Article pubs.acs.org/JAFC
How the Multiple Antioxidant Properties of Ascorbic Acid Affect Lipid Oxidation in Oil-in-Water Emulsions Sibel Uluata,*,†,‡ D. Julian McClements,‡,§ and Eric A. Decker‡,§ †
Food Technology Department, Inonu University, 44280 Malatya, Turkey Department of Food Science, University of Massachusetts Amherst, 228 Chenoweth Laboratory, 100 Holdsworth Way, Amherst, Massachusetts 01003, United States § Department of Biochemistry, Faculty of Science, King Abdulaziz University, Post Office Box 80203, Jeddah 21589, Saudi Arabia ‡
ABSTRACT: Lipid oxidation is a serious problem for oil-containing food products because it negatively affects shelf life and nutritional composition. An antioxidant strategy commonly employed to prevent or delay oxidation in foods is to remove oxygen from the closed food-packaging system. An alternative technique is use of an edible oxygen scavenger to remove oxygen within the food. Ascorbic acid (AA) is a particularly promising antioxidant because of its natural label and multiple antioxidative functions. In this study, AA was tested as an oxygen scavenger in buffer and an oil-in-water (O/W) emulsion. The effects of transition metals on the ability of AA to scavenge oxygen were determined. Headspace oxygen decrease less than 1% in the medium-chain triacylglycerol (MCT) O/W emulsion system (pH 3 and 7). AA was able to almost completely remove dissolved oxygen (DO) in a buffered solution. Transition metals (Fe2+ and Cu+) significantly accelerated the degradation of AA; however, iron and copper only increased DO depletion rates, by 10.6−16.4% from day 1 to 7, compared to the control. AA (2.5−20 mM) decreased DO in a 1% O/W emulsion system 32.0−64.0% and delayed the formation of headspace hexanal in the emulsion from 7 to over 20 days. This research shows that, when AA is used in an O/W emulsion system, oxidation of the emulsion system can be delay by multiple mechanisms. KEYWORDS: oxygen scavenger, ascorbic acid, metal-catalyzed oxidation, antioxidant
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INTRODUCTION L-Ascorbic acid (AA, vitamin C) is a water-soluble vitamin that is a necessary component in human health.1−3 AA has been proposed to be an important antioxidant in both plant and animal tissues by preventing oxidative-induced cellular damage through its ability to scavenge free radicals and regenerate tocopherols.1 In addition, the enediol group of AA on carbons 2 and 3 allows it to oxidize through one- or two-electron transfers, thus making it able to scavenge oxygen4−8 (Figure 1). Preventing lipid oxidation is important for the food industry because it can prolong food-quality properties, such as color, texture, flavor, and nutritive value.9,10 AA offers an attractive antioxidant solution for foods because it satisfies consumer preference for antioxidants from natural sources.8 In foods, AA can inhibit lipid oxidation by acting as a water-soluble free radical scavenger, a synergist to regenerate the activity of other antioxidants (e.g., tocopherol), a means to quench reactive singlet oxygen, and an oxygen scavenger.1,11,12 However, in many situations, it is difficult to determine which of these pathways are important in the antioxidant activity of AA. Further, AA can be prooxidative because it can reduce transition metals, thus increasing their reactivity.13,14 This is especially important at low concentrations, where the capacity of AA to redox cycle metals is more dominant than its free radical scavenging properties.11,15 Therefore, to ensure that AA is antioxidative and not prooxidative, its concentration and factors affecting lipid oxidation, such as transition metal concentration and reactivity, must be considered. Oxygen is a critical factor that can influence the susceptibility of lipid-containing foods to oxidation.2,16,17 Atmospheric triplet © XXXX American Chemical Society
