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Correlation of triacylglycerol oligopolymers and oxidised triacylglycerols to quality parameters in extra virgin olive oil during storage
Food Research International 40 (2007) 855–861 www.elsevier.com/locate/foodres
Correlation of triacylglycerol oligopolymers and oxidised triacylglycerols to quality parameters in extra virgin olive oil during storage Maria Teresa Bilancia a, Francesco Caponio a,*, Ewa Sikorska b, Antonella Pasqualone a, Carmine Summo a a
Dipartimento PROGESA, Sezione di Industrie Agro-Alimentari, Universita` di Bari, Via Amendola 165/A, I-70126 Bari, Italy b Faculty of Commodity Science, Poznan´ University of Economics, al. Niepodleglos´ci 10, 60-967 Poznan´, Poland Received 12 October 2006; accepted 4 February 2007
Abstract The evaluation of the effect of storage on extra virgin olive oil is usually achieved basing on the concurrence of many analytical methods. With the aim of reducing the number of determinations to carry out, the levels of triacylglycerol oligopolymers and oxidised triacylglycerols, determined by high-performance size-exclusion chromatography (HPSEC) analysis of the polar compounds of the oil, have been evaluated. The obtained results highlighted that the oxidised triacylglycerols were significantly correlated (p < 0.001) in a positive way with peroxide value and K232 and in a negative way with total phenols and induction time, hence they can give indications about the primary oxidation level of the oil. The triacylglycerol oligopolymers, instead, were found to be significantly and positively correlated (p < 0.001) with K270 that denotes the secondary oxidation of an oil. On the contrary, significant negative correlations (p < 0.001) between triacylglycerol oligopolymers and both the contents of carotenoids and chlorophylls, whose contents in the oil seriously diminish when the secondary oxidation is high, were found. 2007 Elsevier Ltd. All rights reserved. Keywords: Extra virgin olive oil; Triacylglycerol oligopolymers; Oxidised triacylglycerols; HPSEC analysis
1. Introduction Olive oil is one of the oldest known vegetable oils, and it is widely consumed in the countries bordering on the Mediterranean Sea. It is used almost entirely for edible purposes as cooking and salad oil, and plays a crucial role in the so-called Mediterranean diet (Hrncirik & Fritsche, 2005; Tawfik & Huyghebaert, 1999). Extra virgin olive oil, obtained from the fruit of the olive tree solely by mechanical or other physical means that do not lead to any chemical change, has a high resistance to oxidative deterioration. This is mainly due to its fatty acid
*
Corresponding author. Fax: +39 080 5443467. E-mail address: [email protected] (F. Caponio).
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composition, characterised by a high monounsaturated-topolyunsaturated fatty acid ratio, and to the presence of minor compounds. Some of these compounds have a powerful anti-oxidant activity as well as biological and nutritional functions, and endow added value to extra virgin olive oil in terms of vitamin E content and anticancer properties (Psomiadou & Tsimidou, 2002a; Velasco & Dobarganes, 2002). Despite its anti-oxidant properties, extra virgin olive oil undergoes oxidative processes during storage (Okogeri & Tasioula-Margari, 2002), which affect both its organoleptic properties and the nutritional and commercial value (Rastrelli, Passi, Ippolito, Vacca, & De Simone, 2002). In the last few years, the research about extra virgin olive oil stability during storage has been very intensive: Psomiadou and Tsimidou (2002a, 2002b) evaluated the
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influence of light and oxygen on the stability of the oil stored for different periods of time and under various conditions. The effect of temperature (Gutie´rrez & Ferna´ndez, 2002; Lavelli, Fregapane, & Salvador, 2006), and of the kind and colour of packaging (Rastrelli et al., 2002) on the degradation of some minor compounds having antioxidant properties, both under accelerated and routine storage conditions (Gutie´rrez & Ferna´ndez, 2002; Hrncirik & Fritsche, 2005), were also investigated. Many time-consuming analytical determinations, however, were needed in these studies in order to achieve a full comprehension of the changes affecting the stability of the oils examined. Previous papers of the authors (Caponio & Gomes, 2004; Caponio, Gomes, & Summo, 2003; Gomes, Caponio, & Delcuratolo, 2003) pointed out the possibility to check triacylglycerol oligopolymers and oxidised triacylglycerols contents, by means of high-performance size-exclusion chromatography (HPSEC) analysis of the polar compounds of the oil, to evaluate the oxidative degradation level in edible oils and in the lipid fraction of various foods. Moreover, the HPSEC analysis was utilised to assess the level of degradation of oils, either refined or subjected to treatments that require high process temperatures (Dobarganes, Pe´rez-Camino, & Ma´rquez-Ruiz, 1988; Gomes & Caponio, 1998; Hopia, 1993). The same analysis was used also for monitoring the changes occurring during oil frying (Arroyo, Cuesta, Garrido-Polonio, Lo´pez-Varela, & Sa´nchez-Mun˜iz, 1992; Garrido-Polonio, Sa´nchez-Mun˜iz, Arroyo, & Cuesta, 1994; Lo´pez-Varela, Sa´nchez-Mun˜iz, Garrido-Polonio, Arroyo, & Cuesta, 1995). Moreover, interest in the determination of triacylglycerol oligopolymers and oxidised triacylglycerols was spurred by the observation that they may be potentially toxic when ingested at high doses (Billek, 2000; Saguy & Dana, 2003), and are also thought to have a pro-oxidant activity (Frenkel, Neff, Selke, & Brooks, 1988; Yoon, Jung, & Min, 1988). With the aim to reduce the number of determinations to carry out to evaluate the effect of storage on extra virgin olive oil, the possibility to consider the levels of triacylglycerol oligopolymers and oxidised triacylglycerols was evaluated. In this work the levels of these substances were correlated both to standard quality indices and to minor compounds, whose content varies as a function of storage time and conditions. 2. Materials and methods 2.1. Sampling and experimental design Extra virgin olive oil from the Coratina cultivar was used for the experimental tests. Once in the laboratory, the oil was analysed (0 months) and then transferred into clear and green glass bottles, 150 mL each. The bottles were hermetically sealed. The bottling procedure was the same used in the oil mills. The clear bottles were divided into two series: the first series was placed in a carton box
and stored in darkness, while the second series of clear bottles, and the green ones, were stored under diffuse lighting 10 h per day, simulating the conditions of a supermarket shelf. The average temperature during storage was 20 C. The samples were withdrawn from storage at fixed times: after 1, 2, 4, 6, 8, 10, and 12 months from bottling and storing in the described conditions. Each analysis was performed with two replicates. 