Article pubs.acs.org/JAFC
Influence of Aqueous Phase Emulsifiers on Lipid Oxidation in Waterin-Walnut Oil Emulsions Jianhua Yi,*,†,‡ Zhenbao Zhu,† D. Julian McClements,‡ and Eric A. Decker‡ †
College of Life Science and Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States
‡
ABSTRACT: Effects of selected aqueous phase emulsifiers on lipid oxidative stability of water-in-walnut oil (W/O) emulsions stabilized by polyglycerol polyricinoleate (PGPR) were evaluated. The formation of primary oxidation products (lipid hydroperoxides) and secondary oxidation products (headspace hexanal) increased with increasing dodecyltrimethylammonium bromide (DTAB) concentration (0.1−0.2 wt % of emulsions). In contrast, the addition of sodium dodecyl sulfate (SDS) in the aqueous phase reduced lipid hydroperoxide and hexanal formation. In addition, the presence of Tween 20 in the aqueous phase did not significantly influence lipid oxidation rates in W/O emulsions compared to the control (without Tween 20). Whey protein isolate (WPI) was observed to inhibit lipid oxidation in the W/O emulsions (0.05−0.2 wt % of emulsions). Aqueous phase pH had an important impact on the antioxidant capability of WPI, with higher pH improving its ability to inhibit lipid oxidation. The combination of WPI and DTAB in the aqueous phase suppressed the prooxidant effect of DTAB. The combination of WPI and SDS resulted in improved antioxidant activity, with inhibition being greater at pH 7.0 than at pH 3.0. These results suggest that the oxidative stability of W/O emulsions could be improved by the use of suitable emulsifiers in the aqueous phase. KEYWORDS: lipid oxidation, water-in oil emulsions (W/O emulsions), emulsifiers, walnut oil, whey protein isolates (WPI)
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INTRODUCTION Unsaturated lipids are susceptible to oxidation.1 Although a limited amount of lipid oxidation in some foods is desirable because of the formation of special compounds that have a desirable taste or smell (e.g., cheeses and fried foods), lipid oxidation is generally undesirable in most foods because it results in the production of rancid odors and unpleasant flavor and even compromises the safety of food due to the generation of toxic products.2−4 In food products, naturally occurring and refined lipids are frequently found in the form of emulsions.5 Emulsions are normally categorized in terms of the relative location of the oil and water phases within the system. A system that consists of oil droplets dispersed in an aqueous phase is called an “oil-in-water” (O/W) emulsion, whereas a system that consists of water droplets dispersed in an oil phase is called a “water-in-oil” (W/O) emulsion.6 The O/W type generally includes products such as milk, infant formula, salad dressing, mayonnaise, sauces, soups, beverages, and cream,7 but there are also examples of W/O emulsions, such as spreads and butter.8,9 Unfortunately, emulsions are thermodynamically unstable systems because of differences in density between water and lipids and thermodynamically unfavorable interactions between lipid and water phases. For these reasons, emulsions will eventually destabilize over time.10 To form emulsions that are kinetically stable for a reasonable period of time, chemical substances known as emulsifiers must be added prior to homogenization. Food emulsifiers are surface-active ingredients that readily adsorb to the surface of freshly formed droplets during homogenization.11,12 Once present at the droplet surface, they lower interfacial tension and physically protect the droplets against aggregation during emulsion processing, storage, and utilization.13,14 The most common emulsifiers used © 2014 American Chemical Society
in the food industry are proteins, phospholipids, and small molecule surfactants.15 The nature of the interfacial membrane formed by these emulsifiers can have a large impact on the rate of lipid oxidation in reactions by influencing the location and reactivity of prooxidative transition metals, lipid hydroperoxides, free radical scavengers, and metal chelators.8,16 A great deal of research has demonstrated that the predominant mechanism for the acceleration of lipid oxidation in O/W emulsions is the decomposition of lipid hydroperoxides (ROOH) into highly reactive peroxyl (ROO•) and alkoxyl (RO• ) radicals by transition metals or other prooxidants.6 These radicals react with unsaturated lipids (LH), which leads to the formation of alkyl radicals (L•) and, upon the addition of oxygen, peroxyl radicals (LOO•).17 The lipid oxidation chain reaction propagates as these lipid radicals react with other lipids in their immediate vicinity.14,18 Lipid hydroperoxides are surface-active compounds and thus able to accumulate at the lipid−water interface of emulsion droplets.13,19 Promoting lipid oxidation may therefore require that transition metals come into close contact with the lipids at the emulsion droplet surface. This suggests that oxidation rates would be influenced by the charge of emulsion droplets, which would influence metal−lipid interactions.20 Some emulsifiers used in foods are electrically charged, such as proteins, phospholipids, and anionic and cationic surfactants. As a result, the interface of the droplets may also have an electrical charge with a magnitude and sign that are dependent on the type and Received: Revised: Accepted: Published: 2104