oxygen (present in the headspace and dissolved within foods) is a biradical that can quickly react with lipid radicals at diffusionlimited rates. This reaction produces peroxyl radicals and, upon reaction with another unsaturated fatty acid, produces lipid hydroperoxides. Hydroperoxides are critical in oxidative rancidity because they can decompose, resulting in fatty acid scission, creating low-molecular-weight volatile compounds that are perceived as rancid.18 Thus, one way to decrease lipid oxidation rates is to remove oxygen as a reactant from the food product.16,17 Nowadays, several methods are used to decrease oxygen in food products, such as modified atmosphere packaging, vacuum packaging, flushing with nitrogen, and use of active packaging.19,20 Unfortunately, these techniques do not always achieve desirable levels of oxygen reduction, are not applicable for all foods, and may be cost-prohibitive for certain foods.19−23 In addition, many current, plastic-packaging materials are oxygen-permeable and allow oxygen to reenter the food during storage. An alternative strategy would be to harness the ability of AA to scavenge oxygen and decrease oxygen in a closed food system. AA could potentially be used alone or combined with other oxygen reduction procedures to remove the last portion of oxygen from a food product.19−23 While AA has long been recognized as an oxygen scavenger, very few systematic studies have been conducted to determine its ability to both reduce oxygen concentrations and inhibit Received: November 11, 2014 Revised: January 24, 2015 Accepted: January 26, 2015
A
DOI: 10.1021/jf5053942 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Potential reaction schemes for AA in an emulsion system. homogenizer (M133/1281-0, Biospec Products, Inc., Bartlesville, OK) for 2 min. The coarse emulsion was then homogenized with a microfluidizer (Microfluidics, Newton, MA) at a pressure of 9 kbar for three passes. The MCT O/W emulsion system was used for headspace oxygen analysis, and AA was added to the O/W emulsions at a final concentration ranging from 0.01 to 200 mM. To represent a more realistic food matrix with oxidizable fatty acids, O/W emulsions were prepared using 1.0 wt % commercial soybean oil in 10 mM sodium acetate/imidazole (pH 4.0). Tween 20 was used as an emulsifier at a 1:10 emulsifier/oil ratio. Emulsion preparation was carried out as previously mentioned. The soybean oil O/W system was used to determine lipid oxidation products, and AA was added to the O/W emulsions at a final concentration ranging from 2.5 to 20 mM. Measurement of the Particle Size of Emulsion. The sizes of the emulsion droplets were measured by dynamic light scattering (Zetasizer Nano-ZS, model ZEN3600, Malvern Instruments, Worchester, U.K.). Results were expressed as the Z-average mean diameter. Samples were diluted 50 times with the same buffer as the emulsion, mixed, and immediately measured by transferring the samples into 3 mL plastic cuvettes to determine the size. Measurements were performed on three replicates for each sample at room temperature. The emulsion droplet size averaged 174.1 ± 0.55 nm, and there was no significant change in the droplet size of each emulsion over the course of study (data not shown). In addition, there was no visual observation of creaming during storage in all treatments. Headspace and Dissolved Oxygen (DO) Analysis. To measure headspace oxygen, the MCT O/W emulsion was first mixed with AA solution (final concentration of 0.01−200 mM) and the final emulsion (5 mL) was transferred to 10 mL of gas chromatography (GC) vials, sealed with (tetrafluoroethylene) butyl rubber septa, and then stored at 10, 20, 32, 37, and 45 °C in the dark. Headspace oxygen was determined using a headspace O2/CO2 anaylzer (Benchtop 902D Dualtark, U.S.A.).
lipid oxidation in food systems. These studies are important because AA can act as both an antioxidant and a prooxidant. Oil-in-water (O/W) emulsions, such as salad dressing, beverages, and mayonnaise, are very susceptible to oxidative deterioration24 and represent a food where AA could be added immediately prior to packaging to reduce oxygen concentrations. Therefore, the aim of this study was to investigate the conditions of oxygen reduction by AA in both a buffer system and O/W emulsions. Factors, such as AA concentrations, temperature, and presence of metals, were tested to determine optimum conditions for oxygen reduction. The ability of AA to both decrease oxygen concentrations and inhibit lipid oxidation was determined in O/W emulsions by determining lipid hydroperoxides and headspace hexanal.
