Article pubs.acs.org/JAFC
Complex Role of Monoacylglycerols in the Oxidation of Vegetable Oils: Different Behaviors of Soybean Monoacylglycerols in Different Oils Vito Michele Paradiso, Francesco Caponio,* Giuseppina Bruno, Antonella Pasqualone, Carmine Summo, and Tommaso Gomes Department of Soil, Plant and Food Sciences, Food Science and Technology Unit, University of Bari Aldo Moro, Via Amendola 165/A, I-70126 Bari, Italy ABSTRACT: The relationship between fatty acid composition of oils and their oxidative stability in the presence of monoacylglycerols was investigated. Purified vegetable oils were added at increasing amounts (0.5, 1, 2, and 3%) of monoacylglycerols obtained from purified soybean oil and submitted to an oven test (60 °C for 18 days). The obtained results showed a generally antioxidant effect of monoacylglycerols, with remarkable differences among oils. The antioxidant effect was significantly higher in less unsaturated oils, such as palm and olive oils. Among the more unsaturated vegetable oils, peanut and sunflower oils showed an almost linear slowdown of oxidation, slightly less pronounced in sunflower oil, which was the most susceptible to oxidation due to its high content of linoleic acid. A peculiar trend was highlighted for soybean oil, where the antioxidant effect of high amounts of monoacylglycerols was opposed to a pro-oxidant effect observed up to 1%. KEYWORDS: fatty acid composition, monoacylglycerols, oxidative stability, vegetable oils
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INTRODUCTION
poorer in polyunsaturated fatty acids (PUFA) with respect to soybean oil. The comprehension of the real mechanism of action of MAG in the oxidative phenomena is worthy of attention because they are included in the formulation of many food products as emulsifiers. The aim of the present work was to investigate the relationship between the fatty acid composition of the oils and their oxidative stability in the presence of MAG. For this purpose, to exclude other variables, some of the purified MAG, obtained from purified soybean oil, were added at percentages between 0.5 and 3% to different purified vegetable oils: olive, peanut, sunflower, soybean, and palm. The obtained mixtures were submitted to an oven test at 60 °C for 18 days.
Alterations affecting the edible fats and oils have mainly hydrolytic and oxidative nature. Oxidative deterioration, in particular, decreases the nutritional values and produces potentially toxic compounds,1,2 altering the flavor, color, and safety of edible oils.3−6 Many factors influence lipid oxidation, including pro-oxidant and antioxidant substances. An antioxidant may be defined as a substance that, when present at low concentrations, compared with those of the oxidizable substrate, retards or significantly inhibits the oxidation of that substrate.7 On the other hand, metals,8−10 free fatty acids,11−13 triacylglycerol oligopolymers (TAGP),14 and oxidized triacylglycerols (ox-TAG),15,16 as well as diacylglycerols,17 act as prooxidants and accelerate the oxidation rate of edible oils. With regard to the monoacylglycerols (MAG), the data reported in the literature appear not univocal.17−22 Mistry and Min20 and Colakoglu19 showed a pro-oxidant activity of commercial standards of MAG when added to purified soybean oil. Gomes et al.21 evaluated the effect of MAG obtained from purified olive oil and having the same fatty acid composition as the tested oil they were added to. They observed a significant slowdown of the oxidative process during the oven test at 60 °C. Successively, Caponio et al.,22 by adding increasing amounts of MAG obtained from purified soybean oil to the same oil, highlighted a dose-dependent effect: MAG acted as pro-oxidant when added at low amounts (up to 1%), confirming the data reported in the literature,19 and as antioxidant at higher amounts. The different behaviors of MAG in different oils could be explained by the different fatty acid compositions of the purified oils they were added to, olive oil being richer in monounsatured fatty acids (MUFA) and © 2014 American Chemical Society
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MATERIALS AND METHODS
