Introduction

Lipid oxidation is the major deterioration problem in foodstuffs rich in lipids. During the lipid oxidation process, a variety of compounds formed and most of them influence the food quality parameters including aroma, texture, nutritional value and color. Besides, some of these oxides have toxic impacts on human health [1,2,3,4,5,6].

Antioxidants are used to retard the lipid oxidation process, thus preserve the aroma, color, and nutrition values of foods [7,8,9,10,11]. Synthetic and natural antioxidants are utilized to preserve lipid oxidation. Synthetic antioxidants are effective and less expensive than natural ones. Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are commonly synthetic antioxidants used in the vegetable oil industry [12,13,14]. Natural antioxidants are present in fruits, vegetables, herbs and medicinal plants. Phytochemicals can be used for food preservation and have been applied in food chemistry and pharmacy, demonstrating that these bioactive constituents are innovation hotspot in several technological domains [15]. Natural antioxidants are preferred for consumers than synthetic ones due to safety concerns. Besides, natural antioxidants ensure some health implications [8, 16,17,18].

Phenolics are a potential source for natural antioxidants to protect some diseases induced by free radicals [3]. The most effective natural antioxidants contain phenolic compounds that have strong H-donating activity [14, 19]. Natural compounds such as coumarins, curcumanoids, flavonoids, lignans, tannins, phenolics are found in fruits, vegetables, leaves, and seeds and they are known to protect food ingredents from oxidation [14, 20]. Herbs and spices have been used in food industry as antioxidants and flavoring agents. Phenolics such as thymol, carvacrol, and thymoquinone (TQ) exhibited high antioxidant and health-promoting activities. Thymol and carvacrol are the main bioactive components in the essential oils of the Lamiaceae family [21, 22]. TQ, a biologically active compound in black cumin (Nigella sativa) seeds, is responsible for its health beneficial effects [23,24,25,26,27,28].

Different vegetable oils (palm, corn, canola, soybean, sunflower, etc.) are commonly utilized for frying, each with its specific fatty acids profile, taste, and stability [29]. Some phenolic substances were used to delay the deterioration and oxidation of oils. Studies demonstrated that some phenolic substances could be considered as proper alternatives to synthetic antioxidants. Horuz and Maskan [30] evaluated the effects of thymol and carvacrol on the thermal stability of corn oil during frying. At frying temperature, carvacrol was an effective alternative to BHT for the preservation of corn oil. A study by Karoui et al. [31] showed that refined corn oil mixed with Thymus capitatus improved the oxidative stability of the oil under heating and deep-frying temperatures. Black cumin (Nigella sativa) oil is rich in bioactive compounds such as TQ. The addition of black cumin (Nigella sativa) oil increased the stability of corn oil when heated at 60 °C [24]. Mariod et al. [32] evaluated the effect of Nigella sativa cake methanol extract and its fractions (n-hexane, ethyl acetate, and water) on the oxidation of corn oil at 70 °C. Nigella sativa cake methanol extract and/or fractions improved the oxidative stability of corn oil.

The present study aims to investigate the antioxidant activity of natural antioxidants (thymol, carvacrol, and TQ) compared with commerical ones (α-tocopherol, BHT, and BHA). Morover, the effects of natural phenolics (thymol, carvacrol, and TQ) on the oxidative stability of refined and stripped corn oils using antioxidant tests and tests under thermal storage conditions were invesigated.

Materials and methods

Materials

Commercial refined corn oil (RCO) was obtained from the market (Bolu, Turkey) and stored at − 18 °C until use. BHA, BHT, 1,1-diphenyl-2-picrylhydrazyl (DPPH·), and p-anisidine were purchased from Sigma (St. Louis, MO, USA). All chemicals and solvents used in the study were of analytical reagent grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

Methods

Analytical analyses of refined corn oil

Free fatty acid content (FFA) (Ca 5a-40), p-anisidine value (p-AV) (AOCS Cd 18-90), peroxide value (PV) (Cd 8-53), and specific absorbance values (K232, and K268) (Ch 5-91) were analyzed in the corn oil using AOCS [33] official methods.

