Food Chemistry 167 (2015) 191–196
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Effects of relative humidity on the antioxidant properties of a-tocopherol in stripped corn oil Ji Young Kim, Mi-Ja Kim, BoRa Yi, Sumi Oh, JaeHwan Lee ⇑ Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon, Republic of Korea
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Article history: Received 9 January 2014 Received in revised form 20 June 2014 Accepted 27 June 2014 Available online 5 July 2014 Keywords: a-Tocopherol Relative humidity Antioxidant polar paradox Stripped corn oil
a b s t r a c t The effects of relative humidity (RH) on the antioxidant properties of a-tocopherol (10, 20, 42, and 84 ppm) were determined in stripped corn oil oxidised at 60 °C. The degree of oxidation in oils was determined by analysing headspace oxygen content and conjugated dienoic acids (CDAs). Changes in moisture and a-tocopherol content were also monitored. The oxidative stability of stripped corn oil and stability of a-tocopherol differed significantly depending on the RH. As the concentration of a-tocopherol increased from 10 to 84 ppm, oxidative stability decreased significantly irrespective of RH. The remaining a-tocopherol content decreased as RH increased, suggesting an important role for moisture content in the stability of a-tocopherol. Antioxidant properties of a-tocopherol were greatly influenced by both moisture content in oil and a-tocopherol concentration. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The rate of lipid oxidation depends on the degree of unsaturation of fatty acids, type of oxygen molecules, food matrix type such as oil-in-water (O/W) emulsion or bulk oil, and presence of pro-oxidative metal ions and amphiphilic minor compounds (Chaiyasit, Elias, McClements, & Decker, 2007; Choe & Min, 2006; McClements & Decker, 2000). One of the practical ways to control the rate of lipid oxidation of foods is the addition of antioxidants. Numerous studies in diverse models and real food systems have sought to increase understanding of the mechanisms of and to optimise the conditions of lipid oxidation. Some findings regarding the dependence of antioxidant performance on the type of antioxidant and matrix, including O/W emulsions and bulk oils, have been unexpected (Alamed, Chaiyasit, McClements, & Decker, 2009; Kim, Decker, & Lee, 2012). Lipophilic antioxidants such as tocopherols performed better in O/W emulsions, whereas hydrophilic antioxidants like ascorbic acid performed better in bulk oil systems, referred to as the ‘antioxidant polar paradox’. The antioxidant polar paradox has recently been re-evaluated in O/W emulsions using ‘phenolipids’ (Laguerre et al., 2009, 2010) and in bulk oils according to the concentrations and types of antioxidants (Shahidi & Zhong, 2011). ⇑ Corresponding author. Address: Department of Food Science and Biotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 440746, Republic of Korea. Tel.: +82 31 290 7809; fax: +82 31 290 7882. E-mail address: [email protected] (J. Lee). http://dx.doi.org/10.1016/j.foodchem.2014.06.108 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Shahidi and Zhong (2011) proposed that antioxidant capacities in bulk oils are greatly influenced by the polarity and concentration of antioxidants. Over a certain concentration range, polar compounds such as ascorbic acid and trolox and their corresponding non-polar antioxidants including ascorbyl palmitate and tocopherols show different antioxidant capacities in stripped bulk oils, contrary to expectations based on the antioxidant polar paradox theory. Amphiphilic compounds including free fatty acids (FFAs), diacylglycerols (DAGs), monoacylglycerols (MAGs) and phospholipids (PLs) can migrate in interfacial regions of moisture and triacylglycerols, and form reverse micelles or lamella structures. These association colloids play important roles in the rate of lipid oxidation in bulk oil systems (Chaiyasit et al., 2007; McClements & Decker, 2000; Schwarz et al., 2000). Park, Kim, Kim, and Lee (2014) reported that the rates of lipid oxidation in bulk oils at 100 and 140 °C were higher when carried out under air-tight conditions than non-air-tight conditions up to certain initial period of oxidation. Moisture content was significantly higher in air-tight containers of bulk oils, clearly demonstrating the important role of moisture in bulk oil oxidation. In addition, moisture was shown to actively participate in lipid oxidation at 100 °C. Kim, Kim, and Lee (2014a,b) reported that moisture can participate in the formation of volatile compounds during lipid oxidation of linoleic acid or corn oil in air-tight condition. Despite these insights, the influence of moisture content of stripped bulk oils on the antioxidant capacities of lipophilic compounds has not been reported in the literature. The presence of
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moisture could be a major influencing factor of antioxidant capacity in bulk oils because association colloids may be a major site of lipid oxidation, and active antioxidants are expected to be located near the surface of association colloids. In this study, a-tocopherol was selected as a representing lipophilic antioxidant and moisture content was varied using a saturated salt solution. a-Tocopherol is a representative chain-breaking antioxidant which can donate hydrogen atoms from hydroxyl moieties to peroxyl radicals of lipids (KamaI-Eldin & Appelqvist, 1996). The objective of this study was to determine the effects of relative humidity (RH) and a-tocopherol concentration on the oxidative stability of stripped corn oil. RH was manipulated by using saturated salt solutions and double vials. 