Preparation of Purified Oils. Olive, peanut, sunflower, soybean, and palm oils purchased from local retail were used for the preparation of the respective purified oils (PO) using the method described by Lee and Min23 with some slight modifications. A 50 cm × 4 cm column, packed with 75 g of silica gel 70−230 mesh ASTM (Merck Darmstadt, Germany), 12.6 g of charcoal/Celite (2:1), 37.5 g of sucrose powder/ Celite (2:1), and 75 g of silica gel, was used. A flow rate of 0.20 mL/ min was obtained by applying a slight depression at the outlet of the column with a vacuum pump. The obtained purified oils were, then, free of MAG, free fatty acids, tocopherols, phospholipids, and oxidized compounds.11 Preparation of Purified MAG. Purified MAG were obtained by partial saponification of an aliquot of purified soybean oil. In particular, 10 g of purified soybean oil was added with 100 mL of a 2 N NaOH Received: Revised: Accepted: Published: 10776
June 3, 2014 October 6, 2014 October 13, 2014 October 13, 2014 dx.doi.org/10.1021/jf5025888 | J. Agric. Food Chem. 2014, 62, 10776−10782
Journal of Agricultural and Food Chemistry
Article
Table 1. Percent Fatty Acid Composition of Purified Oils and of Monoacylglycerols (MAG) Obtained from Soybean Purified Oil purified oil fatty acid C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 iodine valuea a
peanut 0.21 11.39 0.23 0.23 0.09 1.57 53.42 30.98 0.22 0.88 0.76 101.55
± ± ± ± ± ± ± ± ± ± ±
0.03 0.75 0.09 0.08 0.02 0.15 1.01 0.78 0.05 0.09 0.05
palm 0.72 36.45 0.52 0.44 0.40 5.05 45.90 8.87 0.46 0.64 0.55 61.88
± ± ± ± ± ± ± ± ± ± ±
0.14 1.65 0.06 0.09 0.05 0.24 0.39 0.08 0.04 0.11 0.10
olive 0.03 9.59 0.36 0.27 0.26 2.53 75.91 9.55 0.63 0.59 0.30 85.99
± ± ± ± ± ± ± ± ± ± ±
sunflower 0.01 0.65 0.06 0.03 0.03 0.11 1.22 0.55 0.12 0.04 0.09
0.12 6.42 0.06 0.05 0.05 3.56 29.29 58.93 0.38 0.21 0.94 115.30
± ± ± ± ± ± ± ± ± ± ±
0.02 0.46 0.01 0.01 0.01 0.22 0.31 0.97 0.09 0.03 0.17
soybean 0.57 12.67 0.39 0.32 0.22 3.68 31.16 44.01 4.71 1.13 1.15 114.03
± ± ± ± ± ± ± ± ± ± ±
0.15 0.33 0.13 0.14 0.09 0.29 1.01 1.14 0.24 0.13 0.16
purified MAG 0.29 14.29 0.35 0.33 0.26 3.02 29.47 47.03 4.18 0.37 0.40
± ± ± ± ± ± ± ± ± ± ±
0.04 0.27 0.08 0.09 0.14 0.43 0.69 1.02 0.23 0.06 0.07
Calculated as reported by Kyriakidis and Katsiloulis.26 double bonds respectively, whereas x, y, and z are the relative coefficients. TAGP and ox-TAG were determined by means of high-performance size exclusion chromatography (HPSEC) of polar compounds (PCs), previously separated from the oil samples by silica gel column chromatography according to AOAC method 982.27.27 After elution of the nonpolar components with 150 mL of petroleum ether/diethyl ether (87:13, v/v), the PCs were recovered with 150 mL of diethyl ether. After removal of the diethyl ether, the PCs were recovered in THF and the efficacy of separation was checked by thin layer chromatography as recommended by the same method. The chromatographic system consisted of a PerkinElmer pump, series 200, a 50 μL injector loop, a PL-gel guard column (PerkinElmer, Beaconsfield, UK) of 5 cm length × 7.5 mm i.d., and a series of two PL-gel columns (PerkinElmer) of 7.5 mm i.d. × 30 cm in length each. The columns were packed with highly cross-linked styrene divinylbenzene copolymers with a particle diameter of 5 μm and pore diameters of 500 Å. The detector was a differential refractometer (series 200A, PerkinElmer). The elution solvent used was THF for HPLC at a flow rate of 1.0 mL min−1. The identification and quantification of individual peaks was carried out as described in previous papers.28,29 The precision of the HPSEC method, expressed as RSD% (n = 10), was 1.9% for PCs, 3.2% for TAGP, and 2.3% for ox-TAG. Three replicates were analyzed per sample for each determination. Statistical Analysis. Analysis of variance (two-way ANOVA) was carried out on the experimental data by using XLStat software (Addinsoft SARL, New York, NY, USA). Two-way ANOVA was performed considering the amount of MAG added to PO (MAG) and the different vegetable oils considered (oil), as well as their first -order interaction, as independent variables; Tukey’s HSD test was applied for multiple comparisons. The surface response regression analysis was carried out by Minitab 16 software (Minitab Inc., State College, PA, USA).
