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Antioxidant dispersions in emulsified olive oils
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Food Research International 41 (2008) 201–207 www.elsevier.com/locate/foodres
Antioxidant dispersions in emulsified olive oils Monica Mosca, Andrea Ceglie, Luigi Ambrosone * Consorzio per lo sviluppo dei Sistemi a Grande Interfase (C.S.G.I.), c/o Department of Food Technology, DISTAAM, Universita` del Molise, via De Sanctis 86100 Campobasso, Italy Received 7 September 2007; accepted 25 November 2007
Abstract In this paper water droplets dispersed in olive oils were used as containers of L-ascorbic acid and its inhibitory effect on lipid oxidation was studied. In detail, olive oil samples were emulsified by adding a certain quantity of vitamin C aqueous solutions and then oxidized by UV light. Data collected from sample analysis by optical microscopy were used to calculate the average droplets parameters and to characterize the droplet size distribution function. The results revealed a strong correlation between the initial rate of hydroperoxide formation and the specific surface area of aqueous dispersed phase. Such correlation has been explained as the consequence of a synergistic interaction between the ascorbic acid contained in the droplets and the natural tocopherols present in olive oil. A key role is played by the water–olive oil interface which is the boundary where the synergisms take place. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Antioxidant; Emulsion; Olive oil; Oxidation; Specific surface area; Vitamin C
1. Introduction Olive oil, the main fat component of the Mediterranean diet, has received great attention since it has been found out its protective role against oxidative stress (Menendez, Vellon, Colomer, & Lupu, 2005). Evaluation and control of lipid oxidation are of concern in the storage of olive oils because it results in the loss of nutritional value and the formation of compounds that may be detrimental to health (Frankel, 1993, 1998). Lipid oxidation leads to the formation of several compounds of different molecular weight and polarity which makes it difficult to evaluate the degree of oxidation. The situation becomes even more complex in the case of veiled olive oils due to additional factors that can be decisive in determining the rate and pathway of lipid oxidation. Many of these factors arise from the presence of a large interfacial area between the dispersed and continuous phases. Veiled oils represent a particular class of olive oils obtained directly from the extraction process and not *
Corresponding author. Tel.: +39 0874 404715; fax: +39 0874 404652. E-mail addresses: [email protected] (M. Mosca), [email protected] (A. Ceglie), [email protected] (L. Ambrosone). 0963-9969/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2007.11.006
filtered. Therefore they are characterized by the presence of water and particulate suspensions. Recently, a great deal of studies on vegetable oil oxidation have shown that water and small particles dispersed in the oil can act as anti-oxidants, allowing longer preservation periods (Lercker, Frega, Bocci, & Servidio, 1994). In this context we have verified that in the absence of particles and in the presence of water only (emulsified oil), the antioxidant function takes place anyway (Ambrosone, Angelico, Cinelli, Di Lorenzo, & Ceglie, 2002; Ambrosone, Mosca, & Ceglie, 2006a; Ambrosone, Cinelli, Mosca, & Ceglie, 2006b) and the oil’s shelf-life depends on the characteristics of the water-in-oil emulsions (Ambrosone, Mosca, & Ceglie, 2007). In general, the inhibitory effect of antioxidants on lipid oxidation is affected by the physico-chemical state of the lipidic substrate and various evaluation systems using different physical conditions are required to provide a good understanding of antioxidant properties in different media (Decker, Warner, Richard, & Shahidi, 2005). Lipid hydroperoxides are more polar than the starting lipids due to the presence of oxygen. We suggested that the high polarity of lipid hydroperoxides would cause them to diffuse towards the water–oil interface
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M. Mosca et al. / Food Research International 41 (2008) 201–207
of emulsions which acts as a ‘‘sink” for hydroperoxides molecules so affecting the course of the oxidation reaction (Ambrosone et al., 2006a; Ambrosone et al., 2006b). The results indicated that the contact time between water droplets and oil is an important parameter both for oxidative stability and for emulsion stability. Therefore, it is interesting to keep working on this topic to find out ways to improve the oxidative stability of water-in-oil emulsions, by acting on the characteristics of the water dispersed phase. The aim of the present study is to analyze the oxidative degradation of emulsified olive oils when L-ascorbic acid (vitamin C) is contained in the water droplets of dispersed phase. Up to now there is no substance which is cheaper, better tolerated by the body, and with less nuisance problems associated with orally administration than ascorbic acid. 2. Experimental procedure 2.1. Materials Chloroform, acetic acid and potassium iodine were reagents of analytical grade of 99.9% purity purchased from Carlo Erba Reagenti (Milan, Italy). L-ascorbic acid was kindly donated by Dr. P. Andrea Lo Nostro (University of Firenze) and used without further purification.
