Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Impact of Phospholipids and Tocopherols on the Oxidative Stability of Soybean Oil-in-Water Emulsions Gautam K. Samdani, D. Julian McClements, and Eric A. Decker* Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States S Supporting Information *
ABSTRACT: Phospholipids have been shown to act synergistically with tocopherols and delay lipid oxidation in bulk oil. The synergistic activity between phospholipids and tocopherols is due to the ability of amino-group-containing phospholipids (e.g., phosphatidylethanolamine (PE) and phosphatidylserine (PS)) to convert oxidized tocopherol back into tocopherols. This study shows the effect of PE and PS on the antioxidant activity of different tocopherol homologues in oil-in-water emulsions. Effect of emulsifier type on the interaction between tocopherols and phospholipids was also studied. δ-Tocopherol and PE exhibited greater antioxidant activity as compared to α-tocopherol and PE. PS displayed 1.5−3 times greater synergism than PE with Tween 20 as emulsifier whereas both PE and PS had a similar antioxidant activity in the presence of α-tocopherol when bovine serum albumin was used as the emulsifier. This study is the first to show that PE and PS can act synergistically with tocopherols to inhibit lipid oxidation in oil-in-water emulsions and can present a new clean label antioxidant strategy for food emulsions. KEYWORDS: lipid oxidation, tocopherol, phospholipid, emulsion, synergism, antioxidant antioxidants partition9 into different places in the food (e.g., lipid vs water-soluble free radical scavengers) and thus could scavenge different radicals. In addition, the antioxidant activity of tocopherols can be improved by compounds that regenerate oxidized tocopherols (e.g., tocopherol quinone) to reform the original active free radical scavenging state of the tocopherols. Synergistic activity of different compounds with endogenous tocopherol have been reported by several investigators. For example, Melo et al.10 reported synergism between αtocopherol and acai seed or grape rachi seed extracts, but this synergism was only observed at low tocopherol to plant extract ratios. Carlotti et al.11 reported synergistic activity between certain amino acids and α-tocopherol at pH 5 but not at pH 7 in emulsions and micellar solutions. Phospholipids form an essential part of all biological membranes and hence are found in practically all foods of both plant and animal origin. Phospholipids consists of a glycerol backbone with a phosphate group attached, at the sn-3 position.12 One of the strategies to enhance the activity of endogenous tocopherols includes the use of phosphatidylethanolamine (PE) or phosphatidylserine (PS), two types of phospholipids naturally found in oil that can regenerate oxidized tocopherols. Doert et al.13 reported that PE and PS regenerated α-tocopherol by reacting with α-tocopherol quinone and hence increased the antioxidant activity of tocopherol. Kashima et al.14 found that the antioxidant activity of phospholipids in perilla oil, specifically PE and PS, was due to the presence of tocopherol, whereas phosphatidylcholine (PC) did not enhance the antioxidant activity of tocopherols. Cui et al.15 reported that PC decreased and PE increased the
1. INTRODUCTION Lipid oxidation is one of the major chemical pathways that negatively affect the quality and nutritive value of lipidcontaining foods. Oxidative deterioration of lipids produces volatile compounds that cause off flavors, and oxidation of lipids can decrease nutritive value by accelerating the degradation of vitamins such as vitamins C and E.1,2 Traditionally, synthetic antioxidants like 2,6-di-tert-butyl-phydroxytoluene (BHT), tert-butyl-4-hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ) or synthetic metal chelators like ethylenediaminetetraaceticacid (EDTA) have been used to increase the shelf life of lipid containing products, but there have been debates regarding deleterious health effects of these compounds.3,4 With an increased demand for cleaner food labels, there