Introduction

Lipid oxidation produces not only rancid odors, unpleasant flavors and discoloration, but it can also decrease the nutritional quality and health safety of food due to the degradation of products, resulting in harmful effects on living cells [1]. Vegetable oils with higher contents of unsaturated fatty acids (UFA), especially polyunsaturated fatty acids (PUFA), are more susceptible to oxidation than lipids of animal origin.

In order to overcome the stability problems of oils and fats, synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ), are widely used as food additives in many countries. Recent reports reveal, however, that these compounds may cause many health risks, including cancer and carcinogenesis [2]. That is why the need for natural antioxidants has recently increased because of questions about the long-term safety and negative consumer perception of synthetic antioxidants [3]. Consumers, who are aware of the impact of synthetic additives on the human, expect an increase in the use of natural compounds to replace these synthetic antioxidants.

In the literature, there are several reports on the use of antioxidants of plant origin, as well as substances of animal origin. Furthermore, some protein hydrolysates from animal and plant sources have also been found to exhibit antioxidant activity [4, 5]. These antioxidants have been investigated mainly with regard to the prevention of lipid oxidation in foods. Shui and Leong [6] found that antioxidants obtained from star fruit (Averrhoa carambola L.) residues slowed the process of rancidity of oil to a greater extent than BHT did.

Egg yolk is widely used as a functional and nutritional ingredient in food products. Egg yolk has been recognized to possess antioxidant properties in a linolenate emulsion [7]. It is reported that egg yolk phospholipids [8] and egg yolk phosvitin [9, 10] have antioxidant effects. Some studies on the use of protein hydrolysates of lecithin-free egg yolk as an antioxidant [11] have been performed. The results from Sakanaka et al. [12] suggest that egg yolk protein hydrolysates could be a suitable natural antioxidant for preventing the oxidation of PUFA and related food ingredients.

Superoxide dismutases (SOD, EC 1.15.1.1) are a family of important antioxidant metallo-enzymes involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide via the dismutation of superoxide. Depending on the ions in the active site, three main metal ion forms are found in living organisms [13–16]. The dimeric, cytosolic Cu, Zn-SOD share a large structural similarity of the two monomers, such as a conserved tertiary structure and arrangement, as well as an almost identical total number of inter- and intra-molecular hydrogen bonds and salt bridges. There are some reports of the preparation of SOD extracts from various sources among others, bacteria, algae, plants, insects, fish and animal tissue [17–21]. However, commercially available preparations are SODs derived from the plasma of animals, mainly cattle. Due to the occurrence of cases of prion disease of cattle as well as foot and mouth disease in cattle, these products seem unattractive. The European Commission has excluded from the definition of food additives products obtained from the plasma of animals [22]. This opens new possibilities to search for other cheap and secure sources of SOD, which can be used as a food additive.

Roginsky and Barsukova [23] showed that the addition of SOD from bovine erythrocytes inhibits the process of lipid oxidation. The specific inhibiting effect of SOD during PUFA oxidation means that O ·−2 (most likely in the protonated form as HO ·2 ) participates in radical chain propagation. The proposed mechanism of formation of superoxide and hydroxyl radicals during the oxidation of PUFA is shown in Eq. 1.

(1)

Benson [24] described that the process of forming a radical (LO ·2 ) of PUFA is thermodynamically easier than the formation of saturated fatty acids (SFA) or mono-unsaturated ones (MUFA). However, even a profound purge of the system from O ·−2 (HO ·2 ) does not completely terminate the radical chain transfer [23].

A hypothesis was proposed that SOD extracted from hen's egg yolk can protect UFA from peroxidative damage and prevent the rancidity of food lipids.

The objective of this study was to examine the effect of the Cu, Zn-SOD extracted from hen's egg yolk on the composition of olive and sunflower oil during storage for 200 days at different temperatures and to compare it with the commonly used synthetic antioxidant BHT.

Materials and methods

Preparation of enzymatic protein

Ten grams of hen's egg yolk was mixed with 10 mL of 0.9 % NaCl and 50 mL of chloroform:ethanol mixture (5:3), vortexed shortly and centrifuged at 3,000×g at 4 °C. Supernatant was collected and evaporated in laboratory evaporator to 50 mL volume. Protein content in supernatant was determined in triplicate using spectrophotometric biuret method [25]. Enzymatic protein was subjected to 2D electrophoresis and Western blotting in order to confirm the presence of SOD in obtained material.

The activity of SOD in egg white was analyzed previously [31] and appeared to be very low, at the level of error of the method. It was the reason why egg white was not considered as the potent source of enzyme in the present study.