Total tocopherols content was determined according to Wong et al. [34]. Oil sample (200 mg) was weighed in a 10-mL flask. Toluene (5 mL) were added then 3.5 mL 2,2′-bipyridine (0.07% w/v in ethanol 95%) and 0.5 mL of FeC13·6H2O (0.2% w/v in ethanol 95%) were added and the solution was made up to 10 mL using ethanol 95%. After 1 min the absorption at 520 nm was determined using as a reference blank solution (without oil). The test was calibrated using standards containing 0–250 µg α-tocopherol in toluene. Total tocopherols in the oil was calculated as follow:

$${\text{Total}}\,{\text{tocopherols}}\,{\text{(ppm)}} = \frac{(A - B)}{\text{M.W.}}$$

where A = sample absorption in 10 mm-cell, B = blank absorption in 10 mm-cell, M = gradient of absorbance vs. weight graph for α-tocopherol calibration, and W = weight of sample (g).

The thermal oxidative stability was tested with the Rancimat apparatus (Metrohm, Herisau, Switzerland). The airflow rate was kept at 10 L/h and the heating block temperature was maintained at 90 °C.

The fatty acid profile was determined by GLC after methylation (Ce 2-66, AOCS method), using an Agilent 7890A fused silica capillary column (J & W Scientific, USA). The column is 100 m long, 0.2 µm film thickness and 0.25 mm inner diameter. The injector temperature was maintained at 250 °C and the detector temperature at 260 °C. Helium was the carrier gas at 1 mL/min flow rate. One µL sample was injected into the column and the split ratio was 1:30. The column temperature was set at 140 °C for 5 min, then programmed to 240 °C at 4 °C/min and held at 240 °C for 10 min. Fatty acid methyl ester (FAME) standard solution (37 FAME mix, Sigma, St. Louis, USA) was used to identify the peaks. Fatty acid composition of oils was given in percentage proportions of FAME using the peak areas.

Evaluation of the antioxidant properties of phenolics and commercial antioxidants

DPPH· radical scavenging test

The solution of phenolic standards was prepared at 25, 50, 100, 250, 500 and 1000 ppm concentrations in methanol and/or acetone. The antiradical potential of the solutions was tested according to Brand-Williams et al. [35]. 3.9 mL of a 0.039 g/L DPPH· methanolic solution was added to 0.1 mL of different concentrations of phenolic standard solutions as well as α-tocopherol, BHA and BHT. The mixture was shaken and left to stand at room temperature for 30 min. The absorbance was measured at 515 nm and the scavenging activity was calculated according to following equation:

$${\text{Scavenging}}\,{\text{activity}}\,{\text{(}}\%{\text{)}} = \left[\frac{\text{A}_{{\text{control}}} - \text{A}_{{\text{smaple}}}}{\text{A}_{{\text{control}}}}\right] \times 100$$

where Acontrol and Asample were the absorbance values of control and sample solutions, respectively. Phenolic concentration providing 50% inhibition (IC50) was calculated from the plot of inhibition % against the certain phenolic concentration. All assays were conducted in triplicate.

Linoleic acid test

The antioxidant activities of phenolics in the linoleic acid emulsion were measured according to Mau et al. [36] and Iqbal et al. [37]. To prepare the 0.02 M linoleic acid emulsion, linoleic acid (1.402 g) and Tween 20 (1.402 g) were dissolved in potassium phosphate buffer (50 mL, pH 7.4, 0.05 M). The mixture was homogenized for 5 min to stabilize the emulsion. Linoleic acid 0.02 M emulsion (2.5 mL), phenolic solution (0.2 mL, at different concentrations) and potassium phosphate buffer (2.3 mL, pH 7, 0.2 M) were mixed in flasks. Methanol and/or acetone were used for the control sample instead of a sample solution. Flasks were incubated for 22 h without a cap at 37 °C in the dark. Before and after incubation, 0.1 mL of samples from every bottle were mixed with 6 mL of methanol solution (60%, v/v). Absorbance differences of each sample before and after incubation were calculated as the absorbance of each sample. These values were compared with those of BHA, BHT, and α-tocopherol at 0.2 mg/mL.

$${\text{Antioxidant}}\,{\text{activity}}\,(\%) = \left[\frac{\Delta {\text{A}}_{{\text{control}}} - \Delta {\text{A}}_{{\text{sample}}}}{\Delta {\text{A}}_{{\text{control}}}}\right] \times 100$$

ΔAcontrol: absorbance difference of control before and after incubation, ΔAsample: absorbance difference of sample before and after incubation.