October 12, 2013 January 13, 2014 January 21, 2014 January 21, 2014 dx.doi.org/10.1021/jf404593f | J. Agric. Food Chem. 2014, 62, 2104−2111
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determine lipid hydroperoxides and hexanal formation. Raw walnut oil also underwent the same emulsification process as the W/O emulsion in the experiment conducted to compare the effect of the existence of water droplet interface on the lipid oxidation. Measurement of Physical Properties of Emulsions. The particle size of the emulsions was measured at 25 °C using a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK). This instrument measures the rate of electrophoretic diffusion of particles. Samples were appropriately diluted with hexadecane (refractive index = 1.434, viscosity = 3.13 mPa s at 25 °C) according to the method of Choi et al., 20 mixed, and immediately measured by transferring the diluted emulsions into 3 mL plastic curvettes. Particle size was reported as the scattering intensity− weighted mean diameter, z-average. The emulsion droplet size ranged from 0.2 to 0.4 μm, and there was no significant change in the droplet size of each emulsion over the course of study (data not shown). Visual observation also showed that there was no phase separation during storage in all treatments. This indicated that the emulsions were stable to droplet coalescence, sedimentation, and phase separation. Measurement of Lipid Hydroperoxides. Lipid hydroperoxides were measured as the primary oxidation products using a method described by Shantha et al.31 with some modifications. Emulsions (0.2 mL) were accurately weighed before they were added to a mixture of methanol/ butanol (2.8 mL, 2:1, v/v) followed by addition of 15 μL of 3.94 M ammonium thiocyanate and 15 μL of ferrous iron solution (prepared by adding equal amounts of 0.132 M BaCl2 and 0.144 M FeSO4). The solution was vortexed, and after 20 min, the absorbance was measured at 510 nm using a Genesys 20 spectrophotometer (ThermoSpectronic, Waltham, MA, USA). The concentration of hydroperoxides was calculated from a cumene hydroperoxide standard curve. Measurement of Hexanal. The secondary oxidation product, headspace hexanal, was monitored using a GC-17A Shimadzu gas chromatograph equipped with an AOC-5000 autosampler (Shimadzu, Kyoto, Japan) according to the method described by Panya et al.32 with some modifications. Samples (1 mL) in 10 mL glass vials capped with aluminum caps with PTFE/silicone septa were heated at 55 °C for 8 min in an autosampler heating block before measurement. A 50/ 30 μm DVB/Carboxen/PDMS solid-phase microextraction (SPME) fiber needle from Supelco (Bellefonte, PA, USA) was injected into the vial for 2 min to absorb volatiles and was then transferred to the injector port (250 °C) for 3 min. The injection port was operated in split mode, and the split ratio was set at 1:7. Volatiles were separated on a 30 m × 0.32 mm Equity DB-1 column from Supelco with a 1 μm film thickness at 65 °C for 10 min. The carrier gas was helium at 15.0 mL/min. A flame ionization detector was used at a temperature of 250 °C. Hexanal concentrations were determined from peak areas using a standard curve prepared from an authentic standard. Measurement of Iron-Binding Capacity of WPI. The ability of WPI to bind iron was determined using a modified method of Bincan et al.33 Dialysis bags (molecular mass cutoff 8 kDa, Shengya Co., Xi’an, China) were cleaned by heating them twice at 80 °C for 30 min in a 2% sodium bicarbonate and 1 mM EDTA solution. The bag was then thoroughly rinsed with deionized water and stored at 5 °C in a 0.1% sodium azide solution. WPI solutions (10% w/v) were prepared with 20 mM phosphate buffer solution at pH 3.0 and 7.0, respectively, using 1.0 M NaOH and HCl to adjust the pH. Two aliquots of 1 mL of WPI solution at pH 3.0 or 7.0 were pipetted into the dialysis bag, equilibrated for 30 min at room temperature in 98 mL of 20 mM phosphate buffer solution at pH 3.0 or 7.0, respectively, and constantly stirred. One milliliter of an 80 μg/mL Fe2+ solution (prepared from Fe2SO4 in 20 mM phosphate buffer solution at pH 3.0 or 7.0) was then added to the flask so that the final concentration of ferrous outside the dialysis bag was 8 μg/mL. After equilibrium for 24 h, the concentrations of Fe2+ outside the dialysis bag were measured by flame atomic absorption spectrophotometer (Sollar-3500, USA). Statistical Analysis. All analyses were performed on triplicate samples. Oxidation lag phases were defined as the first data point significantly greater than the 0 time value. In all cases, comparisons of the means were performed using Duncan’s multiple-range tests. A significance level of p < 0.05 was defined as being statistically different.