2. Materials and methods 2.1. Materials Phosphorus pentoxide, lithium chloride, magnesium chloride, magnesium nitrate, sodium chloride, potassium nitrate and charcoal activated powder were purchased from Daejung Chemical Co. (Seoul, Korea). a-Tocopherol and silicic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Corn oil was purchased from a local grocery market (Suwon, Korea). HPLC grade solvents were purchased from Fisher Scientific (Fairlawn, NJ, USA). Other reagent grade chemicals were obtained from Daejung Chemical Co. (Seoul, Korea). 2.2. Sample preparation A stripping process was performed in corn oil to remove impurities as previously reported by Waraho, Cardenia, RodriguezEstrada, McClements, and Decker (2009). Saturated salt solutions were prepared by dissolving phosphorus pentoxide (RH 0%), lithium chloride (RH 11%), magnesium chloride (RH 32%), magnesium nitrate (RH 52%), sodium chloride (RH 75%), and potassium nitrate (RH 93%) in deionised water. a-Tocopherol was dissolved in n-hexane and added to the stripped oil at a final concentration of 10, 20, 42, and 84 ppm, respectively. n-Hexane solvent was removed under nitrogen gas flow. Three grams of saturated salt solution was placed in each 10-mL bottle. Smaller (1-mL) bottles containing 1 g of stripped corn oil with various concentrations of a-tocopherol were placed inside the 10-mL bottles, which were sealed air-tight with rubber septa and aluminium caps. All sample bottles were incubated at 60 °C in a drying oven (Hysc CO. Ltd, Seoul, Korea) for 62 h. Samples with phosphorus pentoxide (RH 0%), lithium chloride (RH 11%), magnesium chloride (RH 32%), magnesium nitrate (RH 52%), sodium chloride (RH 75%), and potassium nitrate (RH 93%) were labelled PP, LC, MC, MN, SC, and PN, respectively. Samples containing a-tocopherol without addition of salt solution were prepared as control (CON). Samples without addition of a-tocopherol or salt solution were also prepared. The whole experiment was conducted two times. All samples were prepared in triplicate. 2.3. Headspace oxygen analysis Depleted headspace oxygen content can provide information on the degree of oxidation in air-tight samples containing corn oil. The headspace oxygen in air-tight double bottle systems was analysed according to Lee and Min (2009). Headspace oxygen content was withdrawn with an air-tight syringe and 20 lL of headspace gas was analysed in a Hewlett–Packard 7890 gas chromatograph (GC) (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a 60/80 packed column (3.0 m 2 mm ID, Restek Ltd., USA)
and a thermal conductivity detector (TCD) from Agilent Technologies (Palo Alto, CA, USA). The flow rate of helium gas was 20 mL/ min. The temperatures of the oven, injector, and detector were 60, 180, and 180 °C, respectively. 2.4. Conjugated dienoic acid (CDA) analysis CDA was measured according to AOCS (2006) method Ti la-64. 2.5. a-Tocopherol content analysis An oil sample of 0.1 g was directly mixed with 1 mM n-hexane and filtered through a PTFE membrane filter. a-Tocopherol content was analysed by HPLC (Jasco Pu-2089 plus, JASCO International Co. Ltd., Tokyo, Japan) with a fluorescence detector (Jasco Pu-2020 plus, JASCO International Co. Ltd., Tokyo, Japan). The stationary phase was a l-Porasil™ column (3.9 300 mm, 10 lm ID, Waters) and the mobile phase was isocratic n-hexane and isopropanol at a ratio of 99.8–0.2 (v/v) with 2.0 mL/min velocity. The injection volume was 20 lL and the oven temperature was 35 °C. The wavelengths for excitation and emission were 290 and 330 nm, respectively. A calibration curve was constructed from a-tocopherol dissolved in n-hexane. 2.6. Moisture content analysis The moisture content in oils was determined using a coulometric KF titrator (C20, Mettler-Toledo Intl., Columbus, OH, USA). Moisture content in oil samples was analysed according to the manufacturer’s recommendations. 2.7. Statistical analysis Data of headspace oxygen content, CDA, and a-tocopherol content were analysed statistically by ANOVA and Duncan’s multiple range test using SPSS software program (SPSS Inc., Chicago, IL, USA). A p value < 0.05 was considered significant. 3. Results and discussion 3.1. Headspace oxygen content analysis The effects of RH on headspace oxygen content in stripped corn oil with a-tocopherol at 60 °C are shown in Fig. 1. As the concentration of a-tocopherol increased from 10 to 84 ppm, headspace oxygen content decreased significantly irrespective of RH (p < 0.05) (Fig. 1). Headspace oxygen content in the CON were 20.15% and 18.60% for 10 and 84 ppm a-tocopherol, respectively, and 19.64 and 18.56%, respectively, in PP. For SC, headspace oxygen content in samples containing 10 and 84 ppm a-tocopherol were 19.79 and 17.73%, respectively. As the degree of lipid oxidation increased, the depleted headspace oxygen content increased. The highest antioxidant capacity based on headspace oxygen was found for 10 ppm a-tocopherol, followed by 20, 42, and 84 ppm a-tocopherol in decreasing order. A preliminary study showed that the headspace oxygen of stripped oil without addition of a-tocopherol and salt solution was 18.21 ± 0.10% (n = 3) under the same oxidation condition, which implied that 10 ppm a-tocopherol acted as an antioxidant whereas 84 ppm a-tocopherol accelerated the consumption of oxygen by oils. Pro-oxidant properties of high concentrations of a-tocopherol in bulk oils have been reported in the literature (Frankel, Huang, Kanner, & German, 1994; Jerzykiewicz, C´wiela˛gPiasecka, & Jezierski, 2013; Naumov & Vasil’ev, 2003; Ouchi, Ishikura, Konishi, Nagaoka, & Mukai, 2009; Zuta, Simpson, Zhao, & Leclerc, 2007). Zuta et al. (2007) reported that 250 and 500 ppm
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20.5 10 ppm
Headspace oxygen content (%)
a
20.0
ab
20 ppm a
84 ppm
42 ppm b
c cde
e fg
cd
fg
fgh
19.5
cde
de
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i
i
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PP LC MC MN SC PN (RH 0%) (RH 11%) (RH 32%) (RH 52%) (RH 75%) (RH 93%)
Fig. 1. Changes of headspace oxygen content in stripped corn oil containing a-tocopherol (10–84 ppm) at 60 °C with different relative humidity.