is a need to identify alternate antioxidant strategies with natural ingredients that inhibit lipid oxidation. Tocopherols are natural antioxidants found abundantly in vegetable oil. The ability of tocopherols to be effective antioxidants in foods is dependent on several factors. Effectiveness is dependent on tocopherol homologue type with mixed tocopherols being more effective than α-tocopherol in foods such as margarines5 and pecan kernels.6 In food oils, the antioxidant activity of tocopherols does not increase linearly as high concentrations will often have diminished activity.7,8 This means that food manufacturers can only add limited amounts of tocopherols to protect their products. Finally, tocopherols protect fatty acids by being preferentially oxidized and forming antioxidant radicals that are not strong prooxidants.8 This means that eventually tocopherols are consumed and their ability to protect the food is lost. The antioxidant activity of tocopherols could be increased when used in combination with other antioxidants. Combined antioxidants could be more effective because the antioxidants have different mechanisms and thus inhibit different oxidation pathways (e.g., free radical scavenger and chelator)7 or because © XXXX American Chemical Society
Received: February 5, 2018 Revised: March 26, 2018 Accepted: March 30, 2018
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DOI: 10.1021/acs.jafc.8b00677 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
emulsion was prepared by adding phospholipids (15.0 μmol/kg of emulsion) dissolved in chloroform with and without tocopherols (3.0 μmol/kg of emulsion) dissolved in methanol into a beaker and flushing with nitrogen gas to remove the solvents. Stripped soybean oil was then added to the beaker and stirred at room temperature for 30 min. Tween 20 and imidazole-acetate buffer were mixed in a separate beaker and then added to the beaker containing stripped soybean oil, and a coarse emulsion was made by blending with a hand-held homogenizer (M133/1281-0, Biospec Products Inc., Bartlesville, OK) for 2 min. The coarse emulsion was then homogenized with a microfluidizer (Microfluidics, Newton, MA, USA) at a pressure of 9 kpsi for three passes. During homogenization, ice was used to cover the homogenizer chamber and coil, to keep the emulsion cold. One milliliter of each emulsion was transferred into 10 mL GC vials (Supelco, Bellefonte, PA), capped with aluminum lids having PTFE/ silicone septa and stored in the dark at 20 °C. 2.3. Evaluation of Particle Size Distributions and Zeta Potential. Samples for droplet size distribution and ζ-potential measurements were prepared by diluting the emulsion 10 times into 10 mM imidazole-acetate buffer, pH 7.0. Both particle size distributions and ζ-potential of the emulsions were analyzed in a ZetaSizer Nano-ZS (Malvern Instruments, Worcestershire, UK).17 The particle size and ζ-potential were determined right after emulsion preparation and at the end of each experiment. Each measurement was repeated three times at room temperature. 2.4. Evaluation of Lipid Oxidation. Formation of primary and secondary (lipid hydroperoxides and hexanal, respectively) lipid oxidation products were quantified to determine the oxidative stability of the O/W emulsions. Lipid hydroperoxides were quantified using a modified version of the method reported by Shantha and Decker.18 Emulsions (0.3 mL) were vortexed three times (10 s each) with 1.5 mL of an isooctane/isopropanol (3:1, v/v) solution. The samples were then centrifuged for 2 min at 3000 rpm (Centrific TM Centrifuge, Fisher Scientific, Fairlawn, NJ), after which 0.2 mL of the upper organic layer was mixed with 2.8 mL of methanol/butanol solution (2:1, v/v), 15 μL of 3.94 M ammonium thiocyanate, and 15 μL of a ferrous iron solution. The ferrous iron solution was prepared by mixing 0.13 M BaCl2 and 0.14 M FeSO4. Twenty minutes after iron addition, the absorbance of the samples was measured at 510 nm, using a Genesys 20 spectrophotometer (Thermo-Spectronic, Waltham, MA). Hydroperoxide concentrations were quantified