concentration of charged surface-active components present.6,21,22 Several studies have shown that lipid oxidation rates in O/W emulsions stabilized with anionic surfactants are much higher due to the electrostatic attraction of cationic transition metals, whereas O/W emulsions stabilized with cationic surfactants oxidize more slowly because of electrostatic repulsion of metals away from the lipids.23−26 The role of droplet charge has also been demonstrated in the studies of lipid oxidation in protein-stabilized O/W emulsions. In this case, lipid oxidation occurs more rapidly when the pH is above the isoelectric point (pI) of proteins and the emulsion droplet is negatively charged.27−30 Although a great deal of research has focused on the influence of different emulsifiers on lipid oxidation in O/W emulsions, there are fewer studies on the effects of emulsifiers on lipid oxidation in W/O emulsions. For example, little is known about how emulsifier type (e.g., biopolymers, anionic and cationic small molecular surfactants), concentrations, and aqueous phase pH influence lipid oxidation in W/O emulsions. Preliminary experiments showed that among such lipophilic emulsifiers as polyglycerol polyricinoleate (PGPR), lecithin, and span 60, 65, 80, and 85, PGPR was the only one tested capable of forming a stable W/O. Therefore, in the present work, PGPR was used to form stable W/O emulsions. Dodecyltrimethylammonium bromide (DTAB), sodium dodecyl sulfate (SDS), and polyoxyethylene sorbitan monolaurate (Tween 20) were added as small molecular surfactants that are cationic, anionic, and nonionic, respectively. WPI was used as an example of a biopolymer emulsifier. Experiments were then conducted to determine if these emulsifiers influenced lipid oxidation in PGPR-stabilized W/O emulsions. The knowledge gained from these studies could provide fundamental knowledge that could be used to improve the oxidative stability of oils in W/O emulsions by using different emulsifiers.
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MATERIALS AND METHODS
Materials. Walnut oil was purchased from Hain Celestial Group Inc. (Melville, NY, USA) (C16:0 = 7.13%; C16:1 = 0.11%; C18:0 = 3.58%; C18:1 = 17.72%; C18:2 = 61.91%; C18:3 = 9.27%; C20:0 = 0.12%; C20:1 = 0.24%). Ferrous sulfate, cumene hydroperoxide, hexadecane, BaCl2, SDS, Tween 20, DTAB, and sodium phosphate mono- and dibasic were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Methanol, n-hexane, and 1-butanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Polyglycerol polyricinoleate (PGPR, 4175) was gifted by Palsgaard (Morristown, NJ, USA). WPI was obtained from Davisco Food International, Inc. (Eden Praire, MN, USA), and used without further purification. The protein content of WPI was 97.6 wt %. The major protein components of WPI were 55− 61% β-lactoglobulin, 19−22% α-lactoalbumin, and 6−8% bovine serum albumin. All of the chemicals used in these experiments were of analytical grade or better. Glassware was placed in 2 M HCl overnight to remove transition metals followed by rinsing with double-distilled water and then drying before use. Double-distilled, deionized water was used for the preparation of all solutions. Methods. Emulsion Preparation and Storage Condition. PGPRstabilized W/O emulsions were prepared by mixing 0.5 g of PGPR into 100 g of walnut oil. Aqueous phase (2% of a 20 mM phosphate buffer solution at pH 3.0 or 7.0) with different emulsifiers was added to the PGPR−walnut oil blend, and a coarse emulsion was made by homogenization with a hand-held mixer (M133/1281-0, Biospec Products, Inc., Bartlesville, OK, USA) for 2 min. The coarse emulsion was then homogenized with a microfluidizer (Microfluidics, Newton, MA, USA) at a pressure of 12 kbar for three passes. After emulsion preparation, samples were transferred into 10 mL GC vials and sealed with aluminum caps with PTFE/silicone septa and stored at 45 °C in the dark. Three vials of each treatment were taken every day to 2105
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All calculations were performed using the statistical analysis software SPSS 17 (Statistical Package for the Social Sciences).