a-tocopherol were less effective in controlling oxidation in mackerel oils than 50 and 100 ppm a-tocopherol. Frankel et al. (1994) showed that a low concentration of a-tocopherol (232 lM) acted as an antioxidant whereas a high concentration (1161 lM) accelerated the formation of hydroperoxides in bulk corn oil at 60 °C. The antioxidant properties of a-tocopherol are influenced by RH. Oils with 10 and 20 ppm a-tocopherol showed the highest oxidative stability for LC and MC whereas those with 42 and 84 ppm a-tocopherol showed the highest antioxidant capacities for LC, and for both PP and LC, respectively. Therefore, the oxidative stability of oils containing a-tocopherol was highly dependent on the RH. Interestingly, a relatively low concentration of a-tocopherol (10 ppm) showed the strong antioxidant properties in the middle ranges of RH while higher concentration of a-tocopherol (42– 84 ppm) showed better antioxidant capacities at a lower compared to a middle range of RH. This finding is very interesting and important for the control of oxidative stability of oils. Generally, control samples (CON) which contained a-tocopherol and deionised moisture without salt, showed similar oxidative stability to LC samples and higher oxidative stability than MN, SC, and PN. 3.2. CDA analysis The change of CDA in stripped corn oil with a-tocopherol stored at 60 °C are shown in Fig. 2. Oils containing 84 ppm a-tocopherol had significantly higher CDA values than those containing 10, 20, and 42 ppm a-tocopherol regardless of RH (p < 0.05). CDA varied for different RH values at the same concentration of a-tocopherol. Oils under SC, MN, and PN had higher CDA values ranging from 20 to 84 ppm than oils under PP, LC, and MC. Oils under low RH generally had lower CDA values. The oxidative stability patterns from the CDA results were a little different from those determined during measurements of headspace oxygen content (Fig. 1). Stripped oil without addition of a-tocopherol and salt solution had a CDA value of 0.90 ± 0.07% (n = 3), which was substantially higher than samples containing a-tocopherol ranging from 10 to 42 ppm. Similar to the results of analysis of headspace oxygen content, addition of a-tocopherol significantly decreased CDA values. However, some samples containing 84 ppm a-tocopherol showed higher CDA values under MC, MN, SC and PN. The CON generally had higher oxidative stability than samples under SC and PN (Fig. 2).
CDA is one of the assays for determining the content of primary oxidation products from polyunsaturated fatty acids and has been frequently used in bulk oil systems (Lee, Chung, Chang, & Lee, 2007). 3.3. a-Tocopherol content analysis The remaining a-tocopherol in stripped corn oil stored at 60 °C under diverse RH conditions are shown in Fig. 3. The stability of a-tocopherol is critical for the oxidative stability of oils. Generally, as RH increased from PP to PN, less a-tocopherol remained (Fig. 3). In the case of 84 ppm a-tocopherol, LC and PN had significantly higher content of remaining a-tocopherol than other RH conditions (p < 0.05), whereas the content was the lowest under MN, SC, and PN, which implied that stability of a-tocopherol depended on the moisture content of oils. Generally, the moisture content of oils was inversely related to the amount of a-tocopherol that remained. This trend was observed for 20 and 42 ppm a-tocopherol. Significantly more a-tocopherol remained under PP than under other conditions at 20 and 42 ppm a-tocopherol (p < 0.05). Interestingly, no a-tocopherol was detected after storage for a starting concentration of 10 ppm, which may be due to the depletion of a-tocopherol to prevent a free radical chain reaction during autoxidation. CON with 42 and 84 ppm a-tocopherol had a similar remaining content of a-tocopherol to PP and LC, and higher a-tocopherol content than samples under MC, MN, SC and PN (Fig. 3). Considering the high oxidative stability of 10 ppm a-tocopherol based on the results of headspace oxygen content (Fig. 1) and CDA (Fig. 2), 10 ppm a-tocopherol may be enough to protect lipid oxidation. More than 10 ppm a-tocopherol was depleted in samples initially containing 42 and 84 ppm a-tocopherol. For example, the depleted a-tocopherol content in samples containing 84 ppm a-tocopherol under PP, LC, MC, MN, SC, and PN were 24, 22, 34, 45, 47, and 47 ppm, respectively (Fig. 3). Therefore, part of the a-tocopherol was used as a chain breaking antioxidant and another part may have been used to accelerate the consumption of headspace oxygen and formation of conjugated dienes. Decomposition of a-tocopherol under relatively high RH may be closely related to the iron concentration and type (Chen, Panya, McClements, & Decker, 2012). Chen et al. (2012) showed that ferric ion (Fe3+) greatly increases the decomposition of a-tocopherol in a medium chain triacylglycerol model system whereas a-tocopherol in the presence of ferrous ion (Fe2+) had a
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1.4 10 ppm
20 ppm
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Conjugated dienoic acid (%)
1.2 b
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SC (RH 75%)
PN (RH 93%)
i
0.2
0.0 CON
PP (RH 0%)
LC (RH 11%)
MC (RH 32%)
MN (RH 52%)
Fig. 2. Changes of CDA in stripped corn oil containing a-tocopherol (10–84 ppm) at 60 °C with different relative humidity.
80.0
Concentration of α -tocopherol (ppm)
20 ppm
42 ppm
84 ppm
a
70.0 a
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60.0 b
50.0 c
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fg
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0.0 CON
PP (RH 0%)
LC (RH 11%)
MC (RH 32%)
MN (RH 52%)
SC (RH 75%)
PN (RH 93%)
Fig. 3. Changes of a-tocopherol in stripped corn oil containing a-tocopherol (10–84 ppm) at 60 °C with different relative humidity.