2.2. Analytical determinations Free fatty acids, peroxide value, UV spectrophotometry, total phenols, chlorophylls, carotenoids, tocopherols, oxidative stability index (OSI), and polar compounds were determined for each sample. Analyses of free fatty acids, determinations of peroxide value, and UV spectrophotometry were carried out on each sample according to EC Regulation No. 2568/91 (Official Journal of European Communities, 1991). The phenolic compounds were extracted, purified, and determined according to the method described in a previous paper (Caponio, Alloggio, & Gomes, 1999), using Folin–Ciocalteau as a reagent and measuring the absorption at 765 nm. The results obtained were expressed as mg of gallic acid per kg of oil. The chlorophyll fraction was evaluated from the absorption spectrum of each oil sample dissolved in n-hexane, according to the AOCS method (American Oil Chemistry Society, 1993). The chlorophyll content was expressed as mg of pheophytin-a per kg of oil. The concentration of total carotenoids was calculated by measuring the absorption at 449 nm of 0.25 g of oil dissolved in 5 mL n-hexane, using a calibration curve previously obtained by measuring the absorption of solutions of b-carotene having a known concentration. The changes in tocopherol contents were monitored by synchronous scanning fluorescence spectroscopy. The synchronous fluorescence spectra were obtained on a Fluorolog 3-11 spectrofluorometer, Spex-Jobin Yvon S.A. A xenon lamp source was used for excitation. Excitation and emission slit width were 2 nm. The acquisition interval and the integration time were maintained at 1 nm and 0.1 s, respectively. A reference photodiode detector at the excitation monochromator stage compensated for the source intensity fluctuations. The spectra were corrected for the wavelength response of the system. Right-angle geometry was used for oil samples diluted in n-hexane (1% v/v) in a 10 mm fused-quartz cuvette. The synchronous fluorescence spectra were collected by simultaneously scanning the excitation and emission monochromator in the 250–700 nm range, with constant wavelength difference of Dk = 10 nm between them. The changes in fluorescence intensities at excitation wavelength kex = 301 nm and emission wavelength kem = 311 nm were used as indices of changes in tocopherols content in studied oils. It has been shown for different vegetable oils that for diluted samples fluorescence intensity at kex = 301 nm/kem = 311 nm correlates well with
M.T. Bilancia et al. / Food Research International 40 (2007) 855–861
total tocopherol content (Sikorska, Gliszczynska-Swiglo, Khmelinskii, & Sikorski, 2005). The OSI was evaluated by Rancimat apparatus (Methrohm Co., Basel, Switzerland) at 120 C with an air flow of 20 L/h. The results were expressed as induction time (h). The polar compounds were separated from the oils by silica gel column chromatography according to the AOAC method (Association of Official Analytical Chemists, 2003). The polar compounds recovered in CH2Cl2 were then analysed by means of HPSEC using CH2Cl2 as an eluant at a flow rate of 1 mL/min. The HPSEC analysis of the polar compounds enables separating and quantifying the various classes of substances constituting them, due to both oxidation (triacylglycerol oligopolymers and the oxidised triacylglycerols) and hydrolysis (diacylglycerols) of the triacylglycerols. The HPSEC system consisted of a series 200 pump (Perkin–Elmer, Norwalk, CT, USA) with Rheodyne injector, a 50 lL loop, a PL-gel guard column (Perkin–Elmer, Beaconsfield, UK) of 5 cm length · 7.5 mm i.d., and a series of three PL-gel columns (Perkin–Elmer, Beaconsfield, UK) of 30 cm length · 7.5 mm i.d. each. The columns were packed with highly cross-linked styrene-divinylbenzene copolymer with particles of 5 lm and a pore ˚ , respectively. The detector diameter of 500, 500, and 100 A was a series 200 refractive index (Perkin–Elmer, Norwalk, CT, USA) connected to an integrator. Peaks on the chromatograms were identified and quantified as reported in the previous papers (Caponio et al., 2003; Gomes & Caponio, 1999). The precision of the method, expressed as CV%, was 1.2% for triacylglycerol oligopolymers and 1.3% for oxidised triacylglycerols. 2.3. Statistical analysis The analysis of covariance (ANCOVA), performed by XLSTAT (Addinsoft, New York, NY, USA), was used to compare the experimental data. 3. Results and discussion In Table 1, are reported the analytical results obtained on examining the extra virgin olive oil stored in three different conditions; darkness, clear bottles and green bottles, under diffuse lighting are reported. The parameters examined were some standard indices foreseen by the current rules (Official Journal of European Communities, 1991), some minor compounds having interesting nutritional properties, and the triacylglycerol oligopolymers and oxidised triacylglycerols obtained from the polar compounds of the oil. On the whole, the obtained results agreed with those from other authors studying the effect of storage conditions on olive oil stability (Del Caro, Vacca, Poiana, Fenu, & Piga, 2006; Manzi, Panfili, Esti, & Pizzoferrato, 1998; Morello´, Motilva, Tovar, & Romero, 2004; Psomiadou & Tsimidou, 2002a, 2002b; Rastrelli et al., 2002). The analysis of data immediately indicates that the effects of two storage conditions were well distinguishable: the sam-
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ples stored in the dark were different to those stored in clear glass bottles exposed to the light. In particular, the oils kept in the dark showed better qualitative level containing higher levels of minor compounds (carotenoids and tocopherols) and lower amounts of triacylglycerol oligopolymers. The oxidative degradation of the dark-stored samples was merely imputable to primary oxidation products, while in the oils bottled in clear glass, and exposed to the light, a stronger oxidation with formation of secondary products occurred. The oil stored in light-exposed green bottles showed a level of oxidative degradation, which was intermediate with respect to that of the other two series of oils. In particular, the peroxide value was significantly higher (p < 0.001) in the dark-stored oils than in those bottled in clear glass and light-stored. The values of K270 and DK were significantly lower in the oils stored in the dark with respect to those stored in green bottles and exposed to the light (p < 0.05) and, above all, to those stored in the clear glass (p < 0.01). The free fatty acids and the K232 values, indeed, did not show significant differences in comparison to the three series of oils. Among the minor compounds characterised by anti-oxidant activity, the amounts of phenolic compounds and of carotenoids did not show any significant difference in comparison to the oils stored under different conditions. The oils stored in clear glass and exposed to the light showed significant lower (p < 0.05) amounts of tocopherols than those stored in the dark. As far as the chlorophylls are concerned, a definite decrease was observed after just 2 months of storage of clear glass bottles in the light, with an almost total disappearance after 4–6 months. On the contrary, their level remained almost constant in the oils stored in the dark; the oils kept in light-exposed green bottles showing intermediate values of chlorophylls. With the purpose of verifying the possibility to use nonconventional parameters to evaluate the effect of storage, irrespective to its conditions, on extra virgin olive oil, the contents of triacylglycerol oligopolymers and oxidised triacylglycerols were correlated to some routine analytical parameters assessing the oxidation level, foreseen by the European Community, and to some interesting minor components of the oil whose concentration varied as a function of storage time and conditions. Finding a significant correlation of triacylglycerol oligopolymers and oxidised triacylglycerols to examined parameters could render the performance so many determinations unnecessary. In previous investigations, Gomes et al. (2003) found that oxidised triacylglycerols do not represent the final product of triglyceride oxidation, since they can further be involved in polymerisation or degradation reactions and thus are a good index to measure the primary oxidation level. The triacylglycerol oligopolymers, instead, are considered to be a good index of the secondary oxidation level of edible oils and fats, due to their high stability and low volatility (Stevenson, Vaisey-Genser, & Eskim, 1984; White & Wang, 1986).