solution in methanol and vortexed for 3 min. Then, 200 mL of distilled water and 200 mL of diethyl ether were added. After mixing, the aqueous phase was removed while the etheric phase was repeatedly washed with distilled water (80 mL each time), until neutrality of the discarded water. Then, after filtration on sodium sulfate anhydrous, the ether was removed in a rotary evaporator. The adopted saponification method led to the formation of 98% of MAG, as confirmed by the HPSEC analysis. Obtained MAG were added to each PO (olive, peanut, sunflower, soybean, and palm) to obtain the following MAG/ PO proportions: 0.5% (MAG0.5); 1% (MAG1); 2% (MAG2); 3% (MAG3). For each type of oil, as control (C) was used the PO with no added MAG. Oven Test. Five grams of each PO was weighed in 50 mL glass vials and kept for 18 days at 60 °C, which represents the temperature commonly employed in a forced air oven. Fifty vials were prepared for each PO. At 4, 6, 10, 14, and 18 days 10 vials for each PO (two for control and for each MAG/PO proportion) were taken and submitted to analyses. Chemical Analyses. The determinations of the free fatty acids (FFA), peroxide value (PV), and spectrophotometric constants were carried out according to the Of f icial Journal of the European Communities.24 Fatty acids composition was carried out by gas chromatographic analysis of fatty acid methyl esters, according to the official methods.24,25 In particular, the oil was treated with 2 N potassium hydroxide solution in methanol and vortexed for 5 min, and then 1 mL of hexane was added. Then, 1 μL of the hexane fraction was injected. The gas chromatographic system was composed of an Agilent model 7890A gas chromatograph (Cernusco, MI, Italy), equipped with a flame ionization detector (FID) and a WCOT fused-silica capillary column, FFAP-CB coating, 0.32 mm i.d. × 25 m length and 0.30 μm film thickness (Chrompack, Middleburg, The Netherlands). The temperature of the split injector was 210 °C, with a splitting ratio of 1:17; the detector temperature was 220 °C. The oven temperature was isothermally at 180 °C. Helium at flow rate of 1 mL min−1 was utilized as carrier gas. The identification of each fatty acid was carried out by comparing the retention time with that of the corresponding standard methyl ester purchased from Sigma-Aldrich (St. Louis, MO, USA). RSD was about 5%. The iodine value (IV) was determined as proposed by Kyriakidis and Katsiloulis,26 which uses the percentage of fatty acid methyl esters determined from analysis of the fatty acid composition and an equation with coefficients specific for every type of vegetable oil. The equation proposed is
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RESULTS AND DISCUSSION Characterization of Raw Materials. Table 1 reports the fatty acid composition of PO of olive, peanut, sunflower, soybean, and palm oils and of the MAG obtained from the purified soybean oil. The observed values agreed with those of the botanic species they derived from; higher saturated fatty acid levels were detected in palm oil, whereas higher monounsaturated levels were observed in olive, followed by peanut, palm, soybean, and sunflower oils. Linoleic acid was markedly more represented in sunflower, soybean, and peanut oils than in olive and palm oils; soybean oil, finally, showed the highest contents of linolenic acid. The differences in the
IV = xC1 + yC2 + zC3 where C1, C2, and C3 correspond to the sum of the relative percentage concentrations of the unsaturated fatty acids with one, two, and three 10777
dx.doi.org/10.1021/jf5025888 | J. Agric. Food Chem. 2014, 62, 10776−10782
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Table 2. Chemical Characteristics of Purified Oils oil parametera
peanut
−1
FFA (g 100 g ) PV (mequiv O2 kg−1) K232 K270 TAGP (g 100 g−1) ox-TAG (g 100 g−1) DAG (g 100 g−1) PCs (g 100 g−1)
0.00 0.00 0.46 0.09 0.02 0.00 0.08 0.09
± ± ± ± ± ± ± ±
palm
0.00 0.00 0.05 0.01 0.00 0.00 0.01 0.01
0.00 0.00 0.53 0.02 0.00 0.02 0.07 0.10
± ± ± ± ± ± ± ±
olive
0.00 0.00 0.05 0.00 0.00 0.00 0.01 0.01
0.00 0.00 0.59 0.04 0.00 0.00 0.03 0.04
± ± ± ± ± ± ± ±
sunflower
0.00 0.00 0.04 0.01 0.00 0.00 0.00 0.01
0.00 0.00 0.96 0.08 0.03 0.02 0.06 0.11
± ± ± ± ± ± ± ±
0.00 0.00 0.06 0.01 0.00 0.00 0.01 0.01
soybean 0.00 0.00 1.29 0.09 0.00 0.08 0.03 0.11
± ± ± ± ± ± ± ±
0.00 0.00 0.07 0.01 0.00 0.01 0.00 0.01
a FFA, free fatty acids; PV, peroxide value; K232, specific absorption at 232 nm; K270, specific absorption at 270 nm; TAGP, triacylglycerol oligopolymers; ox-TAG, oxidized triacylglycerols; DAG, diacylglycerols; PCs, polar compounds.