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MATERIALS AND METHODS
Chemicals and Materials. Soybean oil was purchased from a local grocer (Amherst, MA). Medium-chain triacylglycerols (MCTs, Miglyol) were obtained from Sasol North America, Inc. (Houston, TX). The fluorescent dye tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) bis(hexafluorophosphate) complex was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). AA was purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of analytical or chromatographic grade. Emulsion Preparation. The current study used two types of O/W emulsions. First, O/W emulsions were prepared using 1.0 wt % MCT in a 10 mM sodium acetate/imidazole buffer solution (pH 3.0 and 7) to produce emulsions that did not have oxidizable fatty acids. Tween 20 was used as an emulsifier at a 1:10 emulsifier/oil ratio. MCT, Tween 20, and sodium acetate/imidazole were added to a beaker, and a coarse emulsion was made by blending with a hand-held B
DOI: 10.1021/jf5053942 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry The fluorescent dye, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) bis(hexafluorophosphate) complex, was used to determine DO in the buffer and O/W emulsion system using a λexcitation = 455 nm and a λemission = 615 nm. Fluorescence intensity of the dye increased with a decrease in the oxygen concentration. A stock dye solution was prepared by dissolving 1 mg of the dye in 1 mL of methanol. This stock solution was then added to buffer and soybean oil O/W emulsions to achieve a final dye concentration of 50 μg/g in oil.25 The samples were mixed with AA (0.01−200 mM final concentration in the solution), and the final samples (2 mL) were transferred to 10 mL GC vials, sealed with (tetrafluoroethylene) butyl rubber septa, and stored at 55 °C in the dark. Three vials of each treatment were sampled at each day of analysis. Standard fluorescence was used in the buffer model. Front-face fluorescence was used to measure DO in the O/W emulsion using steady-state measurements recorded with a PTI spectrofluorometer (PTI, London, Ontario, Canada). Spectral bandwidth for both excitation and emission slits was 2.0 nm; integration time was 30 s; and wavelength increment was 2.5 nm. Quantification of DO was calculated according the Stern−Volmer equation.26 Effect of Transition Metals on AA Degradation in Buffer. The ability of transition metals (e.g., copper and iron) to impact the ability of AA to reduce the DO concentration was determined in an acetic acid/imidazole buffer system (10 mM, pH 3). FeSO4 and CuCl solutions were added to the buffer (final concentration of 2.5 or 10 μM) followed by the addition of AA (75 mM final concentration). The final solution (7 mL) was transferred to 10 mL GC vials, sealed with (tetrafluoroethylene) butyl rubber septa, and stored at 32 °C in the dark. Three vials of each treatment were analyzed for DO at each sampling time. Results were expressed as mean values ± standard deviation. Determination of AA Degradation. Quantification of AA was determined directly in the buffer system or in the aqueous phase of the emulsion. The aqueous phase of the emulsified system was obtained by centrifuging at 38518g (17 000 rpm) for 1 h at 4 °C using a Fiberlite F40L-8 × 100 rotor with a high-speed centrifuge (Thermo Scientific WX Ultra 80, Asheville, NC). After the centrifugation, the lower aqueous phase was carefully removed using a syringe. AA was analyzed according the method described by Acar et al., with minor modifications.27 Briefly, 2,6-dichlorofenolindophenol (approximately 15 mg) was dissolved in double-distilled water until the absorbance was 0.800 ± 0.020 at 520 nm. Samples were diluted (1:100) with oxalic acid solution (0.04%, w/v) and mixed with the 2,6dichlorofenolindophenol solution (1:100). The subsequent reaction results in non-oxidized AA reducing the indicator dye to a colorless compound. The absorbance was detected at 520 nm after vortexing for 10 s using an ultraviolet−visible (UV−vis) spectrophotometer (Genesys 20, Thermo Spectronic). AA concentrations were calculated from a standard curve prepared with reagent-grade AA. Results were expressed as mean values ± standard deviation. Measurements of Lipid Oxidation Products. Lipid hydroperoxides and hexanal were measured as primary and secondary lipid oxidation products, respectively. Lipid hydroperoxide formation in emulsion samples was determined according to the method by Shantha and Decker.28 Emulsion samples (0.3 mL) were mixed with 1.5 mL of isooctane/2-propanol solution (3:1, v/v) and vortexed (10 s, 3 times). The mixed solution was centrifuged at 3400g for 10 min (Centrific Centrifuge, Thermo Fisher Scientific, Inc., Fairlawn, NJ). The upper organic layer (200 μL) was mixed with 2.8 mL of methanol/butanol solution (2:1, v/v), followed by the addition of 15 μL of 3.94 M ammonium thiocyanate and 15 μL of Fe2+ solution. The Fe2+ solution was prepared freshly from the supernatant of a mixture of equal amounts of 0.132 M BaCl2 in 0.4 M HCl and 0.144 M FeSO4. The solution was vortexed and held 20 min at room temperature, and the absorbance was measured at 510 nm in a UV−vis spectrophotometer (Genesys 20, Thermo Fisher Scientific, Inc., Waltham, MA). Hydroperoxide concentrations were determined using a standard curve prepared from cumene hydroperoxide. Headspace hexanal was measured according to the method described by Panya et al.29 using a gas chromatograph (model GC-