from a cumene hydroperoxide standard calibration curve. Headspace hexanal was quantified using a method described by Cardenia et al.19 using solid-phase microextraction headspace gas chromatography with flame ionization detection (SPME-GC-FID). The gas chromatograph was a Shimadzu GC-2014 (Shimadzu, Kyoto, Japan) equipped with an AOC-5000 autosampler (Shimadzu) and a split−splitless injector. An Equity DB-1 column (30 m × 0.32 mm × 1 mm film thickness, Supelco, Bellefonte, PA) was used for separation of volatiles. Samples were shaken and heated at 55 °C for 10 min in an autosampler heating block before injection. A 50/30 mm divinylbenzene/carboxen/polydimethylsiloxane SPME fiber needle (Supelco, Bellefonte, PA) was introduced into the vial for 2 min to absorb volatiles and then was transferred to the injector port to allow volatile desorption for 3 min at 250 °C. Oven temperature was 65 °C, and run time was 6 min. The injector and detector temperatures were both set at 250 °C. Helium was used as carrier gas at a flow rate of 1.0 mL/min with a split ratio of 1:7. Hexanal concentrations were determined from peak areas using a calibration curve prepared with hexanal standard solutions. Both lipid hydroperoxides and headspace hexanal were determined on the day of emulsion preparation (day 0) and then every 24 h. 2.5. Interaction Index. To find out if interactions between antioxidants were synergistic, the interaction index was calculated as {(observed lag phase of combination − lag phase of control)/[(lag phase of tocopherol alone − lag phase of control) + (lag phase of phospholipid alone − lag phase of control)]}.10,20,21 An interaction index value of >1 indicates a synergistic interaction between the antioxidants, a value = 1 indicates additive effect, and a value α. The mixed tocopherols contained only 8.5% α-tocopherol making it more surface active than αtocopherol alone, which could explain its higher antioxidant activity. Mixed tocopherols were less surface active than δtocopherol, but the two had similar antioxidant activities with δtocopherol being slightly better. Mixed tocopherols have been postulated to be better antioxidants than individual tocopherols because the mixtures can partition into multiple locations thus placing more antioxidant near sites of free radical generation. This could be why δ-tocopherol was not a dramatically better antioxidant than the mixed tocopherols. Overall, the best antioxidant activity was observed with combinations of the most surface active tocopherols and phospholipids. For example, δ-tocopherol produced longer lag
to 1 day and hexanal lag phase to 2 days. When PE was added with mixed tocopherol in the emulsion, it extended the hydroperoxide lag phase to 5 days and the hexanal lag phase to 7 days, which produced interaction indexes of 1.3 and 1.5, respectively. The combination of PS and mixed tocopherol extended the hydroperoxide lag phase to 4 days and the hexanal lag phase to 5 days (Figure 6), which produced interaction indexes of 1.3 and 2.5, respectively. 3.4. Discussion. In oil-in-water emulsions, nonpolar antioxidants are more effective as defined in the antioxidant polar paradox hypothesis.26 More recently, the best antioxidants in oil-in-water emulsions are thought to be not only nonpolar and retained in the emulsions droplet but also surface active so they partition at the oil−water interface where lipid oxidation is most prevalent. Tocopherol homologues are nonpolar and have no water solubility. The tocopherol homologues vary in surface activity with δ-tocopherol being more surface active than α-tocopherol.22 It has been reported previously that the more surface active tocopherols were more G
DOI: 10.1021/acs.jafc.8b00677 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 6. Formation of lipid hydroperoxides (A) and hexanal (B) in 1% stripped soybean oil-in-water emulsions stabilized with bovine serum albumin (BSA) containing 3.0 μmol of mixed tocopherol/kg of emulsion, 15.0 μmol/kg of emulsion of PE or PS, or both. Each value represents the mean (n = 3) ± standard deviations.