Reducing power test

Reducing power assay of phenolic compounds was performed according to Mau et al. [36] with slight modifications. Briefly, 1.5 mL phenolic solutions with different concentrations added to 1 mL of 2.4 mM sodium phosphate (pH 6.6) and 2.5 mL potassium ferricyanide solution (1%). The mixture was incubated for 20 min at 50 °C, then 2.5 mL of 10% (w/v) trichloroacetic acid were added, and the mixture was centrifuged for 10 min at 4000 rpm. The upper layer (5 mL) was mixed with 5 mL of deionized water and 0.1% ferric chloride (1 mL). The absorbance, against a blank, was recorded at 700 nm using a Shimadzu spectrophotometer.

Stripping of refined corn oil (RCO)

RCO was purified to obtain stripped corn oil (SCO) using the method described by Karabulut et al. [38] using activated carbon and alumina column chromatography.

Enrichment of RCO and SCO with phenolics and commercial antioxidants

Eight RCO and eight SCO (triglycerides) experimental designs were prepared.

  • Refined corn oil (RCO)

  • RCO supplemented with 100 ppm of BHT.

  • RCO supplemented with 250 ppm of thymol.

  • RCO supplemented with 500 ppm of thymol.

  • RCO supplemented with 250 ppm of carvacrol.

  • RCO supplemented with 500 ppm of carvacrol.

  • RCO supplemented with 250 ppm of TQ.

  • RCO supplemented with 500 ppm of TQ.

  • Stripped corn oil (SCO)

  • SCO supplemented with 100 ppm of BHT.

  • SCO supplemented with 250 ppm of thymol.

  • SCO supplemented with 500 ppm of thymol.

  • SCO supplemented with 250 ppm of carvacrol.

  • SCO supplemented with 500 ppm of carvacrol.

  • SCO supplemented with 250 ppm of TQ.

  • SCO supplemented with 500 ppm of TQ.

.

Accelerated thermal oxidation tests

Rancimat test for RCO

The induction periods of purified corn oils enriched with TQ, thymol or carvacrol were carried out using Rancimat apparatus (Metrohm, Herisau, Switzerland). The oil sample (3 g) was placed in the Rancimat apparatus at 90 °C under 10 L h−1 airflow rate.

Schaal oven test for RCO and SCO

Fifty grams of RCO were weighted in 50-mL open glass bottles and kept 21 days at 60 °C in an oven. Samples were examined at 3-days intervals by collecting sample from the same bottles at a particular period. Besides, 10 g of SCO were used under the same storage conditions for 7 days and examined at everyday intervals. The thermal oxidative stability of the samples was evaluated using PV, p-AV, and CD (K232) tests. All experiments were carried out in two repetitions for each sample.

Statistical analysis

Oxidation experiments were carried out in two replicates. Results were given as mean ± standard deviation. Results were statistically evaluated using the Minitab 17 Statistical Software (v17.3.1) package program. The difference between the group means was determined according to variance analysis technique (ANOVA) (p < 0.05).

Results and discussion

Composition and chemcial characteristics of RCO and SCO

FFA, PV, K232, K268, p-AV, total tocopherol content, induction period and fatty acid compositions of the refined and purified corn oils under study are given in Table 1. FFA, PV, K232, K268, p-AV, and total tocopherols in RCO were 0.10%, 3.90 meq O2/kg, 2.68, 1.84, 8.45 and 842.6 mg/kg, respectively. The initial values of PV, CD, CT, p-AV, and total tocopherols agree with those in the literature [39,40,41]. The PV, K232, K268, and p-AV of the SCO decreased to 1.90 meq O2/kg, 1.84, 1.12 and 0.17, respectively. Besides, tocopherols were absent in the SCO. Tocols are the most active natural antioxidants present in vegetable oils that retard oxidation and the formation of polar compounds [29]. Data in Table 1 demonstrate that RCO have better oxidative stability (39.48 h) than SCO (2.82 h). Linoleic acid was the predominant fatty acid in corn oil (55.1% of total fatty acids), followed by oleic acid (30.72% of total fatty acids), while palmitic acid was the main saturated fatty acid. The fatty acid profile reported in the current study agrees with those reported in the literature [42].