Isoelectric focusing was performed loading 10 µg proteins by in-gel rehydration in a volume of 125 µL denaturating the 2D buffer (8 M urea, 4 % CHAPS, 70 mM DTT, 0.5 % Ampholyte pH 4–7) onto 7 cm IPG Ready Strip linear pH 3–10 (Bio-Rad, Warsaw, Poland) and focused for 30 kVh, using a PROTEAN® IEF system (Bio-Rad, Warsaw, Poland). Before loading onto SDS-polyacrylamide gels, IPG strips were incubated for 10 min in equilibration buffer (50 mM Tris–HCl, pH 8.8, 6 M urea, 30 % glycerol, 2 % SDS) containing 1 % DTT and then for another 5 min in equilibration buffer containing 2.5 % iodoacetamide. The SDS-polyacrylamide gels 15 % were cast according to Laemmli [26]. The second dimension was performed using a Hoefer SE600 (GE Healthcare Life Sciences, Warsaw, Poland). Silver nitrate staining of gels was performed according to Shevchenko et al. [27].

After 2D-PAGE, proteins were transferred to Immun-Blot PVDF (polyvinylidene difluoride; Bio-Rad, Warszawa, Poland) in accordance with the procedure described by Towbin et al. [28] lasting 2 h at 100 V and 4 °C. Specific immune reactions were performed with sheep antibovine Cu, Zn-SOD antibodies (AbDSerotec, Oxford, UK) at a working concentration of 1 μg/mL for 12 h at 4 °C. The visualization of immune reactions was prepared in accordance with Blake et al. [29] with secondary polyclonal donkey antibody directed against sheep IgG conjugated with alkaline phosphatase (Abcam, Cambridge, MA, USA) at a concentration of 0.25 μg/mL.

Images of the silver-stained gel and membranes were digitized using an Image Scanner III (GE Healthcare Life Sciences, Warsaw, Poland).

Enzyme assay

SOD activity was determined using a modified epinephrine assay [30]. The method was adapted and optimized to conditions in eggs [31]. Changes in the absorbance were recorded on Ultrospec 2000 spectrophotometer (Pharmacia, Uppsala, Sweden).

The one unit of SOD (U SOD) activity was equivalent to the quantity of SOD that caused a 50 % inhibition of the auto-oxidation of epinephrine. The SOD activity was expressed as the value of U SOD per g of extracted protein (U SOD·g−1).

FAME determination

Samples of olive oil and sunflower oil (from a local market) were mixed with Cu, Zn-SOD extracted from hen’s egg yolk or BHT (Sigma-Aldrich, Poznań, Poland) at final concentrations of 0, 0.02, 0.04 and 0.06 %, respectively. Samples were stored in the dark at temperatures of 4, 20 and 35 °C for 200 days. Analyses of fatty acid profile were performed at day 1, 50, 100 and 200 of storage.

Samples of oil were saponified with KOH, and the fatty acids esterified (with 10 % solution of BF3 in methanol) in accordance with AOAC methods [32, 33].

The samples were analyzed on a Varian 3800 GC (Varian, Candela, Warsaw, Poland) using 105 m Rtx-2330 column with I.D. 0.25 mm and 0.25 µm film thickness (Restek, Bellefonte, PA, USA) with 1 mL/min helium constant flow, injection temperature of 250 °C, split ratio of 20:1 and temperature range from 60 to 250 °C at 5°/min.

The percentage of the content of the following fatty acids in examined oils was determined: myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), ginkgolic acid (C17:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), α-linolenic acid and γ-linolenic acid (the sum of the results represented linoleic acid isomers, C18:3), arachidic acid (C20:0), gondoic acid (C20:1) and tricosylic acid (C23:0).

The ratio of the total content of unsaturated fatty acids to total saturated fatty acid (RUFA/SFA) was calculated according to Eq. 2.

$${\text{R}}_{\text{UFA/SFA}} = \frac{{\sum [{\text{C}}18:1],[{\text{C}}18:2],[{\text{C}}18:3],[{\text{C}}20:1]}}{{[{\text{C}}14:0][{\text{C}}16:0][{\text{C}}18:0][{\text{C}}20:0][{\text{C}}23:0]}}$$
(2)

Statistical analysis

All of the assays were performed in triplicate. Obtained values were statistically analyzed with SAS 8.0 system (SAS Institute Inc., Cary, NC, USA). A two-way analysis of variance (ANOVA) with p < 0.05 was used to track the effect of the addition of SOD and BHT as an antioxidant of vegetable oil samples.

