. In the systems with a lower solid fat content (0 and 20%), the gels were characterized by large protrusions with irregular shapes, and the protrusions were covered by protein particles. The protrusions were believed to be oil droplets, which had a wide distribution, as determined by CLSM observation and zetasizer analysis. Although the oil droplets were well-connected with the gel network, large gaps between the droplets were present, which could be responsible for the weak gel strength of the systems, as determined by a rheometer and texture analyzer. On the contrary, the gels with a higher solid fat content (20, 50, and 100%) presented a more homogeneous distribution of the protein particles, which connected with each other forming the gel network with pores of high uniformity. Similarly, the oil droplets were also covered by the protein particles, but they presented a finer distribution. Stability of β-Carotene in WPI Emulsion Gels. The βcarotene molecule contains many unsaturated double bonds, which make it sensitive to light and heat.32 Emulsion gels prepared in the current study was through a cold-set process, which was favorable for the delivery of heat-sensitive ingredients.53 Besides, coconut oil mainly contains digestible medium-chain fatty acid,54 which was also reported to be helpful in inhibiting lipid peroxidation and maintaining high antioxidant activity at an elevated temperature.55 Light stability of β-carotene in emulsion gels with different solid fat contents was shown in Figure 6. The inset pictures of the samples show that the gels turned from bright yellow to
Figure 5. Microstructures of emulsions (CLSM, left) and emulsion gels (SEM, right) with different solid fat contents.
dark brown during the light test, indicating different levels of βcarotene degradation. The measurement of the β-carotene content revealed that emulsion gels with a higher solid fat content was capable of retaining more β-carotene in the test. The sample with 100% coconut oil had the lowest level of βcarotene degradation, and about 73% of the original content was retained after the test. On the contrary, the sample without coconut oil had the highest loss of β-carotene and less than 40% of the original content was retained after the test. For the systems with 50 and 80% solid fat, retention ratios were 61.39 ± 0.53 and 60.67 ± 1.02%, respectively. The degradation rate of β-carotene was accelerated with the increase of illumination times, and the solid fat content had a significant effect on the 6471
DOI: 10.1021/acs.jafc.9b01156 J. Agric. Food Chem. 2019, 67, 6466−6475
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Figure 6. Retention of β-carotene in emulsion gels with different solid fat contents exposed to UV light. The sample pictures show the different appearances of the emulsion gel before and after storage.
degradation rate. Under UV light, singlet oxygen reacted with the ground-state carotene to reach an excited state. Singletstate oxygen attacked the double bond of carotenoids to form free radicals, which gradually formed carbonyl cleavage products.56 Studies showed that the light degradation of βcarotene was a reaction initiated at the oil−water interface of an emulsion.57 In our study, the oil droplets in the emulsion gels with higher solid fat contents had a smaller particle size, which allowed for more protein adsorption and, thus, provided better protection for β-carotenes against UV light. More importantly, the compact gel network in the systems with a higher solid fat content created a bigger barrier against the migration of β-carotene and free radicals and inhibited the propagation of oxidation.58 Third, coconut oil mainly contained saturated fatty acids, and their presence could reduce the oxidation level of the liquid oil used, which was helpful to slow the degradation of β-carotene.59 Fourth, solid lipid particles have been proven to be able to improve the stability of the incorporated bioactives.29 Figure 7 shows the stability of β-carotene in emulsion gels when stored at different temperatures. Similarly, the sample with 100% solid fat content had the highest β-carotene retention rate at 20 or 55 °C. During storage, the retention rate of β-carotene was decreased gradually. After 12 days of the test, 63.52 ± 0.78 and 48.51 ± 0.91% of the original content of βcarotene were retained in the gels with 100% coconut oil when stored at 20 and 55 °C, respectively, Coconut oil was melted at 55 °C, and there could be no solid fat in the systems. However, the systems with a higher coconut oil content still had a higher retention ratio of β-carotene during the test, which could be attributed to the higher gel strength of the systems, as indicated by the rheological measurement in Figure 3B. Second, coconut oil was relatively more heat-stable than corn oil, which was helpful for the stability of β-carotene.55 In the current study, β-carotene in the emulsion gel with 100% coconut oil had a retention rate of 68.12% after 9 days of storage at 55 °C, indicating that the gel structure can protect βcarotene more effectively. To better understand the degradation mechanism of βcarotene in emulsion gels, we explored the degradation kinetics of β-carotene by fitting a first-order kinetic model and
Figure 7. Retention of β-carotene in emulsion gels with different solid fat contents at (A) 20 °C and (B) 55 °C.