hydroxytyrosol, for instance) microemulsions might form. Since such systems are thermodynamically stable and not visible to the optical microscope, we verified that all the added water was dispersed in emulsion droplets by centrifuging an emulsified sample at 4000 rpm per 30 min. In this way the quantity of added water was totally recovered indicating that no appreciable amount of water was dispersed in microemulsion droplets. 2.2.3. Oxidizing conditions Aliquots of emulsified oil samples (2.5 g) were put into polyester vessels (40 mm 40 mm 8 mm) and exposed to UV light (wavelength k = 254 nm). The lamp power was 30 W and the direction of irradiation was perpendicular to oil surface. The distance between the lamp and the oil surface was 11.5 cm. This distance was the same for all samples so that such parameter is a constant of the oxidation process. All analyses were performed ‘‘simultaneously” on different samples. Each sample was exposed to oxidation for different time intervals, at 25 °C. The oxidative state of samples was monitored by measuring the peroxide value (PV). The peroxide value of samples was determined according to AOCS official method Cd 8-53 (American Oil Chemist’s Society, 1990). The solution was titrated against standard sodium thiosulfate (0.01 N). PV was calculated and expressed as meq peroxide per kg of oil sample:
2.2. Methods PVðmeqiv=kgÞ ¼ 2.2.1. Oil sampling Olives of mixed cultivars (Cima di Mola, Cima di Bitonto and others) were picked during the 2004/2005 oil campaign and pressed with a discontinuous process (Hoffmann, 1989). The oil samples were taken directly from the crusher and treated for 15 min with an N2 current and then stored at 4 °C in 50 ml flasks. In this way each sample, once it was slowly taken back to room temperature, was completely used for the analyses. The olive oil was characterized by measuring PV, total acidity, spectrophotometric indexes and refractive index, according to the official methods recommended by the European Community (European Community, 1991). The total tocopherols concentration was determined at 520 nm according to the methodology developed by Emmerie–Engel (Alpaslan & Gu¨ndu¨z, 2000). 2.2.2. Preparation of water-in-extra virgin olive oil emulsions Water in olive oil emulsions were prepared by adding distilled water or L-ascorbic acid aqueous solution directly to 15 g of oil. All emulsified samples were prepared so that the aqueous part was always the 3 wt%. As described elsewhere (Ambrosone et al., 2002) the texture of these emulsions, i.e. their stability, is a function of the excess of mechanical energy used for the preparation. For this reason all the samples were shaken for the same length of time (15 min) with Ultraturrax T8-S8NG, 100W, Ika Labortechnik (Janke & Kunkel, Gimbh, Stanfen, Germany). Due to the presence of minor constituents (tyrosol,
ðS BÞ N F 1000 W
ð1Þ
where S is the titer for the sample, B is the titer for the blank, N is the normality of the sodium thiosulfate solution, F is the factor from standardization with potassium dichromate, and W the sample mass. The experimental uncertainty was estimated by calculating the standard deviation (SD) of repeated measurements (not less than three). 2.2.4. Optical microscopy Optical micrographs were obtained by means of an optical microscope Optech B5, at 25 °C. In order to get representative results from the system Video enhanced microscopy (VEM) was used. Such a technique combines the magnification power of the microscope with the digital image acquisition capability of a video camera (Panasonic, model GP-KR222). A series of images (9–10 pictures, 1000–1500 particles) were examined to determine the size polydispersity which was estimated by counting the ‘‘average number” of droplets at different radii in micrographs of a Thoma grating. Image analysis software (Sigma Scan Pro, SPSS Science Software Products) which provides a wide range of analytical features in addition to image enhancement was used to digitize the images. 2.2.5. H2O/vitamin C binary systems The weight fraction of dispersed phase in each emulsified oil sample was converted to volume fraction by means of density data measured by Densito30P (Mettler-Toledo
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GmbH-Switzeland). All ascorbic acid–water solutions were prepared by weight using distilled water as solvent. Density data for H2O/ascorbic acid at (22 ± 0.5) °C, expressed as a function of mass fraction of vitamin C, were fitted to the equation q ¼ 0:9960 þ 0:415W 0:01044W 2
ð2Þ
with standard deviation of 0.00005 gcm3. 3. Results and discussion 3.1. Peroxide measurements Before investigating the antioxidant effect of the aqueous dispersions, the olive oil was characterized analytically. The results are gathered in Table 1. Water-in-olive oil emulsions are complex multiphase systems in which different molecular species interact with each other. Oil oxidation in such systems is greatly affected by the nature of the interface. Because of the complexity of the oxidation process itself and of the difficulties in its accurate quantification, we decided to oxidize the system under controlled conditions. Therefore, natural olive oil samples were emulsified with aqueous solutions of vitamin C at different concentration and then were exposed to UV lamp. In Fig. 1 PV changes with oxidation time for emulsified oils without vitamin C or with different amounts of vitamin C are calculated as the ratio PV/PV0, where PV is the peroxide value at time t and PV0 is the peroxide value at t = 0. It can be seen that the larger the vitamin C amount the smaller the peroxide value of olive oil indicating that vitamin C succeeds in acting as an effective antioxidant even if it is confined into water droplets, out of the reaction site. However, to gather some information about the involvement of vitamin C in the oxidation process of emulsified oils, we analyzed the initial rates of hydroperoxides formation, defined by m ¼ ðdPV Þ . The plot of v0 versus the overdt t¼0 all concentration of vitamin C, displayed in Fig. 2A, exhibits a constant trend up to a value of 1200 mg/Kg of emulsified oil and then decreases for higher vitamin C concentration. Ascorbic acid is a weak diprotic acid (AscH2) with enediol group into a five membered heterocyclic lactone ring. Its dissociation constants in pure water at 22–23° are
Fig. 1. PV/PV0 versus UV oxidation time of extra virgin olive oil emulsified with aqueous solution of L-ascorbic acid. Oil emulsified with water only (s), is compared with dispersion containing an overall concentration of 50 ppm (d), 100 ppm (j), 900 ppm(), 3500 ppm (N), 5000 ppm (.), and 8000 ppm (L) of vitamin C.
