Vegetable and animal oils are susceptible to the oxidation of their lipid components on storage. Polyphenols from apple peels are investigated as potential natural antioxidants used for stabilizing polyunsaturated fatty acid and preventing oxidation. The aim of this study was assessing the antioxidant efficacy of apple pomace as natural antioxidant in linseed and fish oils, stored in anaerobic conditions. Apple pomace was added to the linseed and fish oils stored for eight weeks to evaluate the antioxidant activity of their polyphenolic components. The total phenolic content, activity of DPPH, ABTS•+, FRAP, acid value (AV), peroxide value (PV), and fatty acid profile were analyzed in storage tests. We found that apple pomace, regardless of the oil content of the formulation, was capable of blanking 2,2-diphenyl-1-picrylhydrazyl radicals. The highest ability to reduce Fe3+ ions occurred in the samples containing 30% of the fish oil. The use of apple pomace comprising polyphenolic compounds improves the stability of linseed and fish oils in storage tests. Polyphenols in apple pomace show a high antioxidant potential, as indicated by their values of DPPH, ABST•+, and FRAP. The addition of apple pomace resulted in limiting the acid and peroxide values of the samples during storage.
Lipid oxidation is an essential reason for deterioration in the quality of edible oils [
Cold-pressed vegetable oils, especially those containing
There are earlier reports on the applicability of fruit-processing waste for the protection of lipids in foods against changes resulting from oxidation [
The study involved vegetable (linseed) oil and animal (fish) oil with different fatty acid profiles, especially with respect to their content of long-chain fatty acids. The linseed oil (BiqOIL Laboratorium) and the fish oil (PPHW Tronina, Poland) were purchased on the wholesale market. The “Cortland” variety apple pomace was a waste material from “Maciejowy Sad” juice production. The apple pomace was dried in a laboratory oven at 60°C (Pol-Eko Aparatura, type SLW 115 TOP+, Poland) before being ground and mixed with the oils to obtain samples with 10% and 30% by weight of the oil. Samples of 50 g were packed and sealed using a vacuum packing machine (TEPRO SA Model PP3, Poland) and stored at a room temperature for 56 days. The samples were subjected to storage tests, including determination of total polyphenols by the Follin–Ciocalteu method [
The fatty acid profile of the test samples was analyzed by gas chromatography coupled with mass spectrometry, whereby the compositions of the obtained methyl esters were determined.
The tests were carried out using the methodology developed by Maślak et al. [
The fatty acid profiles of the test samples were analyzed using gas chromatography coupled with mass spectrometry (Saturn Chrompack 2000/2000). Separation was effected using a nonpolar column ZB WAX-MS (30 m × 0.25 mm × 0.25
The acids in the test samples were identified based on three different analytical methods: (1) comparison of Kovac retention indices, as found by the logarithmic method with respect to linear
The phenolic content and antioxidant activity of the formulations were determined using the UV-2401PC spectrophotometer from Shimadzu.
The extract for analyses was prepared by weighing 0.5 g of the formulation and adding enough 50% aqueous solution of methanol (50% aqueous solution of methanol + SO2 (1 ml/l)) to obtain a volume of 5 ml, keeping on water bath for 15 min, and cooling for 12 hours. The resulting solution was filtered. The following parameters were determined for the extracts.
Total phenolic content by the Follin–Ciocalteu method in which phenol compounds, among other ones, form a colored complex with the Follin–Ciocalteu reagent (a mixture of sodium tungstate, sodium molybdate, and lithium sulfate in a medium composed of phosphoric and hydrochloric acids); the complex is green-blue. After oxidation, the complex was analyzed spectrophotometrically at wavelengths starting at 765 nm. The phenolic content was calculated as gallic acid (GA).
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical binding power, which consists in reacting the antioxidants present in the test sample, reduce the stable nitrogen radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), thereby decreasing absorbance measured at 517 nm. The active radical solution is purple, and its discoloration indicates that the previously unpaired electron has been paired. The intensity of discoloration of the DPPH solution after addition of the solution containing the antioxidants is a measure of their free-radical scavenging ability.