Results

The purity of the enzyme

2D electrophoresis of the enzyme extract obtained from egg yolk confirmed the presence of three isoforms of SOD (MW 15.59 ± 0.38 kDa and pI 6.58 ± 0.10, 6.41 ± 0.08 and 6.30 ± 0.15) as the main component, which is consistent with reports by Öztürk-Ürek and Tarhan [34]. Western blotting with antibodies directed against bovine erythrocyte SOD allowed to confirm the presence of the expected enzyme protein (Fig. 1). This is consistent with the reports of Michalski and Prowse [35] related to the immunological compatibility of Cu, Zn-SOD from bovine and chicken erythrocytes.

Fig. 1
figure 1

2D PAGE western blot analysis of Cu, Zn-SOD from hen egg yolk

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SOD activity in extracts obtained from egg yolk was 100.2 ± 9.5 U SOD·g−1.

The content of linoleic acid

The results of the analysis of linoleic acid (C18:2) during storage of the samples of sunflower oil and olive oil with the addition of SOD and BHT at 4 °C are shown in Table 1. A significant difference in the content of linoleic acid has been determined in each period of the analysis. There was no effect of the type and concentration of the additive in the process of peroxidation of linoleic acid at a storage temperature of 4 °C.

Table 1 Changes in the content of linoleic acid (C18:2) in sunflower oil and olive oil during storage at 4 °C with the addition of SOD and BHT
Full size table

The results of the analysis of the content of linoleic acid (C18:2) during storage of the samples of sunflower oil and olive oil with the addition of SOD and BHT at 20 °C are shown in Table 2. A significant difference in the content of linoleic acid has been detected in each period of analysis. With increasing concentration of the additive, an increasing inhibiting effect on the oxidation of linoleic acid was shown. At any time, the addition of SOD inhibited the rate of oxidation at the level similar to BHT.

Table 2 Changes in the content of linoleic acid (C18:2) in sunflower oil and olive oil during storage at 20 °C with the addition of SOD and BHT
Full size table

The results of the analysis of the content of linoleic acid (C18: 2) during storage of the samples of sunflower oil and olive oil with the addition of SOD and BHT at 35 °C are shown in Table 3. Significant increase in the amount of linoleic acid in the samples preserved by the addition of SOD on the 200th day of storage was noticed.

Table 3 Changes in the content of linoleic acid (C18:2) in sunflower oil and olive oil during storage at 35 °C with the addition of SOD and BHT
Full size table

The analysis of remaining fatty acids indicated similar trends, but it has not been shown.

The ratio of saturated to unsaturated fatty acids

Figure 2 shows the results of the comparisons of the ratio of saturated to unsaturated fatty acids (RUFA/SFA) in sunflower oil samples stored under different conditions. The results showed no statistical difference between the addition of SOD and BHT in oils stored at 4 °C. At 20 and 35 °C, BHT presented better antioxidant activity. In both temperatures, there was a clear effect of the concentration of BHT in attempts to increase the ratio of saturated to unsaturated fatty acids (RUFA/SFA). With regard to SOD, there was no impact of the concentration of the additive on the examined ratio.

Fig. 2
figure 2

The ratio of the sum of the percentage of unsaturated fatty acids to saturated fatty acids (RUFA/SFA) in sunflower oil stored at 4, 20 and 35 °C with BHT and SOD. Data are reported as mean ± standard deviation. Different letters indicate significant difference between the components on the same day of analysis (ANOVA with Tukey’s test p < 0.05)

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In any case, the addition of SOD showed positive properties regarding antioxidant protection at a level comparable with BHT.

Figure 3 shows the results of the comparisons of the ratio of saturated to unsaturated fatty acids (RUFA/SFA) in olive oil samples stored under different conditions. The results showed no statistical difference between the addition of SOD and BHT in oils stored at 4 °C.

Fig. 3
figure 3

The ratio of the sum of the percentage of unsaturated fatty acids to saturated fatty acids (RUFA/SFA) in olive oil stored at 4, 20 and 35 °C with BHT and SOD. Data are reported as mean ± standard deviation. Different letters indicate significant difference between the components on the same day of analysis (ANOVA with Tukey’s test p < 0.05)

Full size image

At 20 °C, the ratio of saturated to unsaturated fatty acids (RUFA/SFA) remained statistically higher with the addition of BHT than with the addition of SOD. At 35 °C, there was a clear effect of the addition of SOD in attempts to increase the ratio of saturated to UFA during 200 days of storage. With regard to SOD, there was no impact of the concentration of the additive on the examined ratio.

As in sunflower oil, also in olive oil, the effect of the addition of SOD on the increase of the ratio of saturated to unsaturated fatty acids (RUFA/SFA) at temperatures of 20 and 35 °C was detected.