Table 1 Chemical parameters of refined (RCO) and stripped corn oils
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Antioxidant activities of thymol, carvacrol, TQ and commercial antioxidants

Several natural antioxidants were applied to enhance the stability of vegetable oils during the thermal treatments [43]. No single test is enough to screen the antioxidant potential of phytochemicals or plant extracts, since different methods could yield different results [1, 44]. Thus, different methods based on different mechanisms should be tested [45, 46].

DPPH· assay

Using DPPH· free radicals, the antiradical potential of phenolics and commercial antioxidants was tested in vitro considering that DPPH· radicals are widely used for testing the antioxidant effects. DPPH· are stable free radicals with violet color that gives absorption maxima at 515–528 nm [1, 45,46,47]. DPPH· antiradical activities of thymol, carvacrol, TQ, α-tocopherol, BHA and BHT are presented in Fig. 1. For all tested samples, DPPH· antiradical activities increased with the increase of antioxidant concentration. At 1000 ppm concentration, α-tocopherol had the highest DPPH· antiradical activity (94.9%) among all tested samples, followed by BHA (93.6%). Among the phenolic compounds, TQ had higher DPPH· antiradical activity (65.8%) than thymol (25.0%) and carvacrol (18.3%) at 1000 ppm concentration, which was closed that of BHT (75.0%). Gavaric et al. [48] observed that the antiradical activity of BHT was higher than thymol and carvacrol, as well as the antiradical activity of thymol was higher than carvacrol. In contrast, Milos and Makota [49] demonstrated that thymol had higher antiradical activity than carvacrol and TQ. IC50 values of phenolics and commercial antioxidants are presented in Fig. 2. Antioxidant activities of the samples according to the IC50 values increased in the following order: BHA > α-tocopherol > BHT > TQ > thymol > carvacrol.

Fig. 1
figure 1

DPPH· radical scavenging effects (%) of phenolic compounds, α-tocopherol, BHA, BHT

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Fig. 2
figure 2

IC50 values of phenolic compounds, α-tocopherol, BHA, BHT in DPPH· assay

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The results of the DPPH· radical scavenging test suggest that phenolics under study (thymol, carvacrol, and TQ) are able to scaveng DPPH· radicals through hydrogen-donating mechanism. The antiradical effect of phenolics is due to their redox characteristics, which play a role in quenching singlet and triplet oxygen, neutralizing radicals, and decomposing peroxides [46, 50].

Linoleic acid test

Antioxidant activities of phenolic compounds were tested in linoleic acid emulsion and compared with BHA, BHT, and α-tocopherol as shown in Table 2. BHT and BHA showed strong antioxidant activities (95% and above) at all concentrations. Antioxidant activities of thymol, carvacrol, TQ and BHT increased with the increase of concentration, while there was no increase for BHA and α-tocopherol with the increase of concentration. Among phenolic compounds, thymol and carvacrol showed stronger antioxidant activity than TQ. Besides, α-tocopherol recorded the lowest activity among tested samples. The results presented here agree with previous literature wherein α-tocopherol was reported to be less active than BHA, and BHT [51].

Table 2 Antixoidant activities of phenolic compounds, α-tocopherol, BHA, BHT in linoleic acid system
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Ferric reducing power test

Ferric reducing power of phenolic compounds, BHA, BHT, and α-tocopherol are presented in Table 3. In this assay, commercial antioxidants exhibited stronger reducing power compared to natural phenolic compounds. At 1000 ppm concentration, BHT and α-tocopherol showed similar values and slightly higher values than BHA. Similar results for BHA, BHT, and α-tocopherol reported previously by Elmastas et al. [52]. Regarding phenolic compounds, TQ and thymol exhibited stronger antioxidant activities compared to carvacrol.

Table 3 Antixoidant activity of phenolic compounds, α-tocopherol, BHA, BHT in ferric reducing power test
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Efficiency of phenolics and commercial antioxidants on the stability of RCO and SCO

Oil oxidative stability is commonly tested under thermal conditions (60 °C). To test the antioxidant effects of phenolics and commercial antioxidants in RCO and SCO, PV, p-AV and CD were determined as indices of oxidation [1, 46, 47].