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Table 1 Changes in extra virgin olive oils during storage in different conditions Parameters
Storage time (months) 0
1
2
4
6
8
10
12
0.27 9.1 1.559 0.115 0.000 278 10.57 25.83 45 0.00 0.43 16.0
0.30 13.1 1.915 0.184 0.000 306 9.78 25.82 42 0.00 0.41 14.9
0.28 9.9 1.775 0.165 0.000 289 10.66 25.25 40 0.01 0.55 14.1
0.32 7.5 1.731 0.156 0.000 278 9.67 24.96 40 0.02 0.53 14.2
0.26 11.8 1.834 0.145 0.000 268 8.57 25.20 39 0.02 0.68 11.1
0.29 13.3 1.973 0.164 0.000 184 9.32 25.03 40 0.02 0.71 12.4
0.30 15.3 2.117 0.169 0.000 238 9.88 24.41 37 0.03 0.75 11.2
Extra virgin olive oils stored in the green glass bottles exposed to the light Free fatty acids (%) 0.26 0.29 0.32 Peroxide value (meq O2/kg) 7.5 8.4 9.5 K232 1.388 1.519 1.885 K270 0.130 0.165 0.240 DK 0.000 0.002 0.005 Total phenols (mg/kg) 361 297 304 Carotenoids (mg/kg) 9.80 9.86 11.26 Chlorophylls (mg/kg) 25.70 24.46 24.02 Tocopherolsa 50 42 38 Triacylglycerol oligopolymers (%) 0.00 0.01 0.01 Oxidised triacylglycerols (%) 0.33 0.44 0.51 Induction time (h) 17.3 16.3 15.2
0.28 7.0 1.732 0.208 0.006 312 8.13 21.40 27 0.02 0.48 14.7
0.27 8.9 1.697 0.297 0.007 306 7.26 18.51 24 0.04 0.50 13.5
0.25 9.1 1.692 0.255 0.010 276 9.49 9.56 21 0.06 0.49 13.2
0.33 9.7 1.733 0.248 0.009 226 10.02 7.64 16 0.05 0.69 12.6
0.31 12.3 2.013 0.258 0.008 269 8.12 8.11 18 0.06 0.67 11.7
Extra virgin olive oils stored in the clear glass bottles exposed to the light Free fatty acids (%) 0.26 0.29 0.32 Peroxide value (meq O2/kg) 7.5 5.0 6.7 1.388 1.466 1.782 K232 K270 0.130 0.198 0.250 DK 0.000 0.005 0.009 Total phenols (mg/kg) 361 374 301 Carotenoids (mg/kg) 9.80 9.99 10.57 Chlorophylls (mg/kg) 25.70 10.18 1.82 Tocopherolsa 50 32 29 Triacylglycerol oligopolymers (%) 0.00 0.02 0.03 Oxidised triacylglycerols (%) 0.33 0.39 0.41 Induction time (h) 17.3 17.1 16.0
0.29 4.0 1.527 0.288 0.010 346 8.66 0.45 25 0.04 0.42 15.7
0.29 6.1 1.655 0.309 0.010 288 7.76 0.24 20 0.07 0.43 15.4
0.27 5.5 1.620 0.285 0.010 215 7.79 1.10 20 0.08 0.42 15.2
0.30 5.0 1.587 0.274 0.010 293 7.81 0.11 19 0.08 0.42 15.0
0.32 9.0 1.648 0.278 0.009 295 7.22 0.16 18 0.13 0.53 14.5
Extra virgin olive oils stored in the dark Free fatty acids (%) 0.26 Peroxide value (meq O2/kg) 7.5 K232 1.388 K270 0.130 DK 0.000 Total phenols (mg/kg) 361 Carotenoids (mg/kg) 9.80 Chlorophylls (mg/kg) 25.70 Tocopherolsa 50 Triacylglycerol oligopolymers (%) 0.00 Oxidised triacylglycerols (%) 0.33 Induction time (h) 17.3
a
Fluorescence intensity at 301 nm (a.u.).
As regards the oxidised triacylglycerols, they resulted to be significantly correlated (p < 0.001 for all correlations) with the peroxide value, K232, total phenols, the values of induction time measured by Rancimat, and tocopherols. In particular, as expected, a positive correlation (Fig. 1a and b) was found with peroxide value and K232 that, similarly to oxidised triacylglycerols, are indices of primary oxidation. The linear regressions reported in Figs. 2 and 3 indicate a negative correlation between oxidised triacylglycerols and both total phenol content and tocopherols, respectively. Polyphenols and tocopherols are the two main groups acting as primary anti-oxidant in virgin olive oil (Velasco & Dobarganes, 2002). They mainly act as chain breakers, by donating a radical hydrogen to alkylperoxyl radicals
formed during the propagation step of lipid oxidation, and subsequently forming a stable radical. In virgin olive oil, tocopherols compete with polyphenols at the early stages of oxidation, during which the oxidised triacylglycerols are formed. Most of the papers reporting studies about the influence of different variables on changes in polyphenols also determined the oil stability towards oxidation by the Rancimat test, and good correlation coefficients between the two parameters were found in all the studies (Caponio et al., 1999; Montedoro, Servili, Baldioli, & Miniati, 1992; Tsimidou, 1998), as in the case of the present research (data not shown). The presence of a positive correlation between total phenols and induction times, together with the negative correlation observed between total phenols and oxi-
M.T. Bilancia et al. / Food Research International 40 (2007) 855–861
859
0.9
0.9
Oxidised triacylglycerols (%)
Oxidised triacylglycerols (%)
2
R = 0.5728
0.7
0.5
0.3 3
8
13
2
R = 0.6579 0.7
0.5
0.3
18
10
Peroxide value (meq/kg)
30
40
50
Fig. 3. Oxidised triacylglycerols vs. tocopherols (as fluorescence intensity at 301 nm), n = 21.