Antioxidant activity by the method with ABTS•+ enables the quantitative assessment of free-radical scavenging ability of a given component to quench the stable ABTS•+ radical (2,2′-azine-bis acid (3-ethylenebenzothiazoline ABTS•+). Absorbance was measured at wavelength
The FRAP is based on the reduction of ferric 2,4,6-tris(2-pyridyl)-1,3,5-triazine [Fe(III)-TPTZ] to the ferrous complex at low pH, followed by spectrophotometric analysis. Quantitative analyses were performed by the external standard method using ferrous sulfate as the reference standard and correlating the absorbance (
The results of antioxidant capability of DPPH, ABTS•+, and FRAP were expressed in
The data of apple pomace and linseed oil included in Tables
The content of total polyphenols in the test formulations during storage (mg GA/100 g).
Total phenolic mg GA/100 g (ml) | Storage time (day) | SEM |
| ||||
---|---|---|---|---|---|---|---|
0 | 7 | 14 | 28 | 56 | |||
Pure pomace | 613.74Aa | 585.57 | 555.60 | 538.29 | 530.20 | 16.89 | 0.01 |
Pomace + fish oil 10% | 563.96Ab | 423.81A | 411.36A | 369.4A | 279.99Ba | 17.59 | <0.01 |
Pomace + linseed oil 10% | 571.31Ab | 451A | 375.59B | 298.01Ba | 308.79Bb | 17.46 | <0.01 |
Pomace + fish oil 30% | 441.36B | 354.93Ba | 279.82B | 290.43B | 197.59A | 14.79 | 0.01 |
Pomace + linseed oil 30% | 454.36B | 389.75Bb | 319.9C | 279.3Bb | 218.97A | 17.20 | <0.01 |
The letters A, B, and C in the same columns indicate statistically high significant differences (
The antioxidant activity (DPPH) apple pomace together with the tested oils (
DPPH ( |
Storage time (day) | SEM |
| ||||
---|---|---|---|---|---|---|---|
0 | 7 | 14 | 28 | 56 | |||
Pure pomace | 14.13 | 13.20 | 11.10 | 10.50 | 9.30 | 0.39 | <0.01 |
Pomace + fish oil 10% | 15.51 | 15.20A | 15.10A | 14.90A | 14.55A | 0.42 | 0.93 |
Pomace + linseed oil 10% | 15.60 | 15.47A | 15.95A | 14.11A | 11.28B | 0.44 | <0.01 |
Pomace + fish oil 30% | 16.71 | 15.9A | 10.42B | 11.25B | 12.38 | 0.53 | 0.01 |
Pomace + linseed oil 30% | 14.64 | 13.7B | 13.70A | 11.94B | 9.28B | 0.44 | <0.01 |
The letters A and B in the same columns indicate statistically high significant differences (
The antioxidant activity (FRAP) of apple pomace together with the tested oils (
FRAP ( |
Storage time (day) | SEM |
| ||||
---|---|---|---|---|---|---|---|
0 | 7 | 14 | 28 | 56 | |||
Pure pomace | 12.57A | 11.90Aa | 11.20Aa | 10.50A | 10.40A | 0.68 | 0.07 |
Pomace + fish oil 10% | 21.20 | 20.82B | 18.6B | 16.84B | 13B | 0.79 | 0.03 |
Pomace + linseed oil 10% | 23.41B | 22.2B | 19.36B | 17.06B | 12.57B | 0.91 | <0.01 |
Pomace + fish oil 30% | 22.31 | 22.0B | 17.56b | 17.78B | 10.95A | 0.48 | 0.14 |
Pomace + linseed oil 30% | 23.48B | 17.1Ab | 15.67 | 16.72B | 11.36 | 0.82 | <0.01 |
The letters A and B in the same columns indicate statistically high significant differences (
The antioxidant activity (ABTS•+) of apple pomace together with the tested oils (
ABTS ( |
Storage time (day) | SEM |
| ||||
---|---|---|---|---|---|---|---|
0 | 7 | 14 | 28 | 56 | |||
Pure pomace | 27.93 | 26.89 | 25.62a | 24.10A | 18.54A | 1.45 | 0.01 |
Pomace + fish oil 10% | 36.30 | 35.56A | 24.58a | 17.35Ba | 14.18b | 1,69 | <0.01 |
Pomace + linseed oil 10% | 32.41 | 29.06 | 24.35a | 10.08C | 13.69 | 1.41 | <0.01 |
Pomace + fish oil 30% | 28.75 | 24.8B | 24.1a | 12.38b | 13.87 | 1.56 | <0.01 |
Pomace + linseed oil 30% | 34.62 | 29.65 | 19.93b | 14.74 | 11.54Ba | 1.50 | <0.01 |
The letters A and B in the same columns indicate statistically high significant differences (
Changes in acid number (mg KOH/g) of the test oils stored with the addition of apple pomace.