higher stability at the same conditions. The instability of a-tocopherol in high RH could be due to the reduction of ferric ion (Fe3+) into ferrous ion (Fe2+) by a-tocopherol. 3.4. Effect of concentration and RH on the antioxidant properties of a-tocopherol The effect of a-tocopherol concentration (10–84 ppm) and RH on the antioxidant or pro-oxidant properties of a-tocopherol are shown in Fig. 4. Values measured for stripped corn oil with the addition of a-tocopherol and salt solution were divided by those of stripped oil without addition of a-tocopherol or salt solution. Antioxidative or even pro-oxidative properties of a-tocopherol depended on its concentration and the RH, based on headspace oxygen content (Fig. 4-a) and CDA value (Fig. 4b). For the headspace oxygen content, points over 100% indicate that the oxidative stability of samples were higher than those of control samples because more headspace oxygen was remained in samples by the
antioxidant properties of a-tocopherol. In case of CDA assay, points over 100% indicate more formation of conjugated dienes in samples, which implies that a-tocopherol acted prooxidantly. At 84 ppm and RH under that of MC, a-tocopherol had antioxidant properties, while pro-oxidant properties were observed for RH over MN, based on headspace oxygen content (Fig. 4a). However, CDA results revealed that 84 ppm a-tocopherol showed prooxidative properties at RH over PP (Fig. 4-b). In the range of 10–42 ppm, a-tocopherol showed antioxidant properties irrespective of RH. It is clearly shown that the same concentration of a-tocopherol (84 ppm) had antioxidant or prooxidant properties in stripped oils depending on RH (Fig. 4). Also, antioxidant properties of 10 ppm a-tocopherol were higher than those of 20 and 42 ppm a-tocopherol in stripped oils, which was higher positions in headspace oxygen content (Fig. 4a) and lower positions in CDA values (Fig. 4b). Interestingly, samples under controlled RH conditions from PP to PN had lower oxidative stabilities than control samples with
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2500
(a)
a
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2000 Moisture content (ppm)
Ratio percentage of headspace oxygen content (%)
112
104
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96 10 ppm
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92 PP (RH 0%)
LC (RH 11%)
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SC (RH 75%)
PN (RH 93%)
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Ratio percentage of conjugated dienoic acid (%)
140
CON
(b)
PP
LC
MC
MN
SC
PN
Fig. 5. Moisture content in stripped corn oil containing 84 ppm a-tocopherol at 60 °C after 62-h treatment under different relative humidity.
120 100 80 60 40 20
10 ppm
20 ppm
42 ppm
84 ppm
0 PP (RH 0%)
LC MC MN SC PN (RH 11%) (RH 32%) (RH 52%) (RH 75%) (RH 93%)
Fig. 4. Antioxidative or prooxidative properties of a-tocopherol in stripped corn oil under diverse relative humidity based on headspace oxygen content (a) and conjugated dienoic acid (b) analysis.
addition of deionised water only. Forced conditions to equilibrate for RH could thus be an important factor to consider. For samples at low RH, for example, PP, moisture may be forced to evaporate continuously from oils into the headspace while for those at high RH, like PN, the reverse flux may be found from headspace to oils. Many studies have adapted saturated salt solution as a tool to provide adequate RH to determine the effects of RH and oxidative stability of foods (Gopalakrishna & Prabhakar, 1983; Partanen et al., 2008; Velasco, Holgado, Dobarganes, & Márquez-Ruiz, 2009). However, it is possible that oxidative stability under RH controlled by saturated salt solutions may not reflect the real situation without forced control of RH, and these results should be interpreted with caution. Moisture content in corn oil containing 84 ppm a-tocopherol at 60 °C after 62-h treatment under different RH conditions is shown in Fig. 5. Samples with 10, 20, and 42 ppm a-tocopherol were not determined. As expected, moisture content in PN was the highest, followed in decreasing order by SC, MN, MC, LC, and PP. Moisture content in stripped corn oils under low RH, PP and LC, were 110 and 304 ppm, respectively, whereas those under high RH, SC and PN, were 1389 and 1912 ppm, respectively. Moisture content in CON samples containing tocopherol without addition of water was 606 ppm, which was not significantly different from those in MC (p > 0.05). In addition, moisture content in samples without addition of a-tocopherol and salt solution were not significantly different from those in CON and MC (data not shown). It is clearly confirmed that antioxidant properties of a-tocopherol are greatly influenced by the concentration of a-tocopherol and RH. Although the actual RH of current experimental conditions was not determined, moisture content in oils was determined
using the Karl Fisher method as shown in Fig. 5. Higher moisture content in PN can generate more association colloids in oils, which may provide more places for lipid oxidation. a-Tocopherols have amphiphilic properties due to their phytol chain with 16 carbons and chromanol ring structure. a-Tocopherol may participate in formation of association colloids. Increase in mobility and activity in transition metals could also be a major factor. Chen et al. (2012) reported that iron is a major pro-oxidant in bulk oils. Ferric ion (Fe3+) may enhance the decomposition of a-tocopherol under high RH and an increase in ferrous ion (Fe2+), which can be formed by reduction of ferric ion (Fe3+) by a-tocopherol, can accelerate the rate of lipid oxidation. At low RH conditions, different forces could be driving oxidation compared to high RH, perhaps depending on the number of association colloids. Oxygen flux from headspace to oils could be one of the possible forces driving oxidation at low RH due to the low moisture content in oils. This speculation should be tested in the future. There are several possible theories for the pro-oxidant properties of a-tocopherol at higher concentrations (KamaI-Eldin & Appelqvist, 1996; Naumov & Vasil’ev, 2003). Tocopheroxyl radicals from tocopherols can attack lipids and lipid hydroperoxides to generate lipid radicals and peroxyl radicals, respectively (Naumov & Vasil’ev, 2003). Tocopherols and tocopheroxyl radicals may also reduce metal ions, which could result in more active metal states for pro-oxidant actions through Fenton (or Fenton-like) reactions. Extra tocopherols may be located on the surface of oils, which may help the migration of oxygen into oils (KamaI-Eldin & Appelqvist, 1996). Further research is needed to confirm the proposed pro-oxidative mechanisms of tocopherols in oils.
4. Conclusion The effects of a-tocopherol concentration and RH on the oxidative stability of stripped corn oils were tested. The same concentration of a-tocopherol showed different oxidative stabilities in stripped corn oils under different RH conditions. Generally, lower RH was associated with higher oxidative stability. In addition, a high concentration of a-tocopherol (84 ppm) showed pro-oxidative properties compared to a low concentration (10 ppm) in stripped corn oils. Our findings clearly demonstrate that both concentration and moisture content are important for antioxidant properties of lipophilic antioxidants in stripped corn oil systems.