2014, Shimadzu Co., Tokyo, Japan) equipped with a solid-phase microextraction (SPME) auto injector (model AOC-5000, Shimadzu Co., Tokyo, Japan). Emulsions (1 mL) in 10 mL glass vials capped with aluminum caps with polytetrafluoroethylene (PTFE)/silicone septa were heated at 55 °C for 10 min in the autosampler heating sampler heating block before measurement. A 50/3 μm divinylbenzene (DVB)/carboxen/polydimethylsiloxane (PDMS) stable flex (SPME) fiber (Supelco Co., Bellefonte, PA) was then inserted into the vial headspace for 2 min to absorb volatiles. The fiber was transferred to the GC 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 fused-silica capillary Equity-1 Supelco column (30 × 0.32 mm inner diameter × μm) coated with 100% PDMS at 65 °C for 10 min. A flame ionization detector was used at a temperature of 250 °C. Hexanal concentrations were determined from peak areas using a standard curve made from authentic hexanal. Each measurement was performed in triplicate, and results were expressed as mean values ± standard deviation. Statistical Analysis. All data result were analyzed by analysis of variance (ANOVA) using SPSS 20 (SPSS, Inc., Chicago, IL). The differences between mean values were compared using Duncan’s multiple-range test with a level of significance of p ≤ 0.05.
■
RESULTS AND DISCUSSION Headspace Oxygen Analysis. Headspace oxygen concentrations in MCT O/W emulsions (pH 3 and 7) were only slightly decreased (
How the Multiple Antioxidant Properties of Ascorbic Acid Affect Lipid Oxidation in Oil-in-Water Emulsions Sibel Uluata,*,†,‡ D. Julian McClements,‡,§ and Eric A. Decker‡,§ †
Food Technology Department, Inonu University, 44280 Malatya, Turkey Department of Food Science, University of Massachusetts Amherst, 228 Chenoweth Laboratory, 100 Holdsworth Way, Amherst, Massachusetts 01003, United States § Department of Biochemistry, Faculty of Science, King Abdulaziz University, Post Office Box 80203, Jeddah 21589, Saudi Arabia ‡
ABSTRACT: Lipid oxidation is a serious problem for oil-containing food products because it negatively affects shelf life and nutritional composition. An antioxidant strategy commonly employed to prevent or delay oxidation in foods is to remove oxygen from the closed food-packaging system. An alternative technique is use of an edible oxygen scavenger to remove oxygen within the food. Ascorbic acid (AA) is a particularly promising antioxidant because of its natural label and multiple antioxidative functions. In this study, AA was tested as an oxygen scavenger in buffer and an oil-in-water (O/W) emulsion. The effects of transition metals on the ability of AA to scavenge oxygen were determined. Headspace oxygen decrease less than 1% in the medium-chain triacylglycerol (MCT) O/W emulsion system (pH 3 and 7). AA was able to almost completely remove dissolved oxygen (DO) in a buffered solution. Transition metals (Fe2+ and Cu+) significantly accelerated the degradation of AA; however, iron and copper only increased DO depletion rates, by 10.6−16.4% from day 1 to 7, compared to the control. AA (2.5−20 mM) decreased DO in a 1% O/W emulsion system 32.0−64.0% and delayed the formation of headspace hexanal in the emulsion from 7 to over 20 days. This research shows that, when AA is used in an O/W emulsion system, oxidation of the emulsion system can be delay by multiple mechanisms. KEYWORDS: oxygen scavenger, ascorbic acid, metal-catalyzed oxidation, antioxidant
■
INTRODUCTION L-Ascorbic acid (AA, vitamin C) is a water-soluble vitamin that is a necessary component in human health.1−3 AA has been proposed to be an important antioxidant in both plant and animal tissues by preventing oxidative-induced cellular damage through its ability to scavenge free radicals and regenerate tocopherols.1 In addition, the enediol group of AA on carbons 2 and 3 allows it to oxidize through one- or two-electron transfers, thus making it able to scavenge oxygen4−8 (Figure 1). Preventing lipid oxidation is important for the food industry because it can prolong food-quality properties, such as color, texture, flavor, and nutritive value.9,10 AA offers an attractive antioxidant solution for foods because it satisfies consumer preference for antioxidants from