Table 3. Mean Values of the Analyses Carried out on Purified Oils (POs) and on the Same with Added 0.5, 1, 2, and 3% of Monoacylglycerols (MAG0.5, MAG1, MAG2, MAG3) after 10 Days at 60 °C, as well as Results of Statistical Analysis (Two-Way ANOVA followed by Tukey’s HSD Test for Multiple Comparisons) oil determinationa
MAG %
peanut
palm
olive
PV
PO MAG0.5 MAG1 MAG2 MAG3
0 0.5 1 2 3
Ab B C D E
180.0 146.0 111.3 91.6 85.7
± ± ± ± ±
3.9eb 3.1fgh 2.4i 2.0j 1.8j
79.5 64.2 46.3 17.7 13.8
± ± ± ± ±
1.7j 1.4k 1.0l 0.4m 0.3m
139.9 47.5 13.5 8.7 6.3
± ± ± ± ±
3.0h 1.0l 0.3m 0.2m 0.1m
TAGP
PO MAG0.5 MAG1 MAG2 MAG3
0 0.5 1 2 3
A B C D E
2.53 2.05 1.83 1.66 1.38
± ± ± ± ±
0.02fg 0.02h 0.08i 0.01j 0.03k
0.87 0.63 0.59 0.51 0.31
± ± ± ± ±
0.02l 0.01m 0.01m 0.01m 0.01n
1.01 0.19 0.06 0.04 0.02
± ± ± ± ±
ox-TAG
PO MAG0.5 MAG1 MAG2 MAG3
0 0.5 1 2 3
B B A A B
8.85 8.07 7.39 7.06 6.01
± ± ± ± ±
0.02e 0.06f 0.29ij 0.02k 0.12l
4.26 2.95 2.04 0.85 0.51
± ± ± ± ±
0.04m 0.09n 0.02p 0.01q 0.01rs
7.13 2.42 0.66 0.48 0.32
± ± ± ± ±
sunflower
soybean
389.1 355.6 332.6 329.0 289.6
± ± ± ± ±
8.4a 7.7b 7.2c 7.1c 6.2d
155.9 158.8 159.5 148.0 143.7
± ± ± ± ±
1.2fg 0.2f 0.6f 1.4fgh 1.6gh
0.01l 0.01no 0.01op 0.00op 0.00p
4.22 3.90 3.40 3.27 2.92
± ± ± ± ±
0.03a 0.02b 0.10c 0.13c 0.02d
2.44 2.56 2.72 2.66 2.43
± ± ± ± ±
0.01g 0.01efg 0.01e 0.02ef 0.03g
0.03jk 0.08o 0.01qr 0.01rs 0.01s
12.98 12.22 11.54 11.31 11.20
± ± ± ± ±
0.09a 0.02b 0.07c 0.12cd 0.04d
7.62 7.73 7.99 7.85 7.70
± ± ± ± ±
0.01hi 0.01gh 0.01fg 0.02fgh 0.04ghi
PV, peroxide value (mequiv O2 kg−1); TAGP, triacylglycerol oligopolymers (g 100 g−1); ox-TAG, oxidized triacylglycerols (g 100 g−1). bUpper case letters are used to compare the samples considering the amount of monoacylglycerols added to purified oil; lower case letters are used to compare the samples considering the combined effect of MAG (%), oilm and MAG × oil interaction: different letters mean significant differences at p ≤ 0.05. a
oxidative phenomena were observed when MAG were added to PO derived from olive, palm, peanut, and sunflower, confirming previous results obtained by adding MAG prepared from olive oil to the same olive oil.21 A different and particular trend, instead, was observed when MAG were added to soybean PO. In this case, all of the analytical indices considered, apart from PV, significantly increased when MAG amounts up to 1% were added, confirming other research,19,20,22 and decreased at higher MAG doses. Oven Test: Surface Response Regression. In a recent review, Chen and collaborators30 pointed out the presence of discordant data about MAG behavior toward oxidation. Moreover, the effect of compositional characteristics of MAG (chain length, unsaturation level) have been investigated, but the role of fatty acid composition of the oil to which MAG are added has been scarcely studied, as well as the relationships between the two compositions. We adopted surface response regression analysis to point out the effect of MAG on oxidation in oils having different fatty acid compositions. Table 4 reports the coefficients of the regressions and the corresponding p values. The use of coded units enables
unsaturation degree were paralleled by the IV, which was higher in sunflower and soybean oils than in the others. Table 1 shows also that the fatty acid composition of MAG was similar to that of the corresponding soybean purified oil used for their preparation. POs were constituted by unaltered triacylglycerols (>99.9%) and showed FFA and PV values equal to zero and low values for the spectrophotometric constants (Table 2), similar to those detected in a previous study.14 Oven Test: Oxidation after 10 Days at 60 °C. Table 3 reports the results of the analyses carried out on each PO and on the same PO with added 0.5, 1, 2, and 3% MAG (MAG0.5, MAG1, MAG2, and MAG3, respectively) after 10 days at 60 °C, as well as the results of statistical analysis (two-way ANOVA followed by Tukey’s HSD test for multiple comparisons). The data of oxidation after 10 days were reported as representative of the overall trend during time, to exclude, in the first instance, “time” as a third source of variability, besides MAG amount and type of oil. The addition of increasing amounts of MAG significantly influenced the oxidative process. In particular, less marked 10778
dx.doi.org/10.1021/jf5025888 | J. Agric. Food Chem. 2014, 62, 10776−10782
Journal of Agricultural and Food Chemistry
Article