Changes in peroxide value (meq O2/kg) of the test oils stored with the addition of apple pomace.
A statistical analysis of the numerical values, obtained in the tests, was performed using the Statistica 10.0 software (StatSoft, Tulsa, OK, USA), a two-way analysis of variance (ANOVA). The significance of differences between the means was assessed using the Duncan test. Determined arithmetic means (
Total phenolic content and the antioxidant activity (DPPH, FRAP, and ABTS•+) in the “Cortland” apple pomace before addition of the fats are shown in Table
The content of total polyphenols and antioxidant activity (DPPH, FRAP, and ABTS+) of apple pomace (
Item | Initial sample ( |
---|---|
Total polyphenols | 613.74 ± 19.19 |
DPPH | 14.13 ± 0.89 |
FRAP | 12.57 ± 2.56 |
ABTS | 27.93 ± 6.83 |
Antioxidant activity of the alcoholic extracts of apple pomace is shown in Tables
After seven days, the highest Fe3+ reducing power was observed for the samples of apple pomace mixed with 10% linseed oil (Table
In this paper, dried apple pomace was used as the natural source of antioxidants. Many authors who have tested apples or apple pomace confirm their high antioxidant activity, although its specific value largely depends on the fruit variety [
The metal ion reducing power is one of the mechanisms of action of antioxidants and is typical of secondary antioxidants [
Sea fish and sea animals in general, as well as the oils derived from them, are a rich source of EPA and DHA acids [
The fish oil tested herein was characterized by a higher content of C16:0, C16:1, and C18:1
Fatty acid content (%) in fish and linseed oil or in formulations (oil + apple pomace) before and after 56 days of storage under anaerobic conditions.
Fatty acid | Oils and preparations during the storage period | |||||
---|---|---|---|---|---|---|
Fish oil | Fish oil after 56 d | Fish oil + apple pomace after 56 d | Linseed oil | Linseed oil after 56 d | Linseed oil + apple pomace after 56 d | |
C14:0 | 2.78 | 2.72 | 2.36 | 0.02 | 0.02 | 0.33 |
C15:0 | 0.27 | 0.23 | 0.34 | nd | nd | nd |
C16:0 | 12.85 | 11 | 11.94 | 5.29 | 5.39 | 6.15 |
C16:1 cis | 4.40 | 4.39 | 4.65 | 0.03 | 0.02 | 0.34 |
C17:0 | 1.96 | 0.96 | 1.15 | 0.02 | 0.03 | 0.20 |
C17:1 cis | 0.21 | 0.23 | 0.24 | nd | nd | nd |
C18:0 | 2.97 | 2.92 | 3.05 | 3.97 | 4.37 | 4.58 |
C18:1 |
33.12 | 33.41 | 33.01 | 20.75 | 21.07 | 21.12 |
C18:1 |
3.37 | 3.49 | 3.41 | 0.80 | 0.86 | 1.12 |
C18:2 |
10.90 | 11.29 | 11.6 | 15.81 | 16.49 | 17.07 |
C18:3 |
0.32 | 0.42 | 0.38 | 0.03 | 0.01 | 0.63 |
C18:3 |
4.12 | 382 | 3.97 | 52.73 | 51.01 | 47.35 |
C18:4 |
1.04 | 1.14 | 1.06 | nd | nd | nd |
C20:0 | 0.24 | 0.30 | 0.27 | 0.12 | 0.19 | 0.16 |
C20:1 |
5.03 | 5.39 | 5.26 | 0.15 | 0.18 | 0.26 |
C20:2 |
0.75 | 0.84 | 0.74 | 0.13 | 0.18 | 0.26 |
C20:3 |
0.37 | 0.46 | 0.37 | nd | nd | nd |
C20:3 |