Induction periods of stripped corn oil (SCO)

Induction periods and the protection factor of SCO enriched with phenolics and commercial antioxidants are given in Table 4. A high induction period (15.01 h) at 90 ̊C was reported for SCO with 100 ppm BHT. The addition of phenolic compounds to SCO improved the oxidative stability of the oil (p < 0.05). At 500 ppm concentration, the induction period of thymol, TQ and carvacrol was 5.69, 4.04 and 5.44 h, respectively, while the induction period of SCO was 2.82 h.

Table 4 Induction periods of stripped corn oil (SCO) enriched with phenolics and commercial antioxidants
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Effect of phenolics and commercial antioxidants on the stability of RCO (Schaal oven test)

PV is a measure of the peroxides and hydroperoxides induced in the initial stages of oxidation. PV is a widely used test for the measurement of oils and fats rancidity. The changes of PV of RCO during storage at 60 °C are presented in Fig. 3. At the end of 21 days of storage, PV values of samples enriched with phenolic compounds (except thymol) were lower compared with the control sample. Phenolic compounds improved the oxidative stability of RCO. Among phenolics, TQ showed higher antioxidant effects than thymol and carvacrol. Besides, PV values of samples containing 250 and 500 ppm of TQ (103.7 and 108.0 meq O2/kg, respectively) showed lower PV than that of BHT (128.7 meq O2/kg). Black cumin oil rich in TQ improved the stability of RCO stored under thermal oxidative conditions (60 °C) [53]. The results also showed that thymol does not affect the oxidative stability of RCO. As compared with thymol, carvacrol was an effective phenolic compound in the inhibition of PV during the oxidation process. Horuz and Maskan [30] found that thymol had a slight effect on the induction period of corn oil under frying temperatures, while carvacrol showed strong antioxidant potential on corn oil under the same conditions. These findings are in agreement with the present study.

Fig. 3
figure 3

Changes in PV (meq O2/kg oil) of RCO enriched with phenolics and BHT during Schaal oven test. RCO refined corn oil, BHT butylated hydroxy toluene, T thymol, TQ thymoquinone, C carvacrol

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CD is a good indicator to measure oils and fats stability. During oxidation, methylene-intrupted dienes in lipids show a shift in their double bond position. The induced CD exhibit absorption maxima at 232 nm [1, 46, 47, 54]. Figure 4 shows the CD (K232) values of RCO samples during 21 days of storage. After 21 days, the CD value of the control oil reached to 28.23 from an initial value of 2.95. Except for thymol, the other two phenolic compounds, TQ and carvacrol showed lower CD values than the control, thus showing enhancement of the oxidative stability of RCO. Especially, TQ-enriched samples had a significant (p < 0.05) effect on lowering CD formation among phenolic compounds and also showed a stronger effect on lowering CD compared to BHT.

Fig. 4
figure 4

Changes in K232 of RCO enriched with phenolics and BHT during Schaal oven test. RCO refined corn oil, BHT butylated hydroxy toluene, T thymol, TQ thymoquinone, C carvacrol

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p-AV is an old method to measure secondary lipid oxidation. The test based on the reactiveness of aldehyde carbonyl bond on p-anisidine amine group, that leads to Schiff base formation which absorbs at 350 nm [46, 55]. At 60 °C and after 21 days of storage, changes in the p-AV value of samples are presented in Table 5. The p-AV of the control sample reached 11.1, while p-AV in BHT-enriched RCO was 8.3. Like BHT, TQ showed an effect on the formation of secondary oxidation products and had lower values (7.6 at 250 ppm, and 8.3 at 500 ppm) as compared with those of the phenolics-enriched RCO. However, thymol-enriched oil had a slightly lower p-AV during the later stages of storage.

Table 5 Changes in p-AV of RCO enriched with phenolics and BHT during storage at 60 °C
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Changes in total tocopherol content of RCO samples during storage are given in Table 6. During storage, the total tocopherol content of all RCO samples with or without added antioxidants decreased with increasing the storage time. During the 21-days storage, the total tocopherol content of the control sample decreased sharply (76.7 mg/kg). The addition of phenolic compounds and also BHT to RCO inhibited the reduction in total tocopherols at 60 °C. At the end of storage, the remained tocopherols in oil treated with BHT were 131 mg/kg. Thymoquinone addition was more effective than BHT on RCO. For 250 and 500 ppm TQ concentrations, the total tocopherol content decreased to 176.2 and 189.6 mg/kg, respectively. Besides, the effect of the addition of carvacrol (500 ppm) to RCO was better than that of BHT. Thymol in RCO was more effective than the control sample, while lower than that of oil enriched with BHT.