0.9 2
R = 0.5324
0.9
0.7
0.5
0.3 1.4
1.6
1.8
2.0
2.2
K232
Oxidised triacylglycerols (%)
Oxidised triacylglycerols (%)
20
Fluorescence intensity at 301 nm (a.u.)
2
R = 0.8307 0.7
0.5
0.3
Fig. 1. Oxidised triacylglycerols vs. peroxide value (a) and K232 (b), n = 21.
10
12
14
16
18
Induction time (h) Fig. 4. Oxidised triacylglycerols vs. induction time, n = 21.
2
R = 0.5949 0.7
0.5
0.3 150
200
250
300
350
400
Total phenols (mg/kg) Fig. 2. Oxidised triacylglycerols vs. total phenols, n = 21.
Triacylglycerol oligopolymers (%)
Oxidised triacylglycerols (%)
0.9 0.15 2
R = 0.5308 0.10
0.05
0.00 0.10
0.15
0.20
0.25
0.30
0.35
K270 Fig. 5. Triacylglycerol oligopolymers vs. K270, n = 21.
dised triacylglycerols, clarifies the negative correlation between the latter and the Rancimat induction times (Fig. 4). As regards the triacylglycerol oligopolymers, they were significantly and positively correlated (p < 0.001) to K270 (Fig. 5) that, as for the triacylglycerol oligopolymers, denotes the secondary oxidation of an oil. On the contrary, significant negative correlations (p < 0.001) between triac-
ylglycerol oligopolymers and both the contents of carotenoids and chlorophylls were found (Figs. 6 and 7). These two substances act in a different way, depending on the conditions of storage, and their concentration in the oil varies as a function of many parameters: the presence of oxygen, light, anti-oxidant substances or pro-oxidant ones
Triacylglycerol oligopolymers (%)
860
M.T. Bilancia et al. / Food Research International 40 (2007) 855–861 0.15 2
R = 0.5006 0.10
0.05
0.00 6.00
8.00
10.00
12.00
Carotenoids (mg/kg) Fig. 6. Triacylglycerol oligopolymers vs. carotenoids, n = 21.
Triacylglycerol oligopolymers (%)
0.15 2
R = 0.6160
K270 and to their significant negative correlation to both carotenoids, playing a role in the advanced phases of oxidation, and the chlorophyll content, known to seriously diminish when the oils are exposed to light. Basing on these findings, it could be possible to reduce noticeably the number of determinations needed to evaluate the oxidative stability of extra virgin olive oil, although the total time of analysis of an oil or the lipid fraction of a food product for both oxidised triacylglycerols and triacylglycerol oligopolymers is about 4 h comprising preliminary silica gel column chromatography of polar compounds. Considering that an increasing effort has been devoted in recent years to the development of methods that enable direct rapid analysis of food products without additional sample pre-treatment, the sole determination of triacylglycerol oligopolymers content to evaluate the degradation of an oil is very interesting, because these compounds can also be determined by a direct HPSEC analysis of oil, without preliminary silica gel column chromatography, in less than 30 min (Gomes et al., 2003).
0.10
References 0.05
0.00 0.00
6.00
12.00
18.00
24.00
30.00
Chlorophylls (mg/kg) Fig. 7. Triacylglycerol oligopolymers vs. chlorophylls, n = 21.
(Gutie´rrez & Ferna´ndez, 2002; Psomiadou & Tsimidou, 2002a, 2002b). As reported in the previous papers (Caponio, Bilancia, Pasqualone, Sikorska, & Gomes, 2005; Psomiadou & Tsimidou, 2002a, 2002b), and confirmed in this experimental work (Table 1), the lowest levels of chlorophylls and carotenoids were found in the oil having the highest level of secondary oxidation, so this could explain the negative correlation between them and the triacylglycerol oligopolymers. 4. Conclusion On the basis of the obtained results, it is possible to affirm that the triacylglycerol oligopolymers and the oxidised triacylglycerols can represent valuable analytical indices to evaluate the effect of storage on extra virgin olive oil. As a matter of fact, the oxidised triacylglycerols can give indications about the primary oxidation level, being significantly correlated in a positive way with the peroxide value and K232, and in a negative way with total phenols and induction time. On the other hand, the triacylglycerol oligopolymers point out the entity of secondary oxidation of the oil due to their significant positive correlation to
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Official Journal of European Communities (1991). Commission Regulation, No. 2568/91 (pp. 1–83). L. 248 of September 5th. Okogeri, O., & Tasioula-Margari, M. (2002). Changes occurring in phenolic compounds and a-tocopherol of virgin olive oil during storage. Journal of Agricultural and Food Chemistry, 50, 1077–1080. Psomiadou, E., & Tsimidou, M. (2002a). Stability of virgin olive oil. 1. Autoxidation studies. Journal of Agricultural and Food Chemistry, 50, 716–721. Psomiadou, E., & Tsimidou, M. (2002b). Stability of virgin olive oil. 2. Photo-oxidation studies. Journal of Agricultural and Food Chemistry, 50, 722–727. Rastrelli, L., Passi, S., Ippolito, F., Vacca, G., & De Simone, F. (2002). Rate of degradation of tocopherol, squalene, phenolics, and polyunsaturated fatty acids in olive oil during different storage conditions. Journal of Agricultural and Food Chemistry, 50, 5566–5570. Saguy, I. S., & Dana, D. (2003). Integrated approach to deep fat frying: engineering, nutrition, health and consumer aspects. Journal of Food Engineering, 56, 143–152. Sikorska, E., Gliszczynska-Swiglo, A., Khmelinskii, I. V., & Sikorski, M. (2005). Synchronous fluorescence spectroscopy of edible vegetable oils. Quantification of tocopherols. Journal of Agricultural and Food Chemistry, 53, 6988–6994. Stevenson, S. G., Vaisey-Genser, H., & Eskim, N. A. M. (1984). Quality control in the use of deep frying oils. Journal of the American Oil Chemists’ Society, 61, 1102–1108. Tawfik, M. S., & Huyghebaert, A. (1999). Interaction of packaging materials and vegetable oils: oil stability. Food Chemistry, 64, 451–459. Tsimidou, M. (1998). Polyphenols and quality of virgin olive oil in retrospect. Italian Journal of Food Science, 10, 99–116. Velasco, J., & Dobarganes, C. (2002). Oxidative stability of virgin olive oil. European Journal of Lipid Science and Technology, 104, 661–676. White, P. J., & Wang, Y. C. (1986). A high performance size-exclusion chromatographic method for evaluating heated oils. Journal of the American Oil Chemists’ Society, 63, 914–920. Yoon, S. H., Jung, M. Y., & Min, D. B. (1988). Effect of thermally oxidized triglycerides on the oxidative stability of soybean oil. Journal of the American Oil Chemists’ Society, 65, 1652–1656.