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RESULTS AND DISCUSSION Effects of Type and Concentration of Surfactants on the Oxidative Stability of Water-in-Walnut Oil Emulsions. As far as O/W emulsions are concerned, Boon et al.26 and Haahr et al.34 have proposed that the oxidative stability of oil-in-water emulsions is determined by the ability of the droplet to attract or repel cationic metals. Emulsion droplet charge is dependent on the type of surfactants (cationic, anionic, or neutral) and the emulsion pH that affects surfactant charge.7,17 Many studies have confirmed that lipid oxidation was promoted when positively charged transition metals were electrostatically attracted to the surface of the negatively charged emulsion droplets due to close interaction between the lipid substrate and prooxidative transition metals, whereas lipid oxidation was retarded by cationic surfactants that electrostatically repelled metals away from the lipids.23−30 In the case of W/O emulsions, to obtain a better understanding of the influence of the type of surfactants on the lipid oxidation, a comparison of oxidation rates as determined by lipid hydroperoxide and headspace hexanal was conducted in walnut oil and 2% water-in-walnut oil emulsions stabilized by PGPR alone or with SDS, DTAB, or Tween 20. Emulsions were incubated at 45 °C for up to 25 days, and the results are shown in Figure 1. Interestingly, regardless of the type of emulsifier used, all of the emulsions containing 2% water had higher lipid hydroperoxide and hexanal formation rates than the walnut oil alone. The results were different from the observation by Fritsch, who stated that the rates of lipid oxidation in W/O emulsions would be similar to that in bulk oils if the same amount of surface lipid was exposed to air.35 As stated earlier, the decomposition of lipid hydroperoxides into highly reactive radicals by prooxidants such as transition metals is the main pathway for the promotion of lipid oxidation. Lipid hydroperoxides are surface-active compounds and are thus able to accumulate at the water−lipid interface of emulsion droplets.19,36 In this way, the higher rates of lipid oxidation in W/O emulsions were possibly due to the large surface area that facilitated interactions between the lipid hydroperoxides and water-soluble prooxidants. Similar phenomena have been observed in O/W emulsions.16 The results indicated that the presence of a large interface between oil and water in W/O emulsions might also affect the lipid oxidation. With regard to the type of surfactants, significant differences in oxidation rates were observed among the samples containing different surfactants, suggesting that the type of surfactants in emulsions was an important factor influencing the lipid oxidation in W/O emulsions. However, surprisingly, it could be seen that lipid hydroperoxides increased rapidly in W/O emulsions containing PGPR + DTAB compared to PGPR alone (lag phase 2 and 7 days, respectively), whereas lipid hydroperoxide concentrations were lowest in emulsions containing PGPR + SDS (lag phase 10 days). No differences in lipid hydroperoxide formation were found between the PGPR and PGPR + Tween 20 emulsions (lag phase 7 days). When the secondary lipid oxidation product, hexanal, was measured to monitor lipid oxidation in emulsions containing different surfactants, a similar trend in lipid oxidation rates was observed with PGPR + DTAB > PGPR ≈ PGPR + Tween 20 > PGPR + SDS, indicating that the anionic surfactant again
Figure 1. Effects of different surfactants in the aqueous phase (0.1 wt % of emulsions) on the oxidative stability of 2% water-in-walnut oil at aqueous phase pH 7.0 during storage at 45 °C in the dark for 25 days. Lipid oxidation was monitored by measuring hydroperoxides (A) and hexanal (B). Data points and error bars represent means (n = 3) ± standard deviations.