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Effects of relative humidity on the antioxidant properties of a-tocopherol in stripped corn oil Ji Young Kim, Mi-Ja Kim, BoRa Yi, Sumi Oh, JaeHwan Lee ⇑ Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon, Republic of Korea
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Article history: Received 9 January 2014 Received in revised form 20 June 2014 Accepted 27 June 2014 Available online 5 July 2014 Keywords: a-Tocopherol Relative humidity Antioxidant polar paradox Stripped corn oil
a b s t r a c t The effects of relative humidity (RH) on the antioxidant properties of a-tocopherol (10, 20, 42, and 84 ppm) were determined in stripped corn oil oxidised at 60 °C. The degree of oxidation in oils was determined by analysing headspace oxygen content and conjugated dienoic acids (CDAs). Changes in moisture and a-tocopherol content were also monitored. The oxidative stability of stripped corn oil and stability of a-tocopherol differed significantly depending on the RH. As the concentration of a-tocopherol increased from 10 to 84 ppm, oxidative stability decreased significantly irrespective of RH. The remaining a-tocopherol content decreased as RH increased, suggesting an important role for moisture content in the stability of a-tocopherol. Antioxidant properties of a-tocopherol were greatly influenced by both moisture content in oil and a-tocopherol concentration. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The rate of lipid oxidation depends on the degree of unsaturation of fatty acids, type of oxygen molecules, food matrix type such as oil-in-water (O/W) emulsion or bulk oil, and presence of pro-oxidative metal ions and amphiphilic minor compounds (Chaiyasit, Elias, McClements, & Decker, 2007; Choe & Min, 2006; McClements & Decker, 2000). One of the practical ways to control the rate of lipid oxidation of foods is the addition of antioxidants. Numerous studies in diverse models and real food systems have sought to increase understanding of the mechanisms of and to optimise the conditions of lipid oxidation. Some findings regarding the dependence of antioxidant performance on the type of antioxidant and matrix, including O/W emulsions and bulk oils, have been unexpected (Alamed, Chaiyasit, McClements, & Decker, 2009; Kim, Decker, & Lee, 2012). Lipophilic antioxidants such as tocopherols performed better in O/W emulsions, whereas hydrophilic antioxidants like ascorbic acid performed better in bulk oil systems, referred to as the ‘antioxidant polar paradox’. The antioxidant polar paradox has recently been re-evaluated in O/W emulsions using ‘phenolipids’ (Laguerre et al., 2009, 2010) and in bulk oils according to the concentrations and types of antioxidants (Shahidi & Zhong, 2011). ⇑ Corresponding author. Address: Department of Food Science and Biotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 440746, Republic of Korea. Tel.: +82 31 290 7809; fax: +82 31 290 7882. E-mail address: [email protected] (J. Lee). http://dx.doi.org/10.1016/j.foodchem.2014.06.108 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Shahidi and Zhong (2011) proposed that antioxidant capacities in bulk oils are greatly influenced by the polarity and concentration of antioxidants. Over a certain concentration range, polar compounds such as ascorbic acid and trolox and their corresponding non-polar antioxidants including ascorbyl palmitate and tocopherols show different antioxidant capacities in stripped bulk oils, contrary to expectations based on the antioxidant polar paradox theory. Amphiphilic compounds including free fatty acids (FFAs), diacylglycerols (DAGs), monoacylglycerols (MAGs) and phospholipids (PLs) can migrate in interfacial regions of moisture and triacylglycerols, and form reverse micelles or lamella structures. These association colloids play important roles in the rate of lipid oxidation in bulk oil systems (Chaiyasit et al., 2007; McClements & Decker, 2000; Schwarz et al., 2000). Park, Kim, Kim, and Lee (2014) reported that the rates of lipid oxidation in bulk oils at 100 and 140 °C were higher when carried out under air-tight conditions than non-air-tight conditions up to certain initial period of oxidation. Moisture content was significantly higher in air-tight containers of bulk oils, clearly demonstrating the important role of moisture in bulk oil oxidation. In addition, moisture was shown to actively participate in lipid oxidation at 100 °C. Kim, Kim, and Lee (2014a,b) reported that moisture can participate in the formation of volatile compounds during lipid oxidation of linoleic acid or corn oil in air-tight condition. Despite these insights, the influence of moisture content of stripped bulk oils on the antioxidant capacities of lipophilic compounds has not been reported in the literature. The presence of
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moisture could be a major influencing factor of antioxidant capacity in bulk oils because association colloids may be a major site of lipid oxidation, and active antioxidants are expected to be located near the surface of association colloids. In this study, a-tocopherol was selected as a representing lipophilic antioxidant and moisture content was varied using a saturated salt solution. a-Tocopherol is a representative chain-breaking antioxidant which can donate hydrogen atoms from hydroxyl moieties to peroxyl radicals of lipids (KamaI-Eldin & Appelqvist, 1996). The objective of this study was to determine the effects of relative humidity (RH) and a-tocopherol concentration on the oxidative stability of stripped corn oil. RH was manipulated by using saturated salt solutions and double vials. 