Table 6 Changes in total tocopherols (mg/kg) of RCO enriched with phenolics and BHT during storage at 60 °C
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Effect of phenolics and commercial antioxidants on the stability of SCO (Schaal oven test)

Figure 5 shows the PV development in SCO during the storage at 60 °C for 7 days with various concentrations of phenolic compounds and BHT. The control sample reached the maximum PV (452 meq O2/kg) after 7 days of storage. A significant (p < 0.05) difference in PV was recorded between the control and SCO containing BHT that slowed the rate of peroxide induction. The effect of phenolic compounds was lower on peroxide formation after 7 days of storage compared with BHT-enriched oil. However, PV of all oils enriched with phenolic compounds was lower than that of the control. Among phenolics, TQ was more effective in lowering the PV of SCO during storage. The effect of thymol and carvacrol showed similar effects on peroxide formation in SCO during the storage experiment.

Fig. 5
figure 5

Changes in PV (meq O2/kg oil) of SCO enriched with phenolics and BHT during storage at 60 °C. SCO stripped corn oil, BHT butylated hydroxy toluene, T thymol, TQ thymoquinone, C carvacrol

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CD values of SCO samples were given in Fig. 6. The CD of the control sample increased from 2.05 to 84.5, whereas 100 ppm of BHT could considerably inhibit SCO oxidation and showed an increase from 2.01 to 3.21. However, SCO enriched with phenolic compounds was oxidized fastly compared with BHT-enriched oil. Moreover, the increase in CD of oils including phenolic compounds was lower than the control sample. Thymoquinone exhibited a strong effect on the formation of CD from oil samples among phenolic compounds.

Fig. 6
figure 6

Changes in K232 of of SCO enriched with phenolics and BHT during storage at 60 °C. SCO stripped corn oil, BHT butylated hydroxy toluene, T thymol, TQ thymoquinone, C carvacrol

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Table 7 shows the changes in p-AV of SCO oils during storage experiment. There was an increase in p-AV in all oil samples with increasing the storage time. The p-AV in the control sample increased up to 58.8 at the end of storage. SCO enriched with BHT exhibited lower p-AV (0.3) at the end of storage as compared with the control sample. The phenolic compounds seem to be less effective in inhibition of p-AV than BHT. On the other hand, samples treated with phenolic compounds showed lower p-AV compared with the control sample. Among phenolic compounds, TQ at 250 and 500 ppm inhibited the induction of the secondary oxidation products in comparison with control, while p-AV values seem to be less than that formed in thymol and carvacrol enriched SCO samples.

Table 7 Changes in p-AV of SCO enriched with phenolics and BHT during storage at 60 °C
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Conclusions

Clean label foodstuffes enriched with phytochemicals have attracted more and more attention due to health-promoting effects. Phenolic compounds are known as strong antioxidant compounds. Thymol, carvacrol, and TQ were selected to study its antioxidant potential on the stabilization of refined and stripped corn oils compared with commercial antioxidants. Different tests showed that the free radical scavenging activity of phenolic compounds under study was comparable with that of commercial antioxidants. Phenolic compounds improve the oxidative stability of refined and stripped corn oils stored at 60 ̊C, while its efficacy was less than that of BHT. It could be concluded that phenolics under study (thymol, carvacrol, and TQ) can stabilize RCO and SCO effectively. Thymol, carvacrol, and TQ showed different antioxidant characteristics in different tests in a dose-dependent manner. Thymol, carvacrol, and TQ could inhibit thermal oxidation of corn oil by inhibiting double bond conjugation and enhancing its hydrolytic stability. TQ could be recommended as a potent natural antioxidant for the stabilization of vegetable oils. Further studies are needed to investigate the mechanism by which thymol, carvacrol, and TQ retard the oxidation during thermal treatment of corn oil.