inhibited lipid oxidation, whereas the cationic surfactant promoted lipid oxidation in W/O emulsions. Figures 2 and 3 clearly show that the oxidative stability of emulsions with increasing SDS concentrations did not further increase oxidative stability but increasing DTAB did cause additional promotion of oxidation. These results were completely opposite to the studies in O/W systems by Mei et al.,23,24 Silvestre et al.,25 Boon et al.,26 Hu et al.,27,28 and Mancuso et al.,29,30 who found that lipid oxidation was retarded by cationic surfactants and promoted by anionic surfactants, presumably because the surface of the positively charged emulsion droplets containing cationic surfactants repelled metals away from the water−oil interface where lipid oxidation usually occurs, whereas the surface of the negatively charged emulsion droplets containing anionic surfactants attracted metals to the interface. 2106
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Figure 3. Effects of concentration of DTAB in the aqueous phase on the oxidative stability of 2% water-in-walnut oil at aqueous phase pH 7.0 during storage at 45 °C in the dark for 25 days. Lipid oxidation was monitored by measuring hydroperoxide (A) and hexanal (B). Data points and error bars represent means (n = 3) ± standard deviations.
Figure 2. Effects of concentration of SDS in the aqueous phase on the oxidative stability of 2% water-in-walnut oil at aqueous phase pH 7.0 during storage at 45 °C in the dark for 25 days. Lipid oxidation was monitored by measuring hydroperoxides (A) and hexanal (B). Data points and error bars represent means (n = 3) ± standard deviations.
The reason W/O emulsions containing SDS had higher oxidative stability seemed likely due to the fact that aqueous phase SDS formed negative micelles that were able to electrostatically attract transition metals, thereby reducing the concentration of prooxidants (e.g., transition metals) at the oil−water surface, a location where lipid oxidation reactions primarily occurs (seen in Figure 4). Similar results were found for the hemoglobin-catalyzed oxidation of a safflower oil-inwater emulsion, where the presence of excess anionic surfactant in the aqueous phase of the emulsion increased the oxidative stability.37 Tween 20 is weakly anionic and thus might not be able to remove metals away from the lipid−water interface. It is unclear why a cationic surfactant such as DTAB would promote oxidation in the W/O emulsion. Effects of WPI on the Oxidative Stability of Water-inWalnut Oil Emulsions. Whey protein products such as WPI are commonly used as emulsifiers in O/W emulsions because they have good emulsifying and stabilizing properties.38,39
Figure 4. Schematic demonstration of the ability of SDS in the aqueous phase of W/O emulsions to interact with transition metals such as iron and inhibit lipid oxidation.
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emulsions might have contributed to the better oxidative stability. Hu et al.27 and Kulmyrzaev et al.43 have reported that the isoelectric point (pI) of WPI is 5.1, and pH values below or above the pI can dramatically affect the antioxidant activity of WPI. Therefore, further comparisons of oxidation rates as a function of aqueous phase pH were also determined. Figure 6
Moreover, WPI has been shown to possess antioxidative properties in O/W emulsions when they are either at the emulsion droplet surface or in the aqueous phase.28,40,41 Tong et al.42 reported that the origin of the antioxidant activity of whey proteins was due to free radical scavenging by sulfhydryl and aromatic amino acids and transition metal chelation. To determine if WPI influenced the oxidative stability of W/O emulsions, the oxidative rates were analyzed by monitoring the formation of lipid hydroperoxides and hexanal. Figure 5 shows
Figure 5. Effects of the concentration of WPI in the aqueous phase on the oxidative stability of 2% water-in-walnut oil at aqueous phase pH 7.0 during storage at 45 °C in the dark for 25 days. Lipid oxidation was monitored by measuring hydroperoxide (A) and hexanal (B). Data points and error bars represent means (n = 3) ± standard deviations.
Figure 6. Effects of WPI (0.1 wt % of emulsions) as a function of different aqueous phase pH on the oxidative stability of 2% water-inwalnut oil during storage at 45 °C in the dark for 25 days. Lipid oxidation was monitored by measuring hydroperoxides (A) and hexanal (B). Data points and error bars represent means (n = 3) ± standard deviations.