2. Materials and methods 2.1. Materials Phosphorus pentoxide, lithium chloride, magnesium chloride, magnesium nitrate, sodium chloride, potassium nitrate and charcoal activated powder were purchased from Daejung Chemical Co. (Seoul, Korea). a-Tocopherol and silicic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Corn oil was purchased from a local grocery market (Suwon, Korea). HPLC grade solvents were purchased from Fisher Scientific (Fairlawn, NJ, USA). Other reagent grade chemicals were obtained from Daejung Chemical Co. (Seoul, Korea). 2.2. Sample preparation A stripping process was performed in corn oil to remove impurities as previously reported by Waraho, Cardenia, RodriguezEstrada, McClements, and Decker (2009). Saturated salt solutions were prepared by dissolving phosphorus pentoxide (RH 0%), lithium chloride (RH 11%), magnesium chloride (RH 32%), magnesium nitrate (RH 52%), sodium chloride (RH 75%), and potassium nitrate (RH 93%) in deionised water. a-Tocopherol was dissolved in n-hexane and added to the stripped oil at a final concentration of 10, 20, 42, and 84 ppm, respectively. n-Hexane solvent was removed under nitrogen gas flow. Three grams of saturated salt solution was placed in each 10-mL bottle. Smaller (1-mL) bottles containing 1 g of stripped corn oil with various concentrations of a-tocopherol were placed inside the 10-mL bottles, which were sealed air-tight with rubber septa and aluminium caps. All sample bottles were incubated at 60 °C in a drying oven (Hysc CO. Ltd, Seoul, Korea) for 62 h. Samples with phosphorus pentoxide (RH 0%), lithium chloride (RH 11%), magnesium chloride (RH 32%), magnesium nitrate (RH 52%), sodium chloride (RH 75%), and potassium nitrate (RH 93%) were labelled PP, LC, MC, MN, SC, and PN, respectively. Samples containing a-tocopherol without addition of salt solution were prepared as control (CON). Samples without addition of a-tocopherol or salt solution were also prepared. The whole experiment was conducted two times. All samples were prepared in triplicate. 2.3. Headspace oxygen analysis Depleted headspace oxygen content can provide information on the degree of oxidation in air-tight samples containing corn oil. The headspace oxygen in air-tight double bottle systems was analysed according to Lee and Min (2009). Headspace oxygen content was withdrawn with an air-tight syringe and 20 lL of headspace gas was analysed in a Hewlett–Packard 7890 gas chromatograph (GC) (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a 60/80 packed column (3.0 m 2 mm ID, Restek Ltd., USA)
and a thermal conductivity detector (TCD) from Agilent Technologies (Palo Alto, CA, USA). The flow rate of helium gas was 20 mL/ min. The temperatures of the oven, injector, and detector were 60, 180, and 180 °C, respectively. 2.4. Conjugated dienoic acid (CDA) analysis CDA was measured according to AOCS (2006) method Ti la-64. 2.5. a-Tocopherol content analysis An oil sample of 0.1 g was directly mixed with 1 mM n-hexane and filtered through a PTFE membrane filter. a-Tocopherol content was analysed by HPLC (Jasco Pu-2089 plus, JASCO International Co. Ltd., Tokyo, Japan) with a fluorescence detector (Jasco Pu-2020 plus, JASCO International Co. Ltd., Tokyo, Japan). The stationary phase was a l-Porasil™ column (3.9 300 mm, 10 lm ID, Waters) and the mobile phase was isocratic n-hexane and isopropanol at a ratio of 99.8–0.2 (v/v) with 2.0 mL/min velocity. The injection volume was 20 lL and the oven temperature was 35 °C. The wavelengths for excitation and emission were 290 and 330 nm, respectively. A calibration curve was constructed from a-tocopherol dissolved in n-hexane. 2.6. Moisture content analysis The moisture content in oils was determined using a coulometric KF titrator (C20, Mettler-Toledo Intl., Columbus, OH, USA). Moisture content in oil samples was analysed according to the manufacturer’s recommendations. 2.7. Statistical analysis Data of headspace oxygen content, CDA, and a-tocopherol content were analysed statistically by ANOVA and Duncan’s multiple range test using SPSS software program (SPSS Inc., Chicago, IL, USA). A p value < 0.05 was considered significant. 3. Results and discussion 3.1. Headspace oxygen content analysis The effects of RH on headspace oxygen content in stripped corn oil with a-tocopherol at 60 °C are shown in Fig. 1. As the concentration of a-tocopherol increased from 10 to 84 ppm, headspace oxygen content decreased significantly irrespective of RH (p < 0.05) (Fig. 1). Headspace oxygen content in the CON were 20.15% and 18.60% for 10 and 84 ppm a-tocopherol, respectively, and 19.64 and 18.56%, respectively, in PP. For SC, headspace oxygen content in samples containing 10 and 84 ppm a-tocopherol were 19.79 and 17.73%, respectively. As the degree of lipid oxidation increased, the depleted headspace oxygen content increased. The highest antioxidant capacity based on headspace oxygen was found for 10 ppm a-tocopherol, followed by 20, 42, and 84 ppm a-tocopherol in decreasing order. A preliminary study showed that the headspace oxygen of stripped oil without addition of a-tocopherol and salt solution was 18.21 ± 0.10% (n = 3) under the same oxidation condition, which implied that 10 ppm a-tocopherol acted as an antioxidant whereas 84 ppm a-tocopherol accelerated the consumption of oxygen by oils. Pro-oxidant properties of high concentrations of a-tocopherol in bulk oils have been reported in the literature (Frankel, Huang, Kanner, & German, 1994; Jerzykiewicz, C´wiela˛gPiasecka, & Jezierski, 2013; Naumov & Vasil’ev, 2003; Ouchi, Ishikura, Konishi, Nagaoka, & Mukai, 2009; Zuta, Simpson, Zhao, & Leclerc, 2007). Zuta et al. (2007) reported that 250 and 500 ppm
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20.5 10 ppm
Headspace oxygen content (%)
a
20.0
ab
20 ppm a
84 ppm
42 ppm b
c cde
e fg
cd
fg
fgh
19.5
cde
de
cde
f gh h
i
i
19.0
j
j k
k k l
18.5
18.0
m m n
17.5
17.0
CON
PP LC MC MN SC PN (RH 0%) (RH 11%) (RH 32%) (RH 52%) (RH 75%) (RH 93%)
Fig. 1. Changes of headspace oxygen content in stripped corn oil containing a-tocopherol (10–84 ppm) at 60 °C with different relative humidity.