Correlation of triacylglycerol oligopolymers and oxidised triacylglycerols to quality parameters in extra virgin olive oil during storage Maria Teresa Bilancia a, Francesco Caponio a,*, Ewa Sikorska b, Antonella Pasqualone a, Carmine Summo a a
Dipartimento PROGESA, Sezione di Industrie Agro-Alimentari, Universita` di Bari, Via Amendola 165/A, I-70126 Bari, Italy b Faculty of Commodity Science, Poznan´ University of Economics, al. Niepodleglos´ci 10, 60-967 Poznan´, Poland Received 12 October 2006; accepted 4 February 2007
Abstract The evaluation of the effect of storage on extra virgin olive oil is usually achieved basing on the concurrence of many analytical methods. With the aim of reducing the number of determinations to carry out, the levels of triacylglycerol oligopolymers and oxidised triacylglycerols, determined by high-performance size-exclusion chromatography (HPSEC) analysis of the polar compounds of the oil, have been evaluated. The obtained results highlighted that the oxidised triacylglycerols were significantly correlated (p < 0.001) in a positive way with peroxide value and K232 and in a negative way with total phenols and induction time, hence they can give indications about the primary oxidation level of the oil. The triacylglycerol oligopolymers, instead, were found to be significantly and positively correlated (p < 0.001) with K270 that denotes the secondary oxidation of an oil. On the contrary, significant negative correlations (p < 0.001) between triacylglycerol oligopolymers and both the contents of carotenoids and chlorophylls, whose contents in the oil seriously diminish when the secondary oxidation is high, were found. 2007 Elsevier Ltd. All rights reserved. Keywords: Extra virgin olive oil; Triacylglycerol oligopolymers; Oxidised triacylglycerols; HPSEC analysis
1. Introduction Olive oil is one of the oldest known vegetable oils, and it is widely consumed in the countries bordering on the Mediterranean Sea. It is used almost entirely for edible purposes as cooking and salad oil, and plays a crucial role in the so-called Mediterranean diet (Hrncirik & Fritsche, 2005; Tawfik & Huyghebaert, 1999). Extra virgin olive oil, obtained from the fruit of the olive tree solely by mechanical or other physical means that do not lead to any chemical change, has a high resistance to oxidative deterioration. This is mainly due to its fatty acid
*
Corresponding author. Fax: +39 080 5443467. E-mail address: [email protected] (F. Caponio).
0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2007.02.001
composition, characterised by a high monounsaturated-topolyunsaturated fatty acid ratio, and to the presence of minor compounds. Some of these compounds have a powerful anti-oxidant activity as well as biological and nutritional functions, and endow added value to extra virgin olive oil in terms of vitamin E content and anticancer properties (Psomiadou & Tsimidou, 2002a; Velasco & Dobarganes, 2002). Despite its anti-oxidant properties, extra virgin olive oil undergoes oxidative processes during storage (Okogeri & Tasioula-Margari, 2002), which affect both its organoleptic properties and the nutritional and commercial value (Rastrelli, Passi, Ippolito, Vacca, & De Simone, 2002). In the last few years, the research about extra virgin olive oil stability during storage has been very intensive: Psomiadou and Tsimidou (2002a, 2002b) evaluated the
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influence of light and oxygen on the stability of the oil stored for different periods of time and under various conditions. The effect of temperature (Gutie´rrez & Ferna´ndez, 2002; Lavelli, Fregapane, & Salvador, 2006), and of the kind and colour of packaging (Rastrelli et al., 2002) on the degradation of some minor compounds having antioxidant properties, both under accelerated and routine storage conditions (Gutie´rrez & Ferna´ndez, 2002; Hrncirik & Fritsche, 2005), were also investigated. Many time-consuming analytical determinations, however, were needed in these studies in order to achieve a full comprehension of the changes affecting the stability of the oils examined. Previous papers of the authors (Caponio & Gomes, 2004; Caponio, Gomes, & Summo, 2003; Gomes, Caponio, & Delcuratolo, 2003) pointed out the possibility to check triacylglycerol oligopolymers and oxidised triacylglycerols contents, by means of high-performance size-exclusion chromatography (HPSEC) analysis of the polar compounds of the oil, to evaluate the oxidative degradation level in edible oils and in the lipid fraction of various foods. Moreover, the HPSEC analysis was utilised to assess the level of degradation of oils, either refined or subjected to treatments that require high process temperatures (Dobarganes, Pe´rez-Camino, & Ma´rquez-Ruiz, 1988; Gomes & Caponio, 1998; Hopia, 1993). The same analysis was used also for monitoring the changes occurring during oil frying (Arroyo, Cuesta, Garrido-Polonio, Lo´pez-Varela, & Sa´nchez-Mun˜iz, 1992; Garrido-Polonio, Sa´nchez-Mun˜iz, Arroyo, & Cuesta, 1994; Lo´pez-Varela, Sa´nchez-Mun˜iz, Garrido-Polonio, Arroyo, & Cuesta, 1995). Moreover, interest in the determination of triacylglycerol oligopolymers and oxidised triacylglycerols was spurred by the observation that they may be potentially toxic when ingested at high doses (Billek, 2000; Saguy & Dana, 2003), and are also thought to have a pro-oxidant activity (Frenkel, Neff, Selke, & Brooks, 1988; Yoon, Jung, & Min, 1988). With the aim to reduce the number of determinations to carry out to evaluate the effect of storage on extra virgin olive oil, the possibility to consider the levels of triacylglycerol oligopolymers and oxidised triacylglycerols was evaluated. In this work the levels of these substances were correlated both to standard quality indices and to minor compounds, whose content varies as a function of storage time and conditions. 2. Materials and methods 2.1. Sampling and experimental design Extra virgin olive oil from the Coratina cultivar was used for the experimental tests. Once in the laboratory, the oil was analysed (0 months) and then transferred into clear and green glass bottles, 150 mL each. The bottles were hermetically sealed. The bottling procedure was the same used in the oil mills. The clear bottles were divided into two series: the first series was placed in a carton box