that increasing concentration of WPI in W/O emulsions at pH 7.0 could decrease lipid oxidation rates. The lag phases of lipid hydroperoxide and hexanal formation in the control (without WPI) were 7 and 8 days, respectively. In the presence of 0.05, 0.1, or 0.2 wt % WPI (of emulsions), the lag phase increased to 9, 13, and 15 days for lipid hydroperoxides, respectively, and 10, 13, and 15 days for hexanal, respectively, suggesting that a higher concentration of WPI in the aqueous phase of W/O
shows that lipid oxidation of the controls was more rapid when the aqueous phase was pH 7.0 than when it was pH 3.0. At aqueous phase pH 7.0, lipid oxidation of the emulsions occurred slowly with the lag phases of both lipid hydroperoxide and hexanal formation being 7 and 8 days, respectively. The lag phases of lipid hydroperoxide and hexanal formation in the control at aqueous phase pH 3.0 were the same at 9 days of storage. Mancusco et al.44 also observed that oxidation rates increased with increasing pH in Tween 20-stabilized salmon 2108
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oil-in-water emulsion. The same authors proposed that at pH 7.0, low iron solubility resulted in precipitation of metal onto the lipid droplet surface, thereby bringing iron in closer contact with the lipid compared to pH 3.0, where the water solubility of iron is dramatically higher. If the same was true in the W/O emulsions, where iron could precipitate onto the lipid surface, it was not surprising that high pH in the aqueous phase microenvironment promoted lipid oxidation in W/O emulsions. In addition, it was reported that at pH 7.0, free fatty acids in the oil are negatively charged and thus can attract transition metals and increase lipid oxidation rates.45,46 At pH 3.0 the free fatty acids lose their charge and are less prooxidative. Because all refined oils contain some free fatty acids, it is possible that the decrease in lipid oxidation rates with decreasing pH observed in this study could be due to the presence of free fatty acids. The ability of WPI to inhibit lipid oxidation in W/O emulsions was greater at pH 7.0 than at pH 3.0 (Figure 6). For example, the lag phase of hexanal formation increased from 8 days (control) to 13 days in the presence of 0.1 wt % WPI at pH 7.0. This compares to an increase the lag phase of hexanal formation from 9 days (control) to 12 days in the presence of 0.1 wt % WPI at pH 3.0. The antioxidant activity of proteins in the continuous phase of O/W emulsions has also been found to be greater at pH values above the pI of the protein.47 The reason for increased antioxidant activity of proteins at pH values above the pI is thought to be the increased ability of proteins to bind transition metals when they are negatively charged. For example, WPI bound over 3 times more iron at aqueous phase pH 7.0 than at aqueous phase pH 3.0 (Figure 7).
The impact of combinations of between WPI and DTAB or SDS on lipid oxidation rates in the W/O emulsion were tested at both pH 3.0 and 7.0 because the antioxidant activity of WPI was strongly influenced by pH. The oxidative stability of the controls was observed to be pH dependent, with higher pH increasing lipid oxidation rates in the W/O emulsions as also observed in Figure 6. WPI was again found to be a stronger antioxidant at pH 7.0 than at pH 3.0 as first reported in Figure 6. Table 1 shows that emulsions Table 1. Effects of the Interaction of WPI with SDS on Lag Phases of Lipid Hydroperoxide and Hexanal Formation in 2% Water-in-Walnut Oil Emulsions during Storage at 45 °C in the Dark for 25 Days lag phasesa (days) treatment
hydroperoxide formation
hexanal formation
WPI (pH 3.0) WPI (pH 7.0) SDS (pH 3.0) SDS (pH 7.0) WPI + SDS (pH 3.0) WPI + SDS (pH 7.0) control (pH 3.0) control (pH 7.0)
11 13 10 10 12 15 9 7
12 13 10 10 13 15 9 7
a Oxidation lag phases were defined as the first data point significantly greater than the 0 time value at the level of p < 0.05. All concentrations of emulsifiers were 0.1 wt % of emulsions.