calculating the reaction rate constant (k) and half-life time (t1/2) (Table 3).60,61 The correlation coefficients (R2) of the results were between 0.8882 and 0.9981, indicating that the 6472
DOI: 10.1021/acs.jafc.9b01156 J. Agric. Food Chem. 2019, 67, 6466−6475
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Journal of Agricultural and Food Chemistry
Table 3. Degradation Rate Constant (k), Half-Life (t1/2), and Regression Coefficient of the First-Order Kinetic Model (R2) for β-Carotene in Emulsion Gels at Different Storage Conditionsa k proportion of coconut oil in the oil phase (%) 0 20 50 80 100
UV light (×10−4, min−1) 1.88 1.45 1.03 1.03 0.69
± ± ± ± ±
0.24 0.19 0.08 0.09 0.08
a b c c d
25 °C (×10−4, h−1) 2.70 2.54 1.93 1.77 1.58
± ± ± ± ±
R2
t1/2
0.16 0.25 0.14 0.18 0.27
a b c d e
55 °C (×10−4, h−1)
UV light (min)
25 °C (h)
55 °C (h)
UV light
25 °C
55 °C
3.47 ± 0.08 a 3.26 ± 0.17 b 2.9 ± 0.27 c 2.56 ± 0.26 c 2.36 ± 0.36 a
368.62 477.93 672.82 672.82 1008.99
256.67 272.83 359.07 391.53 438.61
199.71 212.58 238.97 270.70 293.64
0.9372 0.9318 0.9766 0.9694 0.9510
0.9863 0.9633 0.9802 0.9591 0.8882
0.9981 0.9884 0.9651 0.9593 0.9152
a
Values were means of three determinations, and different lowercase letters in the same column indicated a significant difference (p < 0.05).
first-order kinetic model was appropriate to describe the degradation of β-carotene. Generally, β-carotene had lower k and higher t1/2 in the systems with a higher coconut oil content. For example, the sample with 50% coconut oil content had a k of 1.03 ± 0.08 × 10−4 min−1 when exposed to UV light and 2.9 ± 0.27 × 10−4 h−1 when stored at 55 °C. When the coconut oil content was increased to 100%, the k value was decreased to 0.69 ± 0.08 × 10−4 min−1 and 2.36 ± 0.36 × 10−4 h−1 for the two tests. These results also indicated that the effect of UV light on β-carotene degradation was greater than that of a high temperature. In summary, the incorporation of solid fat in the oil phase of emulsion gels contributed to the systems with compact gel structures and high gel strength, which were helpful to improve the stability of β-carotene against light irritation and thermal treatment. The study also indicated the big roles of the oil droplets (behaved as active fillers in the current study) in the structures of the gels and their protection performance, highlighting the significance of structural design in food systems. As a result of the heath concern of the consumption of saturated fat (e.g., coconut oil), it is important to reduce the level of saturated fat in food products and new approaches to structure emulsion gels should be explored.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-10-62737034. Fax: +86-10-62737986. Email:
[email protected]. ORCID
Like Mao: 0000-0002-1418-5224 Funding
This research was funded by the National Natural Science Foundation of China (31701648) and the Beijing Natural Science Foundation (6182028). Notes
The authors declare no competing financial interest.
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