and stored in darkness, while the second series of clear bottles, and the green ones, were stored under diffuse lighting 10 h per day, simulating the conditions of a supermarket shelf. The average temperature during storage was 20 C. The samples were withdrawn from storage at fixed times: after 1, 2, 4, 6, 8, 10, and 12 months from bottling and storing in the described conditions. Each analysis was performed with two replicates. 2.2. Analytical determinations Free fatty acids, peroxide value, UV spectrophotometry, total phenols, chlorophylls, carotenoids, tocopherols, oxidative stability index (OSI), and polar compounds were determined for each sample. Analyses of free fatty acids, determinations of peroxide value, and UV spectrophotometry were carried out on each sample according to EC Regulation No. 2568/91 (Official Journal of European Communities, 1991). The phenolic compounds were extracted, purified, and determined according to the method described in a previous paper (Caponio, Alloggio, & Gomes, 1999), using Folin–Ciocalteau as a reagent and measuring the absorption at 765 nm. The results obtained were expressed as mg of gallic acid per kg of oil. The chlorophyll fraction was evaluated from the absorption spectrum of each oil sample dissolved in n-hexane, according to the AOCS method (American Oil Chemistry Society, 1993). The chlorophyll content was expressed as mg of pheophytin-a per kg of oil. The concentration of total carotenoids was calculated by measuring the absorption at 449 nm of 0.25 g of oil dissolved in 5 mL n-hexane, using a calibration curve previously obtained by measuring the absorption of solutions of b-carotene having a known concentration. The changes in tocopherol contents were monitored by synchronous scanning fluorescence spectroscopy. The synchronous fluorescence spectra were obtained on a Fluorolog 3-11 spectrofluorometer, Spex-Jobin Yvon S.A. A xenon lamp source was used for excitation. Excitation and emission slit width were 2 nm. The acquisition interval and the integration time were maintained at 1 nm and 0.1 s, respectively. A reference photodiode detector at the excitation monochromator stage compensated for the source intensity fluctuations. The spectra were corrected for the wavelength response of the system. Right-angle geometry was used for oil samples diluted in n-hexane (1% v/v) in a 10 mm fused-quartz cuvette. The synchronous fluorescence spectra were collected by simultaneously scanning the excitation and emission monochromator in the 250–700 nm range, with constant wavelength difference of Dk = 10 nm between them. The changes in fluorescence intensities at excitation wavelength kex = 301 nm and emission wavelength kem = 311 nm were used as indices of changes in tocopherols content in studied oils. It has been shown for different vegetable oils that for diluted samples fluorescence intensity at kex = 301 nm/kem = 311 nm correlates well with
M.T. Bilancia et al. / Food Research International 40 (2007) 855–861
total tocopherol content (Sikorska, Gliszczynska-Swiglo, Khmelinskii, & Sikorski, 2005). The OSI was evaluated by Rancimat apparatus (Methrohm Co., Basel, Switzerland) at 120 C with an air flow of 20 L/h. The results were expressed as induction time (h). The polar compounds were separated from the oils by silica gel column chromatography according to the AOAC method (Association of Official Analytical Chemists, 2003). The polar compounds recovered in CH2Cl2 were then analysed by means of HPSEC using CH2Cl2 as an eluant at a flow rate of 1 mL/min. The HPSEC analysis of the polar compounds enables separating and quantifying the various classes of substances constituting them, due to both oxidation (triacylglycerol oligopolymers and the oxidised triacylglycerols) and hydrolysis (diacylglycerols) of the triacylglycerols. The HPSEC system consisted of a series 200 pump (Perkin–Elmer, Norwalk, CT, USA) with Rheodyne injector, a 50 lL loop, a PL-gel guard column (Perkin–Elmer, Beaconsfield, UK) of 5 cm length · 7.5 mm i.d., and a series of three PL-gel columns (Perkin–Elmer, Beaconsfield, UK) of 30 cm length · 7.5 mm i.d. each. The columns were packed with highly cross-linked styrene-divinylbenzene copolymer with particles of 5 lm and a pore ˚ , respectively. The detector diameter of 500, 500, and 100 A was a series 200 refractive index (Perkin–Elmer, Norwalk, CT, USA) connected to an integrator. Peaks on the chromatograms were identified and quantified as reported in the previous papers (Caponio et al., 2003; Gomes & Caponio, 1999). The precision of the method, expressed as CV%, was 1.2% for triacylglycerol oligopolymers and 1.3% for oxidised triacylglycerols. 2.3. Statistical analysis The analysis of covariance (ANCOVA), performed by XLSTAT (Addinsoft, New York, NY, USA), was used to compare the experimental data. 3. Results and discussion In Table 1, are reported the analytical results obtained on examining the extra virgin olive oil stored in three different conditions; darkness, clear bottles and green bottles, under diffuse lighting are reported. The parameters examined were some standard indices foreseen by the current rules (Official Journal of European Communities, 1991), some minor compounds having interesting nutritional properties, and the triacylglycerol oligopolymers and oxidised triacylglycerols obtained from the polar compounds of the oil. On the whole, the obtained results agreed with those from other authors studying the effect of storage conditions on olive oil stability (Del Caro, Vacca, Poiana, Fenu, & Piga, 2006; Manzi, Panfili, Esti, & Pizzoferrato, 1998; Morello´, Motilva, Tovar, & Romero, 2004; Psomiadou & Tsimidou, 2002a, 2002b; Rastrelli et al., 2002). The analysis of data immediately indicates that the effects of two storage conditions were well distinguishable: the sam-