a-tocopherol were less effective in controlling oxidation in mackerel oils than 50 and 100 ppm a-tocopherol. Frankel et al. (1994) showed that a low concentration of a-tocopherol (232 lM) acted as an antioxidant whereas a high concentration (1161 lM) accelerated the formation of hydroperoxides in bulk corn oil at 60 °C. The antioxidant properties of a-tocopherol are influenced by RH. Oils with 10 and 20 ppm a-tocopherol showed the highest oxidative stability for LC and MC whereas those with 42 and 84 ppm a-tocopherol showed the highest antioxidant capacities for LC, and for both PP and LC, respectively. Therefore, the oxidative stability of oils containing a-tocopherol was highly dependent on the RH. Interestingly, a relatively low concentration of a-tocopherol (10 ppm) showed the strong antioxidant properties in the middle ranges of RH while higher concentration of a-tocopherol (42– 84 ppm) showed better antioxidant capacities at a lower compared to a middle range of RH. This finding is very interesting and important for the control of oxidative stability of oils. Generally, control samples (CON) which contained a-tocopherol and deionised moisture without salt, showed similar oxidative stability to LC samples and higher oxidative stability than MN, SC, and PN. 3.2. CDA analysis The change of CDA in stripped corn oil with a-tocopherol stored at 60 °C are shown in Fig. 2. Oils containing 84 ppm a-tocopherol had significantly higher CDA values than those containing 10, 20, and 42 ppm a-tocopherol regardless of RH (p < 0.05). CDA varied for different RH values at the same concentration of a-tocopherol. Oils under SC, MN, and PN had higher CDA values ranging from 20 to 84 ppm than oils under PP, LC, and MC. Oils under low RH generally had lower CDA values. The oxidative stability patterns from the CDA results were a little different from those determined during measurements of headspace oxygen content (Fig. 1). Stripped oil without addition of a-tocopherol and salt solution had a CDA value of 0.90 ± 0.07% (n = 3), which was substantially higher than samples containing a-tocopherol ranging from 10 to 42 ppm. Similar to the results of analysis of headspace oxygen content, addition of a-tocopherol significantly decreased CDA values. However, some samples containing 84 ppm a-tocopherol showed higher CDA values under MC, MN, SC and PN. The CON generally had higher oxidative stability than samples under SC and PN (Fig. 2).
CDA is one of the assays for determining the content of primary oxidation products from polyunsaturated fatty acids and has been frequently used in bulk oil systems (Lee, Chung, Chang, & Lee, 2007). 3.3. a-Tocopherol content analysis The remaining a-tocopherol in stripped corn oil stored at 60 °C under diverse RH conditions are shown in Fig. 3. The stability of a-tocopherol is critical for the oxidative stability of oils. Generally, as RH increased from PP to PN, less a-tocopherol remained (Fig. 3). In the case of 84 ppm a-tocopherol, LC and PN had significantly higher content of remaining a-tocopherol than other RH conditions (p < 0.05), whereas the content was the lowest under MN, SC, and PN, which implied that stability of a-tocopherol depended on the moisture content of oils. Generally, the moisture content of oils was inversely related to the amount of a-tocopherol that remained. This trend was observed for 20 and 42 ppm a-tocopherol. Significantly more a-tocopherol remained under PP than under other conditions at 20 and 42 ppm a-tocopherol (p < 0.05). Interestingly, no a-tocopherol was detected after storage for a starting concentration of 10 ppm, which may be due to the depletion of a-tocopherol to prevent a free radical chain reaction during autoxidation. CON with 42 and 84 ppm a-tocopherol had a similar remaining content of a-tocopherol to PP and LC, and higher a-tocopherol content than samples under MC, MN, SC and PN (Fig. 3). Considering the high oxidative stability of 10 ppm a-tocopherol based on the results of headspace oxygen content (Fig. 1) and CDA (Fig. 2), 10 ppm a-tocopherol may be enough to protect lipid oxidation. More than 10 ppm a-tocopherol was depleted in samples initially containing 42 and 84 ppm a-tocopherol. For example, the depleted a-tocopherol content in samples containing 84 ppm a-tocopherol under PP, LC, MC, MN, SC, and PN were 24, 22, 34, 45, 47, and 47 ppm, respectively (Fig. 3). Therefore, part of the a-tocopherol was used as a chain breaking antioxidant and another part may have been used to accelerate the consumption of headspace oxygen and formation of conjugated dienes. Decomposition of a-tocopherol under relatively high RH may be closely related to the iron concentration and type (Chen, Panya, McClements, & Decker, 2012). Chen et al. (2012) showed that ferric ion (Fe3+) greatly increases the decomposition of a-tocopherol in a medium chain triacylglycerol model system whereas a-tocopherol in the presence of ferrous ion (Fe2+) had a
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1.4 10 ppm
20 ppm
84 ppm
42 ppm
Conjugated dienoic acid (%)
1.2 b
ab
a ab
1.0
c c
0.8
d
e ef
fef
ef
0.6
d
d d
d d
d
ef ef
g hi
hi
0.4
i
hi
h
h
SC (RH 75%)
PN (RH 93%)
i
0.2
0.0 CON
PP (RH 0%)
LC (RH 11%)
MC (RH 32%)
MN (RH 52%)
Fig. 2. Changes of CDA in stripped corn oil containing a-tocopherol (10–84 ppm) at 60 °C with different relative humidity.
80.0
Concentration of α -tocopherol (ppm)
20 ppm
42 ppm
84 ppm
a
70.0 a
a
60.0 b
50.0 c
40.0
30.0
20.0
d
fg
de f
de f
c
c
e fg
fg
fg
fg
fg g
g
10.0
0.0 CON
PP (RH 0%)
LC (RH 11%)
MC (RH 32%)
MN (RH 52%)
SC (RH 75%)
PN (RH 93%)
Fig. 3. Changes of a-tocopherol in stripped corn oil containing a-tocopherol (10–84 ppm) at 60 °C with different relative humidity.