natural sources.8 In foods, AA can inhibit lipid oxidation by acting as a water-soluble free radical scavenger, a synergist to regenerate the activity of other antioxidants (e.g., tocopherol), a means to quench reactive singlet oxygen, and an oxygen scavenger.1,11,12 However, in many situations, it is difficult to determine which of these pathways are important in the antioxidant activity of AA. Further, AA can be prooxidative because it can reduce transition metals, thus increasing their reactivity.13,14 This is especially important at low concentrations, where the capacity of AA to redox cycle metals is more dominant than its free radical scavenging properties.11,15 Therefore, to ensure that AA is antioxidative and not prooxidative, its concentration and factors affecting lipid oxidation, such as transition metal concentration and reactivity, must be considered. Oxygen is a critical factor that can influence the susceptibility of lipid-containing foods to oxidation.2,16,17 Atmospheric triplet © XXXX American Chemical Society
oxygen (present in the headspace and dissolved within foods) is a biradical that can quickly react with lipid radicals at diffusionlimited rates. This reaction produces peroxyl radicals and, upon reaction with another unsaturated fatty acid, produces lipid hydroperoxides. Hydroperoxides are critical in oxidative rancidity because they can decompose, resulting in fatty acid scission, creating low-molecular-weight volatile compounds that are perceived as rancid.18 Thus, one way to decrease lipid oxidation rates is to remove oxygen as a reactant from the food product.16,17 Nowadays, several methods are used to decrease oxygen in food products, such as modified atmosphere packaging, vacuum packaging, flushing with nitrogen, and use of active packaging.19,20 Unfortunately, these techniques do not always achieve desirable levels of oxygen reduction, are not applicable for all foods, and may be cost-prohibitive for certain foods.19−23 In addition, many current, plastic-packaging materials are oxygen-permeable and allow oxygen to reenter the food during storage. An alternative strategy would be to harness the ability of AA to scavenge oxygen and decrease oxygen in a closed food system. AA could potentially be used alone or combined with other oxygen reduction procedures to remove the last portion of oxygen from a food product.19−23 While AA has long been recognized as an oxygen scavenger, very few systematic studies have been conducted to determine its ability to both reduce oxygen concentrations and inhibit Received: November 11, 2014 Revised: January 24, 2015 Accepted: January 26, 2015
A
DOI: 10.1021/jf5053942 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Potential reaction schemes for AA in an emulsion system. homogenizer (M133/1281-0, Biospec Products, Inc., Bartlesville, OK) for 2 min. The coarse emulsion was then homogenized with a microfluidizer (Microfluidics, Newton, MA) at a pressure of 9 kbar for three passes. The MCT O/W emulsion system was used for headspace oxygen analysis, and AA was added to the O/W emulsions at a final concentration ranging from 0.01 to 200 mM. To represent a more realistic food matrix with oxidizable fatty acids, O/W emulsions were prepared using 1.0 wt % commercial soybean oil in 10 mM sodium acetate/imidazole (pH 4.0). Tween 20 was used as an emulsifier at a 1:10 emulsifier/oil ratio. Emulsion preparation was carried out as previously mentioned. The soybean oil O/W system was used to determine lipid oxidation products, and AA was added to the O/W emulsions at a final concentration ranging from 2.5 to 20 mM. Measurement of the Particle Size of Emulsion. The sizes of the emulsion droplets were measured by dynamic light scattering (Zetasizer Nano-ZS, model ZEN3600, Malvern Instruments, Worchester, U.K.). Results were expressed as the Z-average mean diameter. Samples were diluted 50 times with the same buffer as the emulsion, mixed, and immediately measured by transferring the samples into 3 mL plastic cuvettes to determine the size. Measurements were performed on three replicates for each sample at room temperature. The emulsion droplet size averaged 174.1 ± 0.55 nm, and there was no significant change in the droplet size of each emulsion over the course of study (data not shown). In addition, there was no visual observation of creaming during storage in all treatments. Headspace and Dissolved Oxygen (DO) Analysis. To measure headspace oxygen, the MCT O/W emulsion was first mixed with AA solution (final concentration of 0.01−200 mM) and the final emulsion (5 mL) was transferred to 10 mL of gas chromatography (GC) vials, sealed with (tetrafluoroethylene) butyl rubber septa, and then stored at 10, 20, 32, 37, and 45 °C in the dark. Headspace oxygen was determined using a headspace O2/CO2 anaylzer (Benchtop 902D Dualtark, U.S.A.).