857
ples stored in the dark were different to those stored in clear glass bottles exposed to the light. In particular, the oils kept in the dark showed better qualitative level containing higher levels of minor compounds (carotenoids and tocopherols) and lower amounts of triacylglycerol oligopolymers. The oxidative degradation of the dark-stored samples was merely imputable to primary oxidation products, while in the oils bottled in clear glass, and exposed to the light, a stronger oxidation with formation of secondary products occurred. The oil stored in light-exposed green bottles showed a level of oxidative degradation, which was intermediate with respect to that of the other two series of oils. In particular, the peroxide value was significantly higher (p < 0.001) in the dark-stored oils than in those bottled in clear glass and light-stored. The values of K270 and DK were significantly lower in the oils stored in the dark with respect to those stored in green bottles and exposed to the light (p < 0.05) and, above all, to those stored in the clear glass (p < 0.01). The free fatty acids and the K232 values, indeed, did not show significant differences in comparison to the three series of oils. Among the minor compounds characterised by anti-oxidant activity, the amounts of phenolic compounds and of carotenoids did not show any significant difference in comparison to the oils stored under different conditions. The oils stored in clear glass and exposed to the light showed significant lower (p < 0.05) amounts of tocopherols than those stored in the dark. As far as the chlorophylls are concerned, a definite decrease was observed after just 2 months of storage of clear glass bottles in the light, with an almost total disappearance after 4–6 months. On the contrary, their level remained almost constant in the oils stored in the dark; the oils kept in light-exposed green bottles showing intermediate values of chlorophylls. With the purpose of verifying the possibility to use nonconventional parameters to evaluate the effect of storage, irrespective to its conditions, on extra virgin olive oil, the contents of triacylglycerol oligopolymers and oxidised triacylglycerols were correlated to some routine analytical parameters assessing the oxidation level, foreseen by the European Community, and to some interesting minor components of the oil whose concentration varied as a function of storage time and conditions. Finding a significant correlation of triacylglycerol oligopolymers and oxidised triacylglycerols to examined parameters could render the performance so many determinations unnecessary. In previous investigations, Gomes et al. (2003) found that oxidised triacylglycerols do not represent the final product of triglyceride oxidation, since they can further be involved in polymerisation or degradation reactions and thus are a good index to measure the primary oxidation level. The triacylglycerol oligopolymers, instead, are considered to be a good index of the secondary oxidation level of edible oils and fats, due to their high stability and low volatility (Stevenson, Vaisey-Genser, & Eskim, 1984; White & Wang, 1986).
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Table 1 Changes in extra virgin olive oils during storage in different conditions Parameters
Storage time (months) 0
1
2
4
6
8
10
12
0.27 9.1 1.559 0.115 0.000 278 10.57 25.83 45 0.00 0.43 16.0
0.30 13.1 1.915 0.184 0.000 306 9.78 25.82 42 0.00 0.41 14.9
0.28 9.9 1.775 0.165 0.000 289 10.66 25.25 40 0.01 0.55 14.1
0.32 7.5 1.731 0.156 0.000 278 9.67 24.96 40 0.02 0.53 14.2
0.26 11.8 1.834 0.145 0.000 268 8.57 25.20 39 0.02 0.68 11.1
0.29 13.3 1.973 0.164 0.000 184 9.32 25.03 40 0.02 0.71 12.4
0.30 15.3 2.117 0.169 0.000 238 9.88 24.41 37 0.03 0.75 11.2
Extra virgin olive oils stored in the green glass bottles exposed to the light Free fatty acids (%) 0.26 0.29 0.32 Peroxide value (meq O2/kg) 7.5 8.4 9.5 K232 1.388 1.519 1.885 K270 0.130 0.165 0.240 DK 0.000 0.002 0.005 Total phenols (mg/kg) 361 297 304 Carotenoids (mg/kg) 9.80 9.86 11.26 Chlorophylls (mg/kg) 25.70 24.46 24.02 Tocopherolsa 50 42 38 Triacylglycerol oligopolymers (%) 0.00 0.01 0.01 Oxidised triacylglycerols (%) 0.33 0.44 0.51 Induction time (h) 17.3 16.3 15.2
0.28 7.0 1.732 0.208 0.006 312 8.13 21.40 27 0.02 0.48 14.7
0.27 8.9 1.697 0.297 0.007 306 7.26 18.51 24 0.04 0.50 13.5
0.25 9.1 1.692 0.255 0.010 276 9.49 9.56 21 0.06 0.49 13.2
0.33 9.7 1.733 0.248 0.009 226 10.02 7.64 16 0.05 0.69 12.6
0.31 12.3 2.013 0.258 0.008 269 8.12 8.11 18 0.06 0.67 11.7
Extra virgin olive oils stored in the clear glass bottles exposed to the light Free fatty acids (%) 0.26 0.29 0.32 Peroxide value (meq O2/kg) 7.5 5.0 6.7 1.388 1.466 1.782 K232 K270 0.130 0.198 0.250 DK 0.000 0.005 0.009 Total phenols (mg/kg) 361 374 301 Carotenoids (mg/kg) 9.80 9.99 10.57 Chlorophylls (mg/kg) 25.70 10.18 1.82 Tocopherolsa 50 32 29 Triacylglycerol oligopolymers (%) 0.00 0.02 0.03 Oxidised triacylglycerols (%) 0.33 0.39 0.41 Induction time (h) 17.3 17.1 16.0
0.29 4.0 1.527 0.288 0.010 346 8.66 0.45 25 0.04 0.42 15.7
0.29 6.1 1.655 0.309 0.010 288 7.76 0.24 20 0.07 0.43 15.4
0.27 5.5 1.620 0.285 0.010 215 7.79 1.10 20 0.08 0.42 15.2
0.30 5.0 1.587 0.274 0.010 293 7.81 0.11 19 0.08 0.42 15.0
0.32 9.0 1.648 0.278 0.009 295 7.22 0.16 18 0.13 0.53 14.5
Extra virgin olive oils stored in the dark Free fatty acids (%) 0.26 Peroxide value (meq O2/kg) 7.5 K232 1.388 K270 0.130 DK 0.000 Total phenols (mg/kg) 361 Carotenoids (mg/kg) 9.80 Chlorophylls (mg/kg) 25.70 Tocopherolsa 50 Triacylglycerol oligopolymers (%) 0.00 Oxidised triacylglycerols (%) 0.33 Induction time (h) 17.3
a
Fluorescence intensity at 301 nm (a.u.).