phases for both hydroperoxide and hexanal formation than αtocopherol and the mixed tocopherols in the presence of both PE and PS. The phospholipids are surface active, and this was observed in the reduction of ζ-potential by PS in the Tween 20stabilized oil-in-water emulsion. The ability of PS but not PE to decrease ζ-potential in the Tween emulsions could be due to the great negative charge of PS compared to PE as seen in lipid vesicles29 and the greater negative charge of PS compared to Tween 20 in oil-in-water emulsions at pH 7.0.19,29 PS did not change surface charge in the protein stabilized emulsions. This does not necessary mean that it was not able to concentrate at the surface of the BSA-stabilized emulsions since the BSA and PS could have similar negative charges at pH 7.0. The combination of phospholipids and tocopherols resulted in synergistic antioxidant activity (interaction index >1.0) with the exception of α-tocopherol and PE. Again, the most surface
active tocopherols were the most effective when used in combination with PE and PS. This suggests that the phospholipid and tocopherol combinations were most effective when both concentrated at the emulsion droplet interface. Conversely, the inability of α-tocopherol to produce synergistic activity could be due to its lower surface activity and thus less interactions with phospholipids. Synergistic activity between PS and the tocopherols was greater in the Tween-stabilized emulsions than PE and tocopherols, whereas both had a similar activity in the BSAstabilized emulsions. This again could be due to differences in interfacial concentrations. PS is more negatively charged than PE.29 Since BSA is also negatively charged at pH 7.0, it is possible that charge repulsion could decrease the concentration of PS at the interface decreasing its ability to interact with tocopherols. H
DOI: 10.1021/acs.jafc.8b00677 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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(6) King, C. C., Jr. Effects of antioxidants and modified atmospheres on the storage stability of the pecan kernel (tocopherol, vacuum, nitrogen flush, food quality). Ph.D. Thesis, Texas A&M University, 1986. (7) Naumov, V. V.; Vasilev, R. F. Antioxidant and prooxidant effects of tocopherol. Kinet. Catal. 2003, 44, 101−105. (8) Choe, E.; Min, D. B. Mechanisms of antioxidants in the oxidation of foods. Compr. Rev. Food Sci. Food Saf. 2009, 8, 345−358. (9) Kiralan, S. S.; Doğ u -Baykut, E.; Kittipongpittaya, K.; McClements, D. J.; Decker, E. A. Increased antioxidant efficacy of tocopherols by surfactant solubilization in oil-in-water emulsions. J. Agric. Food Chem. 2014, 62, 10561−10566. (10) Melo, P. S.; Arrivetti, L. d. O. R.; Alencar, S. M. d.; Skibsted, L. H. Antioxidative and prooxidative effects in food lipids and synergism with α-tocopherol of açai ́ seed extracts and grape rachis extracts. Food Chem. 2016, 213, 440−449. (11) Carlotti, M. E.; Gallarate, M.; Gasco, M. R.; Morel, S.; Serafino, A.; Ugazio, E. Synergistic action of vitamin C and amino acids on vitamin E in inhibition of the lipoperoxidation of linoleic acid in disperse systems. Int. J. Pharm. 1997, 155, 251−261. (12) Cui, L.; Decker, E. A. Phospholipids in foods: Prooxidants or antioxidants? J. Sci. Food Agric. 2016, 96, 18−31. (13) Doert, M.; Jaworska, K.; Moersel, J.; Kroh, L. Synergistic effect of lecithins for tocopherols: Lecithin-based regeneration of αtocopherol. Eur. Food Res. Technol. 2012, 235, 915−928. (14) Kashima, M.; Cha, G. S.; Isoda, Y.; Hirano, J.; Miyazawa, T. The antioxidant effects of phospholipids on perilla oil. J. Am. Oil Chem. Soc. 1991, 68, 119−122. (15) Cui, L.; McClements, D. J.; Decker, E. A. Impact of phosphatidylethanolamine on the antioxidant activity of α-tocopherol and trolox in bulk oil. J. Agric. Food Chem. 2015, 63, 3288. (16) Bandarra, N.; Campos, R.; Batista, I.; Nunes, M.; Empis, J. Antioxidant synergy of α-tocopherol and phospholipids. J. Am. Oil Chem. Soc. 1999, 76, 905−913. (17) Johnson, D. R.; Tian, F.; Roman, M. J.; Decker, E. A.; Goddard, J. M. Development of iron-chelating poly(ethylene terephthalate) packaging for inhibiting lipid oxidation in