Discussion

The present study has shown that the addition of SOD to vegetable oils has a positive influence on the content of linoleic acid (18:2) during storage of sunflower oil and olive oil at 20 and 35 °C. During storage of sunflower oil for a period of 200 days, better antioxidant properties of SOD (with regard to the ratio of UFA to SFA) than for similar concentrations of BHT were demonstrated. No effect of SOD concentration on the efficiency of inhibition of the peroxidation of UFA with regard to both sunflower oil and olive oil was also detected. Such relationship was clearly visible with the addition of BHT. During the storage of oil samples for 200 days at different temperatures, SOD showed similar properties in the prevention of fatty acid oxidative damage as BHT.

Worldwide vegetable oil consumption is expected to grow by 2 % per year as a result of increasing edible oil and renewable energy demands [36] and that is why appropriate protection of fatty acids is of vital importance. A striking feature of sunflower oil was the relative high level of PUFA and MUFA. From the health point of view, MUFA have been shown to lower “bad” LDL cholesterol (low density lipoproteins) and retain “good” HDL cholesterol (high density lipoproteins). This is in fact the major benefit of olive oil over the highly polyunsaturated seed oils, wherein PUFA reduce both the “bad” as well as the “good” serum cholesterol levels in our blood [37].

Oxidative reactions limit the half-life of fresh and processed foodstuff and are of serious concern in food industry. ROS easily attack multiple bonds in UFA, lead to a number of subsequent reactions which result in changes in the quantitative composition of the fat, and produce others compounds which can be dangerous to the consumer [38]. That is why antioxidants are often added to food to prevent the radical chain reactions and they act by inhibiting the initiation and propagation steps, consequently delaying the oxidation process.

BHT is a synthetic phenolic antioxidant widely used as a food additive. It is very effective in the protection of unsaturated oils and is therefore used as potential inhibitor of oxidative reaction. It is assumed that the mechanism of its action is based on neutralizing free radicals [39]. However, this results in the formation of potentially dangerous BHT metabolites, mainly 3,5-di-tert-butyl-4-hydroxybenzoic acid and its derivatives which can cause damage in a number of organs such as lung, kidney, liver, and muscle [40, 41]. The use of BHT is limited by law; therefore, it is necessary to search for efficient natural antioxidants without the aforementioned disadvantages [42]. Molecules of protein origin seem to be perfect as antioxidants because they are hydrolyzed in the digestive tract to amino acids, lose their properties and do not exert any harmful effect.

Olive oil and sunflower oil are often used in studies of oxidative changes in fat [43, 44]. Changes in the content of linoleic acid (C18:2) are often monitored as a good indicator of the oxidative degradation of vegetable oils, during long storage or when subjected to elevated temperatures [45].

Sakanaka et al. [12] tested antioxidant properties of the protein hydrolysates of egg yolk. The enzymatically hydrolyzed protein with mass distribution of less than 1 kDa from skimmed egg yolk showed potent antioxidant activity with respect to linoleic acid (C18:2). The effect was dependent on the concentration of the hydrolysate in the sample.

The results of present experiment showed better antioxidant properties of SOD extracted from hen’s egg yolk compared with the same concentrations of BHT at elevated temperatures (at 20 and 35 °C) in oil with a high content of UFA. Low SOD activity as an antioxidant in the oil stored at 4 °C can be explained by the low activity of the enzyme protein outside the optimum temperature. In the literature, there is no information available about the optimum temperature for the action of SOD in hens (Gallus gallus domesticus). There are, however, a number of reports describing the optimum temperature for the activity of Cu, Zn-SOD from other species ranging between 25 and 37 °C [21, 34].

Comparing the sunflower oil sample, the relationship between the concentration of BHT and the ratio of the total content of UFA to total SFA content in the analyzed samples was observed. It confirmed the results obtained by Fujisawa et al. [39]. In the case of the addition of SOD, there was no direct link between the concentration of the additive and the ratio of unsaturated to saturated fatty acids in the oil samples, which is consistent with reports by Roginsky and Barsukova [23].

The corresponding results in olive oil similarly showed the effect of the concentration of BHT on the sum of the content of UFA to total saturated fatty acid in the analyzed samples. Such a relationship was not observed with regard to the addition of SOD from chicken eggs.

Our results confirm the statement that SOD is a second type (secondary) hen’s egg oxidant, which does not directly interrupt the free radical chain reaction, but acts on the intermediate products and is not consumed during the reaction [23, 46].

Conclusions

SOD isolated from hen’s egg yolk can protect fatty acids from peroxidative damage and prevent the process of rancidity in food lipids in a similar or even better way than BHT.