Table 4. Results of the Surface Response Regression: Regression Coefficients and Respective p Values peroxide valuea term constant time MAG time × time MAG × MAG MAG × time constant time MAG time × time MAG × MAG MAG × time constant time MAG time × time MAG × MAG MAG × time constant time MAG time × time MAG × MAG MAG × time constant days MAG time × time MAG × MAG MAG × time
coefficient Soybean Oil 176.511 119.497 −2.023 110.945 −9.711 6.544 Sunflower Oil 345.23 286.83 −28.58 −9.54 5.38 −3.22 Peanut Oil 138.85 162.99 −25.25 34.68 15.50 −11.87 Palm Oil 38.64 56.44 −42.94 13.23 20.88 −42.79 Olive Oil 5.640 47.130 −68.435 20.049 65.648 −54.630
ox-TAGa
TAGPa
p value
coefficient
p value
coefficient
p value
Complex Role of Monoacylglycerols in the Oxidation of Vegetable Oils: Different Behaviors of Soybean Monoacylglycerols in Different Oils Vito Michele Paradiso, Francesco Caponio,* Giuseppina Bruno, Antonella Pasqualone, Carmine Summo, and Tommaso Gomes Department of Soil, Plant and Food Sciences, Food Science and Technology Unit, University of Bari Aldo Moro, Via Amendola 165/A, I-70126 Bari, Italy ABSTRACT: The relationship between fatty acid composition of oils and their oxidative stability in the presence of monoacylglycerols was investigated. Purified vegetable oils were added at increasing amounts (0.5, 1, 2, and 3%) of monoacylglycerols obtained from purified soybean oil and submitted to an oven test (60 °C for 18 days). The obtained results showed a generally antioxidant effect of monoacylglycerols, with remarkable differences among oils. The antioxidant effect was significantly higher in less unsaturated oils, such as palm and olive oils. Among the more unsaturated vegetable oils, peanut and sunflower oils showed an almost linear slowdown of oxidation, slightly less pronounced in sunflower oil, which was the most susceptible to oxidation due to its high content of linoleic acid. A peculiar trend was highlighted for soybean oil, where the antioxidant effect of high amounts of monoacylglycerols was opposed to a pro-oxidant effect observed up to 1%. KEYWORDS: fatty acid composition, monoacylglycerols, oxidative stability, vegetable oils
■
INTRODUCTION
poorer in polyunsaturated fatty acids (PUFA) with respect to soybean oil. The comprehension of the real mechanism of action of MAG in the oxidative phenomena is worthy of attention because they are included in the formulation of many food products as emulsifiers. The aim of the present work was to investigate the relationship between the fatty acid composition of the oils and their oxidative stability in the presence of MAG. For this purpose, to exclude other variables, some of the purified MAG, obtained from purified soybean oil, were added at percentages between 0.5 and 3% to different purified vegetable oils: olive, peanut, sunflower, soybean, and palm. The obtained mixtures were submitted to an oven test at 60 °C for 18 days.
Alterations affecting the edible fats and oils have mainly hydrolytic and oxidative nature. Oxidative deterioration, in particular, decreases the nutritional values and produces potentially toxic compounds,1,2 altering the flavor, color, and safety of edible oils.3−6 Many factors influence lipid oxidation, including pro-oxidant and antioxidant substances. An antioxidant may be defined as a substance that, when present at low concentrations, compared with those of the oxidizable substrate, retards or significantly inhibits the oxidation of that substrate.7 On the other hand, metals,8−10 free fatty acids,11−13 triacylglycerol oligopolymers (TAGP),14 and oxidized triacylglycerols (ox-TAG),15,16 as well as diacylglycerols,17 act as prooxidants and accelerate the oxidation rate of edible oils. With regard to the monoacylglycerols (MAG), the data reported in the literature appear not univocal.17−22 Mistry and Min20 and Colakoglu19 showed a pro-oxidant activity of commercial standards of MAG when added to purified soybean oil. Gomes et al.21 evaluated the effect of MAG obtained from purified olive oil and having the same fatty acid composition as the tested oil they were added to. They observed a significant slowdown of the oxidative process during the oven test at 60 °C. Successively, Caponio et al.,22 by adding increasing amounts of MAG obtained from purified soybean oil to the same oil, highlighted a dose-dependent effect: MAG acted as pro-oxidant when added at low amounts (up to 1%), confirming the data reported in the literature,19 and as antioxidant at higher amounts. The different behaviors of MAG in different oils could be explained by the different fatty acid compositions of the purified oils they were added to, olive oil being richer in monounsatured fatty acids (MUFA) and © 2014 American Chemical Society