higher stability at the same conditions. The instability of a-tocopherol in high RH could be due to the reduction of ferric ion (Fe3+) into ferrous ion (Fe2+) by a-tocopherol. 3.4. Effect of concentration and RH on the antioxidant properties of a-tocopherol The effect of a-tocopherol concentration (10–84 ppm) and RH on the antioxidant or pro-oxidant properties of a-tocopherol are shown in Fig. 4. Values measured for stripped corn oil with the addition of a-tocopherol and salt solution were divided by those of stripped oil without addition of a-tocopherol or salt solution. Antioxidative or even pro-oxidative properties of a-tocopherol depended on its concentration and the RH, based on headspace oxygen content (Fig. 4-a) and CDA value (Fig. 4b). For the headspace oxygen content, points over 100% indicate that the oxidative stability of samples were higher than those of control samples because more headspace oxygen was remained in samples by the
antioxidant properties of a-tocopherol. In case of CDA assay, points over 100% indicate more formation of conjugated dienes in samples, which implies that a-tocopherol acted prooxidantly. At 84 ppm and RH under that of MC, a-tocopherol had antioxidant properties, while pro-oxidant properties were observed for RH over MN, based on headspace oxygen content (Fig. 4a). However, CDA results revealed that 84 ppm a-tocopherol showed prooxidative properties at RH over PP (Fig. 4-b). In the range of 10–42 ppm, a-tocopherol showed antioxidant properties irrespective of RH. It is clearly shown that the same concentration of a-tocopherol (84 ppm) had antioxidant or prooxidant properties in stripped oils depending on RH (Fig. 4). Also, antioxidant properties of 10 ppm a-tocopherol were higher than those of 20 and 42 ppm a-tocopherol in stripped oils, which was higher positions in headspace oxygen content (Fig. 4a) and lower positions in CDA values (Fig. 4b). Interestingly, samples under controlled RH conditions from PP to PN had lower oxidative stabilities than control samples with
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2500
(a)
a
108
2000 Moisture content (ppm)
Ratio percentage of headspace oxygen content (%)
112
104
100
96 10 ppm
20 ppm
42 ppm
84 ppm
b
1500 c
1000 d
d e
500
92 PP (RH 0%)
LC (RH 11%)
MC (RH 32%)
MN (RH 52%)
SC (RH 75%)
PN (RH 93%)
f
0
Ratio percentage of conjugated dienoic acid (%)
140
CON
(b)
PP
LC
MC
MN
SC
PN
Fig. 5. Moisture content in stripped corn oil containing 84 ppm a-tocopherol at 60 °C after 62-h treatment under different relative humidity.
120 100 80 60 40 20
10 ppm
20 ppm
42 ppm
84 ppm
0 PP (RH 0%)
LC MC MN SC PN (RH 11%) (RH 32%) (RH 52%) (RH 75%) (RH 93%)
Fig. 4. Antioxidative or prooxidative properties of a-tocopherol in stripped corn oil under diverse relative humidity based on headspace oxygen content (a) and conjugated dienoic acid (b) analysis.
addition of deionised water only. Forced conditions to equilibrate for RH could thus be an important factor to consider. For samples at low RH, for example, PP, moisture may be forced to evaporate continuously from oils into the headspace while for those at high RH, like PN, the reverse flux may be found from headspace to oils. Many studies have adapted saturated salt solution as a tool to provide adequate RH to determine the effects of RH and oxidative stability of foods (Gopalakrishna & Prabhakar, 1983; Partanen et al., 2008; Velasco, Holgado, Dobarganes, & Márquez-Ruiz, 2009). However, it is possible that oxidative stability under RH controlled by saturated salt solutions may not reflect the real situation without forced control of RH, and these results should be interpreted with caution. Moisture content in corn oil containing 84 ppm a-tocopherol at 60 °C after 62-h treatment under different RH conditions is shown in Fig. 5. Samples with 10, 20, and 42 ppm a-tocopherol were not determined. As expected, moisture content in PN was the highest, followed in decreasing order by SC, MN, MC, LC, and PP. Moisture content in stripped corn oils under low RH, PP and LC, were 110 and 304 ppm, respectively, whereas those under high RH, SC and PN, were 1389 and 1912 ppm, respectively. Moisture content in CON samples containing tocopherol without addition of water was 606 ppm, which was not significantly different from those in MC (p > 0.05). In addition, moisture content in samples without addition of a-tocopherol and salt solution were not significantly different from those in CON and MC (data not shown). It is clearly confirmed that antioxidant properties of a-tocopherol are greatly influenced by the concentration of a-tocopherol and RH. Although the actual RH of current experimental conditions was not determined, moisture content in oils was determined
using the Karl Fisher method as shown in Fig. 5. Higher moisture content in PN can generate more association colloids in oils, which may provide more places for lipid oxidation. a-Tocopherols have amphiphilic properties due to their phytol chain with 16 carbons and chromanol ring structure. a-Tocopherol may participate in formation of association colloids. Increase in mobility and activity in transition metals could also be a major factor. Chen et al. (2012) reported that iron is a major pro-oxidant in bulk oils. Ferric ion (Fe3+) may enhance the decomposition of a-tocopherol under high RH and an increase in ferrous ion (Fe2+), which can be formed by reduction of ferric ion (Fe3+) by a-tocopherol, can accelerate the rate of lipid oxidation. At low RH conditions, different forces could be driving oxidation compared to high RH, perhaps depending on the number of association colloids. Oxygen flux from headspace to oils could be one of the possible forces driving oxidation at low RH due to the low moisture content in oils. This speculation should be tested in the future. There are several possible theories for the pro-oxidant properties of a-tocopherol at higher concentrations (KamaI-Eldin & Appelqvist, 1996; Naumov & Vasil’ev, 2003). Tocopheroxyl radicals from tocopherols can attack lipids and lipid hydroperoxides to generate lipid radicals and peroxyl radicals, respectively (Naumov & Vasil’ev, 2003). Tocopherols and tocopheroxyl radicals may also reduce metal ions, which could result in more active metal states for pro-oxidant actions through Fenton (or Fenton-like) reactions. Extra tocopherols may be located on the surface of oils, which may help the migration of oxygen into oils (KamaI-Eldin & Appelqvist, 1996). Further research is needed to confirm the proposed pro-oxidative mechanisms of tocopherols in oils.
4. Conclusion The effects of a-tocopherol concentration and RH on the oxidative stability of stripped corn oils were tested. The same concentration of a-tocopherol showed different oxidative stabilities in stripped corn oils under different RH conditions. Generally, lower RH was associated with higher oxidative stability. In addition, a high concentration of a-tocopherol (84 ppm) showed pro-oxidative properties compared to a low concentration (10 ppm) in stripped corn oils. Our findings clearly demonstrate that both concentration and moisture content are important for antioxidant properties of lipophilic antioxidants in stripped corn oil systems.
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