containing WPI alone oxidized significantly more slowly than emulsions containing SDS alone, indicating that WPI had stronger antioxidant activity than SDS. From Table 1, it can also be seen that pH had no significant influence on the lag phases of both lipid hydroperoxide and hexanal formation in emulsions containing SDS (p > 0.05). The combination of WPI and SDS increased the lag phases of both lipid hydroperoxide and hexanal formation greater than either WPI or SDS alone at both pH 3.0 and 7.0. This increase in antioxidant activity by the WPI/SDS combination could simply due to an increase in the total metal chelating capacity in the emulsion. However, it is also well-known that SDS can induce denaturation of proteins and that exposing amino acids in the core of proteins can increase the ability of proteins to inhibit lipid oxidation.49,50 The greater inhibition of lipid oxidation by the combination of WPI/SDS at pH 7.0 than at pH 3.0 could be due to WPI’s greater antioxidant activity when it is negatively charged. The influence of interactions between WPI and DTAB on lag phases of lipid hydroperoxide and hexanal formation in W/O emulsions was also investigated (Table 2). DTAB alone consistently promoted lipid oxidation, whereas WPI alone consistently inhibited lipid oxidation in W/O emulsions, similar to the results above (Figures 3 and 6). The combination of WPI and DTAB resulted in lag phases for 5 and 6 days for hydroperoxides and hexanal formation, respectively, at pH 3.0 (Table 2). At pH 7.0, lag phases for hydroperoxides and hexanal formation were 7 and 8 days, respectively, for the DTAB/WPI combination (Table 2). These lag phases are greater than those for DTAB alone but less than for WPI. This is likely because the antioxidant capability of WPI was able to counteract some of the prooxidant activity of DTAB but not to the extent that the lag phases were equal to WPI alone. The lag phases for hydroperoxides and hexanal formation for the WPI/ DTAB combination were greater at pH 7.0 than at pH 3.0,
Figure 7. Iron bonding constant per gram WPI during incubation of ferrous ion (8 mg/L) in WPI solution at different pH values for 24 h. Data points and error bars represent means (n = 3) ± standard deviations.
Thus, it was possible that in the microenvironment of the aqueous phase in the W/O emulsions, WPI exhibited better antioxidant activity at pH 7.0 than at pH 3.0 due to binding more transition metals. However, it should be noted that WPI did inhibit lipid oxidation at pH 3.0 when the protein was cationic. This could still be due to its weak chelating activity but also could be due to its free radical scavenging activity. Effects of WPI and Surfactants Combinations on the Oxidative Stability of Water-in-Walnut Oil Emulsions. Combinations of molecules in emulsion can often interact and synergistically or antagonistically impact lipid oxidation rates.48 2109
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Table 2. Effects of the Interaction of WPI with DTAB on Lag Phases of Lipid Hydroperoxide and Hexanal Formation in 2% Water-in-Walnut Oil Emulsions during Storage at 45 °C in the Dark for 25 Days treatment
hydroperoxide formation
hexanal formation
11 13 2 2 5 7 9 7
12 13 4 4 6 8 9 7
Oxidation lag phases were defined as the first data point significantly greater than the 0 time value at the level of p < 0.05. All concentrations of emulsifiers were 0.1 wt % of emulsions. a
which again could be attributed to the increase antioxidant activity of WPI when it is anionic and better able to chelate metals. In conclusion, the current study shows that formation of a W/O emulsion increased oxidation rates compared to bulk oil. This acceleration of oxidation could be due to the increase in surface area that could facilitate interactions between lipids and water-soluble transition metals. The present work also indicates that Tween 20 had no impact on lipid oxidation rates, whereas DTAB increased oxidation in an unknown manner. SDS and WPI inhibited lipid oxidation in the W/O emulsions, which could be due to metal chelation by these anionic surfactants. WPI was a better antioxidant at pH 7.0 than at pH 3.0 presumably due to its higher transition metal-binding capacity at pH values above the pI of the proteins, where the proteins would be anionic. However, WPI was also able to inhibit oxidation at pH 3.0 when it would be cationic, suggesting that it may also be able to inhibit oxidation by scavenging free radicals. Only the combination of WPI and SDS resulted in increased antioxidant activity compared to the individual compounds, which could be due to increased metal chelating capacity or combining both metal chelating and free radical scavenging activities. These results suggest that compounds that chelate transition metal could be used to increase the oxidative stability of W/O emulsions. The ability of WPI to inhibit lipid oxidation in W/O emulsions suggests that it could be used as a natural antioxidant in foods such as spreads.
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REFERENCES
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lag phasesa (days) WPI (pH 3.0) WPI (pH 7.0) DTAB (pH 3.0) DTAB (pH 7.0) WPI + DTAB (pH 3.0) WPI + DTAB (pH 7.0) control (pH 3.0) control (pH 7.0)
Article
AUTHOR INFORMATION
Corresponding Author
*(J.Y.) E-mail:
[email protected]. Funding
This study was supported by the Cultivation of Academic Program of Shaanxi University of Science and Technology (XS11-07). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Yuhua Chang, Jean Alamed, and Leann Barden for assistance. 2110
dx.doi.org/10.1021/jf404593f | J. Agric. Food Chem. 2014, 62, 2104−2111
Journal of Agricultural and Food Chemistry
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