■
MATERIALS AND METHODS
Preparation of Purified Oils. Olive, peanut, sunflower, soybean, and palm oils purchased from local retail were used for the preparation of the respective purified oils (PO) using the method described by Lee and Min23 with some slight modifications. A 50 cm × 4 cm column, packed with 75 g of silica gel 70−230 mesh ASTM (Merck Darmstadt, Germany), 12.6 g of charcoal/Celite (2:1), 37.5 g of sucrose powder/ Celite (2:1), and 75 g of silica gel, was used. A flow rate of 0.20 mL/ min was obtained by applying a slight depression at the outlet of the column with a vacuum pump. The obtained purified oils were, then, free of MAG, free fatty acids, tocopherols, phospholipids, and oxidized compounds.11 Preparation of Purified MAG. Purified MAG were obtained by partial saponification of an aliquot of purified soybean oil. In particular, 10 g of purified soybean oil was added with 100 mL of a 2 N NaOH Received: Revised: Accepted: Published: 10776
June 3, 2014 October 6, 2014 October 13, 2014 October 13, 2014 dx.doi.org/10.1021/jf5025888 | J. Agric. Food Chem. 2014, 62, 10776−10782
Journal of Agricultural and Food Chemistry
Article
Table 1. Percent Fatty Acid Composition of Purified Oils and of Monoacylglycerols (MAG) Obtained from Soybean Purified Oil purified oil fatty acid C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 iodine valuea a
peanut 0.21 11.39 0.23 0.23 0.09 1.57 53.42 30.98 0.22 0.88 0.76 101.55
± ± ± ± ± ± ± ± ± ± ±
0.03 0.75 0.09 0.08 0.02 0.15 1.01 0.78 0.05 0.09 0.05
palm 0.72 36.45 0.52 0.44 0.40 5.05 45.90 8.87 0.46 0.64 0.55 61.88
± ± ± ± ± ± ± ± ± ± ±
0.14 1.65 0.06 0.09 0.05 0.24 0.39 0.08 0.04 0.11 0.10
olive 0.03 9.59 0.36 0.27 0.26 2.53 75.91 9.55 0.63 0.59 0.30 85.99
± ± ± ± ± ± ± ± ± ± ±
sunflower 0.01 0.65 0.06 0.03 0.03 0.11 1.22 0.55 0.12 0.04 0.09
0.12 6.42 0.06 0.05 0.05 3.56 29.29 58.93 0.38 0.21 0.94 115.30
± ± ± ± ± ± ± ± ± ± ±
0.02 0.46 0.01 0.01 0.01 0.22 0.31 0.97 0.09 0.03 0.17
soybean 0.57 12.67 0.39 0.32 0.22 3.68 31.16 44.01 4.71 1.13 1.15 114.03
± ± ± ± ± ± ± ± ± ± ±
0.15 0.33 0.13 0.14 0.09 0.29 1.01 1.14 0.24 0.13 0.16
purified MAG 0.29 14.29 0.35 0.33 0.26 3.02 29.47 47.03 4.18 0.37 0.40
± ± ± ± ± ± ± ± ± ± ±
0.04 0.27 0.08 0.09 0.14 0.43 0.69 1.02 0.23 0.06 0.07
Calculated as reported by Kyriakidis and Katsiloulis.26 double bonds respectively, whereas x, y, and z are the relative coefficients. TAGP and ox-TAG were determined by means of high-performance size exclusion chromatography (HPSEC) of polar compounds (PCs), previously separated from the oil samples by silica gel column chromatography according to AOAC method 982.27.27 After elution of the nonpolar components with 150 mL of petroleum ether/diethyl ether (87:13, v/v), the PCs were recovered with 150 mL of diethyl ether. After removal of the diethyl ether, the PCs were recovered in THF and the efficacy of separation was checked by thin layer chromatography as recommended by the same method. The chromatographic system consisted of a PerkinElmer pump, series 200, a 50 μL injector loop, a PL-gel guard column (PerkinElmer, Beaconsfield, UK) of 5 cm length × 7.5 mm i.d., and a series of two PL-gel columns (PerkinElmer) of 7.5 mm i.d. × 30 cm in length each. The columns were packed with highly cross-linked styrene divinylbenzene copolymers with a particle diameter of 5 μm and pore diameters of 500 Å. The detector was a differential refractometer (series 200A, PerkinElmer). The elution solvent used was THF for HPLC at a flow rate of 1.0 mL min−1. The identification and quantification of individual peaks was carried out as described in previous papers.28,29 The precision of the HPSEC method, expressed as RSD% (n = 10), was 1.9% for PCs, 3.2% for TAGP, and 2.3% for ox-TAG. Three replicates were analyzed per sample for each determination. Statistical Analysis. Analysis of variance (two-way ANOVA) was carried out on the experimental data by using XLStat software (Addinsoft SARL, New York, NY, USA). Two-way ANOVA was performed considering the amount of MAG added to PO (MAG) and the different vegetable oils considered (oil), as well as their first -order interaction, as independent variables; Tukey’s HSD test was applied for multiple comparisons. The surface response regression analysis was carried out by Minitab 16 software (Minitab Inc., State College, PA, USA).