As regards the oxidised triacylglycerols, they resulted to be significantly correlated (p < 0.001 for all correlations) with the peroxide value, K232, total phenols, the values of induction time measured by Rancimat, and tocopherols. In particular, as expected, a positive correlation (Fig. 1a and b) was found with peroxide value and K232 that, similarly to oxidised triacylglycerols, are indices of primary oxidation. The linear regressions reported in Figs. 2 and 3 indicate a negative correlation between oxidised triacylglycerols and both total phenol content and tocopherols, respectively. Polyphenols and tocopherols are the two main groups acting as primary anti-oxidant in virgin olive oil (Velasco & Dobarganes, 2002). They mainly act as chain breakers, by donating a radical hydrogen to alkylperoxyl radicals
formed during the propagation step of lipid oxidation, and subsequently forming a stable radical. In virgin olive oil, tocopherols compete with polyphenols at the early stages of oxidation, during which the oxidised triacylglycerols are formed. Most of the papers reporting studies about the influence of different variables on changes in polyphenols also determined the oil stability towards oxidation by the Rancimat test, and good correlation coefficients between the two parameters were found in all the studies (Caponio et al., 1999; Montedoro, Servili, Baldioli, & Miniati, 1992; Tsimidou, 1998), as in the case of the present research (data not shown). The presence of a positive correlation between total phenols and induction times, together with the negative correlation observed between total phenols and oxi-
M.T. Bilancia et al. / Food Research International 40 (2007) 855–861
859
0.9
0.9
Oxidised triacylglycerols (%)
Oxidised triacylglycerols (%)
2
R = 0.5728
0.7
0.5
0.3 3
8
13
2
R = 0.6579 0.7
0.5
0.3
18
10
Peroxide value (meq/kg)
30
40
50
Fig. 3. Oxidised triacylglycerols vs. tocopherols (as fluorescence intensity at 301 nm), n = 21.
0.9 2
R = 0.5324
0.9
0.7
0.5
0.3 1.4
1.6
1.8
2.0
2.2
K232
Oxidised triacylglycerols (%)
Oxidised triacylglycerols (%)
20
Fluorescence intensity at 301 nm (a.u.)
2
R = 0.8307 0.7
0.5
0.3
Fig. 1. Oxidised triacylglycerols vs. peroxide value (a) and K232 (b), n = 21.
10
12
14
16
18
Induction time (h) Fig. 4. Oxidised triacylglycerols vs. induction time, n = 21.
2
R = 0.5949 0.7
0.5
0.3 150
200
250
300
350
400
Total phenols (mg/kg) Fig. 2. Oxidised triacylglycerols vs. total phenols, n = 21.
Triacylglycerol oligopolymers (%)
Oxidised triacylglycerols (%)
0.9 0.15 2
R = 0.5308 0.10
0.05
0.00 0.10
0.15
0.20
0.25
0.30
0.35
K270 Fig. 5. Triacylglycerol oligopolymers vs. K270, n = 21.
dised triacylglycerols, clarifies the negative correlation between the latter and the Rancimat induction times (Fig. 4). As regards the triacylglycerol oligopolymers, they were significantly and positively correlated (p < 0.001) to K270 (Fig. 5) that, as for the triacylglycerol oligopolymers, denotes the secondary oxidation of an oil. On the contrary, significant negative correlations (p < 0.001) between triac-
ylglycerol oligopolymers and both the contents of carotenoids and chlorophylls were found (Figs. 6 and 7). These two substances act in a different way, depending on the conditions of storage, and their concentration in the oil varies as a function of many parameters: the presence of oxygen, light, anti-oxidant substances or pro-oxidant ones
Triacylglycerol oligopolymers (%)
860
M.T. Bilancia et al. / Food Research International 40 (2007) 855–861 0.15 2
R = 0.5006 0.10
0.05
0.00 6.00
8.00
10.00
12.00
Carotenoids (mg/kg) Fig. 6. Triacylglycerol oligopolymers vs. carotenoids, n = 21.
Triacylglycerol oligopolymers (%)
0.15 2
R = 0.6160
K270 and to their significant negative correlation to both carotenoids, playing a role in the advanced phases of oxidation, and the chlorophyll content, known to seriously diminish when the oils are exposed to light. Basing on these findings, it could be possible to reduce noticeably the number of determinations needed to evaluate the oxidative stability of extra virgin olive oil, although the total time of analysis of an oil or the lipid fraction of a food product for both oxidised triacylglycerols and triacylglycerol oligopolymers is about 4 h comprising preliminary silica gel column chromatography of polar compounds. Considering that an increasing effort has been devoted in recent years to the development of methods that enable direct rapid analysis of food products without additional sample pre-treatment, the sole determination of triacylglycerol oligopolymers content to evaluate the degradation of an oil is very interesting, because these compounds can also be determined by a direct HPSEC analysis of oil, without preliminary silica gel column chromatography, in less than 30 min (Gomes et al., 2003).
0.10
References 0.05
0.00 0.00
6.00
12.00
18.00
24.00
30.00
Chlorophylls (mg/kg) Fig. 7. Triacylglycerol oligopolymers vs. chlorophylls, n = 21.
(Gutie´rrez & Ferna´ndez, 2002; Psomiadou & Tsimidou, 2002a, 2002b). As reported in the previous papers (Caponio, Bilancia, Pasqualone, Sikorska, & Gomes, 2005; Psomiadou & Tsimidou, 2002a, 2002b), and confirmed in this experimental work (Table 1), the lowest levels of chlorophylls and carotenoids were found in the oil having the highest level of secondary oxidation, so this could explain the negative correlation between them and the triacylglycerol oligopolymers. 4. Conclusion On the basis of the obtained results, it is possible to affirm that the triacylglycerol oligopolymers and the oxidised triacylglycerols can represent valuable analytical indices to evaluate the effect of storage on extra virgin olive oil. As a matter of fact, the oxidised triacylglycerols can give indications about the primary oxidation level, being significantly correlated in a positive way with the peroxide value and K232, and in a negative way with total phenols and induction time. On the other hand, the triacylglycerol oligopolymers point out the entity of secondary oxidation of the oil due to their significant positive correlation to
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