lipid oxidation in food systems. These studies are important because AA can act as both an antioxidant and a prooxidant. Oil-in-water (O/W) emulsions, such as salad dressing, beverages, and mayonnaise, are very susceptible to oxidative deterioration24 and represent a food where AA could be added immediately prior to packaging to reduce oxygen concentrations. Therefore, the aim of this study was to investigate the conditions of oxygen reduction by AA in both a buffer system and O/W emulsions. Factors, such as AA concentrations, temperature, and presence of metals, were tested to determine optimum conditions for oxygen reduction. The ability of AA to both decrease oxygen concentrations and inhibit lipid oxidation was determined in O/W emulsions by determining lipid hydroperoxides and headspace hexanal.
■
MATERIALS AND METHODS
Chemicals and Materials. Soybean oil was purchased from a local grocer (Amherst, MA). Medium-chain triacylglycerols (MCTs, Miglyol) were obtained from Sasol North America, Inc. (Houston, TX). The fluorescent dye tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) bis(hexafluorophosphate) complex was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). AA was purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of analytical or chromatographic grade. Emulsion Preparation. The current study used two types of O/W emulsions. First, O/W emulsions were prepared using 1.0 wt % MCT in a 10 mM sodium acetate/imidazole buffer solution (pH 3.0 and 7) to produce emulsions that did not have oxidizable fatty acids. Tween 20 was used as an emulsifier at a 1:10 emulsifier/oil ratio. MCT, Tween 20, and sodium acetate/imidazole were added to a beaker, and a coarse emulsion was made by blending with a hand-held B
DOI: 10.1021/jf5053942 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry The fluorescent dye, tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) bis(hexafluorophosphate) complex, was used to determine DO in the buffer and O/W emulsion system using a λexcitation = 455 nm and a λemission = 615 nm. Fluorescence intensity of the dye increased with a decrease in the oxygen concentration. A stock dye solution was prepared by dissolving 1 mg of the dye in 1 mL of methanol. This stock solution was then added to buffer and soybean oil O/W emulsions to achieve a final dye concentration of 50 μg/g in oil.25 The samples were mixed with AA (0.01−200 mM final concentration in the solution), and the final samples (2 mL) were transferred to 10 mL GC vials, sealed with (tetrafluoroethylene) butyl rubber septa, and stored at 55 °C in the dark. Three vials of each treatment were sampled at each day of analysis. Standard fluorescence was used in the buffer model. Front-face fluorescence was used to measure DO in the O/W emulsion using steady-state measurements recorded with a PTI spectrofluorometer (PTI, London, Ontario, Canada). Spectral bandwidth for both excitation and emission slits was 2.0 nm; integration time was 30 s; and wavelength increment was 2.5 nm. Quantification of DO was calculated according the Stern−Volmer equation.26 Effect of Transition Metals on AA Degradation in Buffer. The ability of transition metals (e.g., copper and iron) to impact the ability of AA to reduce the DO concentration was determined in an acetic acid/imidazole buffer system (10 mM, pH 3). FeSO4 and CuCl solutions were added to the buffer (final concentration of 2.5 or 10 μM) followed by the addition of AA (75 mM final concentration). The final solution (7 mL) was transferred to 10 mL GC vials, sealed with (tetrafluoroethylene) butyl rubber septa, and stored at 32 °C in the dark. Three vials of each treatment were analyzed for DO at each sampling time. Results were expressed as mean values ± standard deviation. Determination of AA Degradation. Quantification of AA was determined directly in the