0.65 | 1.45 | 1.7 | nd | nd | nd |
C20:4 |
0.53 | 0.58 | 0.31 | 0.06 | 0.09 | 0.34 |
C20:5 |
3.03 | 3.11 | 3.15 | nd | nd | nd |
C21:0 | 0.14 | 0.18 | 0.14 | nd | nd | nd |
C22:0 | 4.50 | 4.97 | 4.65 | nd | nd | nd |
C22:1 |
0.40 | 0.11 | 0.12 | nd | nd | nd |
C22:5 |
0.93 | 1.06 | 1.15 | nd | nd | nd |
C22:6 |
5.12 | 5.53 | 4.98 | nd | nd | nd |
Saturated FA | 25.71 | 23.28 | 23.90 | 9.42 | 10.00 | 11.42 |
Unsaturated FA | 74.29 | 76.72 | 76.10 | 90.49 | 89.91 | 88.49 |
Monounsaturated FA | 46.53 | 47.02 | 46.69 | 21.73 | 22.13 | 22.84 |
Polyunsaturated FA | 27.76 | 29.70 | 29.41 | 68.76 | 67.78 | 65.65 |
Total |
13.80 | 14.65 | 14.55 | 16.03 | 16.77 | 18.30 |
Total |
13.96 | 15.05 | 14.86 | 52.73 | 51.01 | 47.35 |
The formulation tested in our study is applicable in animal feed production for modification of the fatty acid profile in animal tissues and milk. Supplementation of cow feeds with fish oil or a lipid complex based on fish oil and vegetable oils caused a considerable increase in the content of CLA in milk fat [
On the starting day of the experiment, AV was 16.17 mg KOH/g and 1.05 mg KOH/g in the fish oil and linseed oil, respectively (Figure
The fat oxidation process intensity, as measured with their PV, indicates the current amount of peroxides. Before using the apple pomace, the PV was 1.7 meq O2 per 1 kg of the linseed oil and 3.99 meq O2 per 1 kg of the fish oil (Figure
Anaerobic storage of the fish or linseed oils mixed with the apple pomace inhibited the increase of AV and PV in the stored samples. Aerobic conditions tended to result in higher increases in AV and PV, according to earlier reports [
The apple pomace was the source of phenolic compounds enhancement of oils stability, evidenced by peroxide and hydroxide value at the end of the study. After 56 days of storage, the total polyphenol contents in all tested combinations of fish or linseed oils with pomace were reduced (46 to 55%). However, apple pomace used in connection with oils, storage under anaerobic conditions did not significantly increased oxidation or degradation of fatty acids. The highest antioxidant capacity against DPPH• radicals and ABTS radicals cation was found in combination of apple pomace with 10% of linseed oil content, and the lowest capacity was found in samples with 30% of linseed oil content. In animals, feeding could be solved in the proposed approach, which makes the product acceptable for livestock breeding. Pomace, as a relative rich source of polyphenols, could improve metabolic and oxidative processes in animals. Therefore, the further research in this direction is necessary.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This project was supported by Wroclaw Centre of Biotechnology through the programme “The Leading National Research Centre” (KNOW) for years 2014–2018.