solution in methanol and vortexed for 3 min. Then, 200 mL of distilled water and 200 mL of diethyl ether were added. After mixing, the aqueous phase was removed while the etheric phase was repeatedly washed with distilled water (80 mL each time), until neutrality of the discarded water. Then, after filtration on sodium sulfate anhydrous, the ether was removed in a rotary evaporator. The adopted saponification method led to the formation of 98% of MAG, as confirmed by the HPSEC analysis. Obtained MAG were added to each PO (olive, peanut, sunflower, soybean, and palm) to obtain the following MAG/ PO proportions: 0.5% (MAG0.5); 1% (MAG1); 2% (MAG2); 3% (MAG3). For each type of oil, as control (C) was used the PO with no added MAG. Oven Test. Five grams of each PO was weighed in 50 mL glass vials and kept for 18 days at 60 °C, which represents the temperature commonly employed in a forced air oven. Fifty vials were prepared for each PO. At 4, 6, 10, 14, and 18 days 10 vials for each PO (two for control and for each MAG/PO proportion) were taken and submitted to analyses. Chemical Analyses. The determinations of the free fatty acids (FFA), peroxide value (PV), and spectrophotometric constants were carried out according to the Of f icial Journal of the European Communities.24 Fatty acids composition was carried out by gas chromatographic analysis of fatty acid methyl esters, according to the official methods.24,25 In particular, the oil was treated with 2 N potassium hydroxide solution in methanol and vortexed for 5 min, and then 1 mL of hexane was added. Then, 1 μL of the hexane fraction was injected. The gas chromatographic system was composed of an Agilent model 7890A gas chromatograph (Cernusco, MI, Italy), equipped with a flame ionization detector (FID) and a WCOT fused-silica capillary column, FFAP-CB coating, 0.32 mm i.d. × 25 m length and 0.30 μm film thickness (Chrompack, Middleburg, The Netherlands). The temperature of the split injector was 210 °C, with a splitting ratio of 1:17; the detector temperature was 220 °C. The oven temperature was isothermally at 180 °C. Helium at flow rate of 1 mL min−1 was utilized as carrier gas. The identification of each fatty acid was carried out by comparing the retention time with that of the corresponding standard methyl ester purchased from Sigma-Aldrich (St. Louis, MO, USA). RSD was about 5%. The iodine value (IV) was determined as proposed by Kyriakidis and Katsiloulis,26 which uses the percentage of fatty acid methyl esters determined from analysis of the fatty acid composition and an equation with coefficients specific for every type of vegetable oil. The equation proposed is
■
RESULTS AND DISCUSSION Characterization of Raw Materials. Table 1 reports the fatty acid composition of PO of olive, peanut, sunflower, soybean, and palm oils and of the MAG obtained from the purified soybean oil. The observed values agreed with those of the botanic species they derived from; higher saturated fatty acid levels were detected in palm oil, whereas higher monounsaturated levels were observed in olive, followed by peanut, palm, soybean, and sunflower oils. Linoleic acid was markedly more represented in sunflower, soybean, and peanut oils than in olive and palm oils; soybean oil, finally, showed the highest contents of linolenic acid. The differences in the
IV = xC1 + yC2 + zC3 where C1, C2, and C3 correspond to the sum of the relative percentage concentrations of the unsaturated fatty acids with one, two, and three 10777
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Table 2. Chemical Characteristics of Purified Oils oil parametera
peanut
−1
FFA (g 100 g ) PV (mequiv O2 kg−1) K232 K270 TAGP (g 100 g−1) ox-TAG (g 100 g−1) DAG (g 100 g−1) PCs (g 100 g−1)
0.00 0.00 0.46 0.09 0.02 0.00 0.08 0.09
± ± ± ± ± ± ± ±
palm
0.00 0.00 0.05 0.01 0.00 0.00 0.01 0.01
0.00 0.00 0.53 0.02 0.00 0.02 0.07 0.10
± ± ± ± ± ± ± ±
olive
0.00 0.00 0.05 0.00 0.00 0.00 0.01 0.01
0.00 0.00 0.59 0.04 0.00 0.00 0.03 0.04
± ± ± ± ± ± ± ±
sunflower
0.00 0.00 0.04 0.01 0.00 0.00 0.00 0.01
0.00 0.00 0.96 0.08 0.03 0.02 0.06 0.11
± ± ± ± ± ± ± ±
0.00 0.00 0.06 0.01 0.00 0.00 0.01 0.01
soybean 0.00 0.00 1.29 0.09 0.00 0.08 0.03 0.11
± ± ± ± ± ± ± ±
0.00 0.00 0.07 0.01 0.00 0.01 0.00 0.01
a FFA, free fatty acids; PV, peroxide value; K232, specific absorption at 232 nm; K270, specific absorption at 270 nm; TAGP, triacylglycerol oligopolymers; ox-TAG, oxidized triacylglycerols; DAG, diacylglycerols; PCs, polar compounds.