oil-in-water emulsions. J. Agric. Food Chem. 2015, 63, 5055−5060. (18) Shantha, N. C.; Decker, E. A. Rapid, sensitive, iron-based spectrophotometric methods for determination of peroxide values of food lipids. J. AOAC Int. 1994, 77, 421−424. (19) Cardenia, V.; Waraho, T.; Rodriguez-Estrada, M.; Julian McClements, D.; Decker, E. Antioxidant and prooxidant activity behavior of phospholipids in stripped soybean oil-in-water emulsions. J. Am. Oil Chem. Soc. 2011, 88, 1409−1416. (20) Kittipongpittaya, K.; Panya, A.; Phonsatta, N.; Decker, E. A. Effects of environmental pH on antioxidant interactions between rosmarinic acid and α-tocopherol in oil-in-water (O/W) emulsions. J. Agric. Food Chem. 2016, 64, 6575−6583. (21) Peyrat-Maillard, M.; Cuvelier, M.; Berset, C. Antioxidant activity of phenolic compounds in 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidation: Synergistic and antagonistic effects. J. Am. Oil Chem. Soc. 2003, 80, 1007−1012. (22) Chaiyasit, W.; McClements, D. J.; Decker, E. A. The relationship between the physicochemical properties of antioxidants and their ability to inhibit lipid oxidation in bulk oil and oil-in-water emulsions. J. Agric. Food Chem. 2005, 53, 4982−4988. (23) Shahidi, F.; de Camargo, A. Tocopherols and tocotrienols in common and emerging dietary sources: Occurrence, applications, and health benefits. Int. J. Mol. Sci. 2016, 17, 1745−1774. (24) Dubbs, M. D.; Gupta, R. B. Solubility of vitamin E (alphatocopherol) and vitamin K-3 (menadione) in ethanol-water mixture. J. Chem. Eng. Data 1998, 43, 590−591. (25) Berton-Carabin, C. C.; Ropers, M.; Genot, C. Lipid oxidation in oil-in-water emulsions: Involvement of the interfacial layer. Compr. Rev. Food Sci. Food Saf. 2014, 13, 945−977. (26) Porter, W. L.; Black, E. D.; Drolet, A. M. Use of polyamide oxidative fluorescence test on lipid emulsions: Contrast in relative
The synergistic activity between the phospholipids and tocopherols could be due to several factors. Phospholipids have been reported to inhibit lipid oxidation by metal chelation.19,30,31 Chelators can decrease the ability of transition metals to decompose lipid hydroperoxide into free radicals. Decreased production of free radicals will decrease tocopherol degradation, meaning that it can be an effective antioxidant for longer periods of time. PE and PS can also convert the oxidized form of tocopherols, the quinone, back to tocopherol regenerating tocopherols back to their active state.13 Regeneration of α-tocopherol by PE resulted in synergistic antioxidant in bulk oil.13,15 The observation that the synergistic activity of tocopherols and PE and PS combinations was greater with the most surface active tocopherols (e.g., δ-tocopherol and mixed tocopherols) suggests that when both were at the interface, more tocopherol regeneration occurred. While it is difficult to know the exact reasons for the observed synergistic activity, the results of this work suggest that regeneration is involved. This is the first research that shows that the combination of tocopherols and PE or PS produce synergistic antioxidant activity in oil-in-water emulsions. Antioxidant combinations were able to increase the lag phase of lipid oxidation from 1.3to 2.75-fold. This suggests that the combination of tocopherols and PE or PS could be a good clean label antioxidant strategy. Mixed tocopherols would be the most viable commercial strategy since it was more effective than α-tocopherol and δtocopherol is currently too expensive for food applications. The biggest challenge in making this strategy viable in foods will be identifying economical sources of PE and PS. High PE lecithins are commercially available, but they have not been tested for their ability to synergistically interact with tocopherols.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00677. Emulsion droplet size and ζ potential and interaction index (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Eric A. Decker E-mail:
[email protected]. ORCID
Gautam K. Samdani: 0000-0002-2967-5437 D. Julian McClements: 0000-0002-9016-1291 Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.jafc.8b00677 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