buffer system or in the aqueous phase of the emulsion. The aqueous phase of the emulsified system was obtained by centrifuging at 38518g (17 000 rpm) for 1 h at 4 °C using a Fiberlite F40L-8 × 100 rotor with a high-speed centrifuge (Thermo Scientific WX Ultra 80, Asheville, NC). After the centrifugation, the lower aqueous phase was carefully removed using a syringe. AA was analyzed according the method described by Acar et al., with minor modifications.27 Briefly, 2,6-dichlorofenolindophenol (approximately 15 mg) was dissolved in double-distilled water until the absorbance was 0.800 ± 0.020 at 520 nm. Samples were diluted (1:100) with oxalic acid solution (0.04%, w/v) and mixed with the 2,6dichlorofenolindophenol solution (1:100). The subsequent reaction results in non-oxidized AA reducing the indicator dye to a colorless compound. The absorbance was detected at 520 nm after vortexing for 10 s using an ultraviolet−visible (UV−vis) spectrophotometer (Genesys 20, Thermo Spectronic). AA concentrations were calculated from a standard curve prepared with reagent-grade AA. Results were expressed as mean values ± standard deviation. Measurements of Lipid Oxidation Products. Lipid hydroperoxides and hexanal were measured as primary and secondary lipid oxidation products, respectively. Lipid hydroperoxide formation in emulsion samples was determined according to the method by Shantha and Decker.28 Emulsion samples (0.3 mL) were mixed with 1.5 mL of isooctane/2-propanol solution (3:1, v/v) and vortexed (10 s, 3 times). The mixed solution was centrifuged at 3400g for 10 min (Centrific Centrifuge, Thermo Fisher Scientific, Inc., Fairlawn, NJ). The upper organic layer (200 μL) was mixed with 2.8 mL of methanol/butanol solution (2:1, v/v), followed by the addition of 15 μL of 3.94 M ammonium thiocyanate and 15 μL of Fe2+ solution. The Fe2+ solution was prepared freshly from the supernatant of a mixture of equal amounts of 0.132 M BaCl2 in 0.4 M HCl and 0.144 M FeSO4. The solution was vortexed and held 20 min at room temperature, and the absorbance was measured at 510 nm in a UV−vis spectrophotometer (Genesys 20, Thermo Fisher Scientific, Inc., Waltham, MA). Hydroperoxide concentrations were determined using a standard curve prepared from cumene hydroperoxide. Headspace hexanal was measured according to the method described by Panya et al.29 using a gas chromatograph (model GC-
2014, Shimadzu Co., Tokyo, Japan) equipped with a solid-phase microextraction (SPME) auto injector (model AOC-5000, Shimadzu Co., Tokyo, Japan). Emulsions (1 mL) in 10 mL glass vials capped with aluminum caps with polytetrafluoroethylene (PTFE)/silicone septa were heated at 55 °C for 10 min in the autosampler heating sampler heating block before measurement. A 50/3 μm divinylbenzene (DVB)/carboxen/polydimethylsiloxane (PDMS) stable flex (SPME) fiber (Supelco Co., Bellefonte, PA) was then inserted into the vial headspace for 2 min to absorb volatiles. The fiber was transferred to the GC 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 fused-silica capillary Equity-1 Supelco column (30 × 0.32 mm inner diameter × μm) coated with 100% PDMS at 65 °C for 10 min. A flame ionization detector was used at a temperature of 250 °C. Hexanal concentrations were determined from peak areas using a standard curve made from authentic hexanal. Each measurement was performed in triplicate, and results were expressed as mean values ± standard deviation. Statistical Analysis. All data result were analyzed by analysis of variance (ANOVA) using SPSS 20 (SPSS, Inc., Chicago, IL). The differences between mean values were compared using Duncan’s multiple-range test with a level of significance of p ≤ 0.05.
■
RESULTS AND DISCUSSION Headspace Oxygen Analysis. Headspace oxygen concentrations in MCT O/W emulsions (pH 3 and 7) were only slightly decreased (