Table 3. Mean Values of the Analyses Carried out on Purified Oils (POs) and on the Same with Added 0.5, 1, 2, and 3% of Monoacylglycerols (MAG0.5, MAG1, MAG2, MAG3) after 10 Days at 60 °C, as well as Results of Statistical Analysis (Two-Way ANOVA followed by Tukey’s HSD Test for Multiple Comparisons) oil determinationa
MAG %
peanut
palm
olive
PV
PO MAG0.5 MAG1 MAG2 MAG3
0 0.5 1 2 3
Ab B C D E
180.0 146.0 111.3 91.6 85.7
± ± ± ± ±
3.9eb 3.1fgh 2.4i 2.0j 1.8j
79.5 64.2 46.3 17.7 13.8
± ± ± ± ±
1.7j 1.4k 1.0l 0.4m 0.3m
139.9 47.5 13.5 8.7 6.3
± ± ± ± ±
3.0h 1.0l 0.3m 0.2m 0.1m
TAGP
PO MAG0.5 MAG1 MAG2 MAG3
0 0.5 1 2 3
A B C D E
2.53 2.05 1.83 1.66 1.38
± ± ± ± ±
0.02fg 0.02h 0.08i 0.01j 0.03k
0.87 0.63 0.59 0.51 0.31
± ± ± ± ±
0.02l 0.01m 0.01m 0.01m 0.01n
1.01 0.19 0.06 0.04 0.02
± ± ± ± ±
ox-TAG
PO MAG0.5 MAG1 MAG2 MAG3
0 0.5 1 2 3
B B A A B
8.85 8.07 7.39 7.06 6.01
± ± ± ± ±
0.02e 0.06f 0.29ij 0.02k 0.12l
4.26 2.95 2.04 0.85 0.51
± ± ± ± ±
0.04m 0.09n 0.02p 0.01q 0.01rs
7.13 2.42 0.66 0.48 0.32
± ± ± ± ±
sunflower
soybean
389.1 355.6 332.6 329.0 289.6
± ± ± ± ±
8.4a 7.7b 7.2c 7.1c 6.2d
155.9 158.8 159.5 148.0 143.7
± ± ± ± ±
1.2fg 0.2f 0.6f 1.4fgh 1.6gh
0.01l 0.01no 0.01op 0.00op 0.00p
4.22 3.90 3.40 3.27 2.92
± ± ± ± ±
0.03a 0.02b 0.10c 0.13c 0.02d
2.44 2.56 2.72 2.66 2.43
± ± ± ± ±
0.01g 0.01efg 0.01e 0.02ef 0.03g
0.03jk 0.08o 0.01qr 0.01rs 0.01s
12.98 12.22 11.54 11.31 11.20
± ± ± ± ±
0.09a 0.02b 0.07c 0.12cd 0.04d
7.62 7.73 7.99 7.85 7.70
± ± ± ± ±
0.01hi 0.01gh 0.01fg 0.02fgh 0.04ghi
PV, peroxide value (mequiv O2 kg−1); TAGP, triacylglycerol oligopolymers (g 100 g−1); ox-TAG, oxidized triacylglycerols (g 100 g−1). bUpper case letters are used to compare the samples considering the amount of monoacylglycerols added to purified oil; lower case letters are used to compare the samples considering the combined effect of MAG (%), oilm and MAG × oil interaction: different letters mean significant differences at p ≤ 0.05. a
oxidative phenomena were observed when MAG were added to PO derived from olive, palm, peanut, and sunflower, confirming previous results obtained by adding MAG prepared from olive oil to the same olive oil.21 A different and particular trend, instead, was observed when MAG were added to soybean PO. In this case, all of the analytical indices considered, apart from PV, significantly increased when MAG amounts up to 1% were added, confirming other research,19,20,22 and decreased at higher MAG doses. Oven Test: Surface Response Regression. In a recent review, Chen and collaborators30 pointed out the presence of discordant data about MAG behavior toward oxidation. Moreover, the effect of compositional characteristics of MAG (chain length, unsaturation level) have been investigated, but the role of fatty acid composition of the oil to which MAG are added has been scarcely studied, as well as the relationships between the two compositions. We adopted surface response regression analysis to point out the effect of MAG on oxidation in oils having different fatty acid compositions. Table 4 reports the coefficients of the regressions and the corresponding p values. The use of coded units enables
unsaturation degree were paralleled by the IV, which was higher in sunflower and soybean oils than in the others. Table 1 shows also that the fatty acid composition of MAG was similar to that of the corresponding soybean purified oil used for their preparation. POs were constituted by unaltered triacylglycerols (>99.9%) and showed FFA and PV values equal to zero and low values for the spectrophotometric constants (Table 2), similar to those detected in a previous study.14 Oven Test: Oxidation after 10 Days at 60 °C. Table 3 reports the results of the analyses carried out on each PO and on the same PO with added 0.5, 1, 2, and 3% MAG (MAG0.5, MAG1, MAG2, and MAG3, respectively) after 10 days at 60 °C, as well as the results of statistical analysis (two-way ANOVA followed by Tukey’s HSD test for multiple comparisons). The data of oxidation after 10 days were reported as representative of the overall trend during time, to exclude, in the first instance, “time” as a third source of variability, besides MAG amount and type of oil. The addition of increasing amounts of MAG significantly influenced the oxidative process. In particular, less marked 10778
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Table 4. Results of the Surface Response Regression: Regression Coefficients and Respective p Values peroxide valuea term constant time MAG time × time MAG × MAG MAG × time constant time MAG time × time MAG × MAG MAG × time constant time MAG time × time MAG × MAG MAG × time constant time MAG time × time MAG × MAG MAG × time constant days MAG time × time MAG × MAG MAG × time
coefficient Soybean Oil 176.511 119.497 −2.023 110.945 −9.711 6.544 Sunflower Oil 345.23 286.83 −28.58 −9.54 5.38 −3.22 Peanut Oil 138.85 162.99 −25.25 34.68 15.50 −11.87 Palm Oil 38.64 56.44 −42.94 13.23 20.88 −42.79 Olive Oil 5.640 47.130 −68.435 20.049 65.648 −54.630
ox-TAGa
TAGPa
p value
coefficient
p value
coefficient
p value