Ka1 = 7.58106 Ka2 = 3.09 1012, respectively (Kumler and Daniels,1935). It is well known that ascorbate anion (AscH) is the species directly involved in the oxidation process through the formation of the ascorbyl radical anion. Assuming the inner of droplet to be pure water and neglecting the second acid dissociation reaction we calculated the local concentration of ascorbate anion as 2 a solution of the algebraic equation ½AscH þ k a1 ½AscH k a1 ½AscH2 0 ¼ 0; where [AscH2]0 is the initial concentration of ascorbic acid. The perfect linearity between the hydroperoxides (ROOH) formation rate and the local concentration of the ascorbate anion, displayed in Fig. 2B, demonstrates the direct involvement of this anion in the oxidative process also in water-in-olive oil emulsions. It is interesting the presence of a region in Fig. 2A where the formation rate of hydroperoxides is poorly sensitive to vitamin C concentration. This is an
Table 1 Chemical characteristics of olive oil samples. The results are expressed as mean of repeated measurements ± standard deviation (n P 3) Peroxide valuea (meq O2Kg1) 13.19 ± 0.02 a b c d e
Acidity valueb (% oleic acid)
Total tocopherolsc (mg Kg1 as a-tocopherol)
Refractive indexc
Spectrophotometric indexes k232d
k270d
Dke
0.850 ± 0.004
232 ± 9
1.4675 ± 0.0005
1.74 ± 0.01
0.27 ± 0.01
0.085 ± 0.02
n = 18. n = 3. n = 4. kk = specific extinction coefficient at wavelength k. Dk = k2700.5 (k266 + k274).
b
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itoring the texture measurements.
of
emulsions
through
VEM
3.2. Video enhanced microscopy measurements
Fig. 2. Initial rate of hydroperoxides formation as a function of the (A) overall L-ascorbic acid concentration and (B) local ascorbate monoanion concentration.
indication of some particular mechanism of action for scavenging free radicals. To understand this aspect it is worthwhile remarking that in olive oils there are different substances with antioxidant properties, like tocopherols and poliphenols, which play a key role in determining oil resistance to oxidation. Although the content of phenolic derivates is dependent on olive cultivar, climatic conditions, oil extraction process and storage conditions, the average amount of total tocopherols is quite constant and specific for olive oils and it is around 200–300 mg per kg of oil. Among tocopherols, a-tocopherol is the most abundant in olive oils and it is the molecule with the highest antioxidant activity (Hoffmann, 1989). This datum is particularly noteworthy since it has been demonstrated the synergic interaction between tocopherols and ascorbic acid in homogeneous systems (Lambelet & Lo¨liger, 1985; Packer, Slater, & Wilson, 1979). However, there have been some controversly on whether such an interaction can occur in heterogeneous systems in which tocopherols are located in the lipidic environment whereas ascorbic acid is located in aqueous environment (Niki, 1991a; Packer, 1997). Ascorbic acid, due to its high lipophobicity, is not able to exert its antioxidant function when the radicals are primarily formed within the oil phase where tocopherols are active. On the other hand a-tocopherol molecules are slightly surface active and therefore they might be located at the water–oil interphase especially when present in oxidized form. Indeed, once at the interface the tocopheroxyl radicals would be able to interact with vitamin C and to be regenerated back to the reduced form. This surface–mediated interaction between vitamin C and vitamin E was studied in oil-in-water emulsions by Frankel et al. and in liposomal membranes by Niki (Frankel, Huang, Kanner, & German, 1994; Niki, 1991b). Herein we have studied the role of the water–oil interface in the antioxidant action of vitamin C with mon-
Emulsion structure of freshly prepared samples is clearly identified through optical microscopy which shows a large size polydispersity of dispersed phase. Droplet size distributions of samples containing increasing amounts of vitamin C are shown in Fig. 3. The experimental VEM results were processed according to the technique we described in detail elsewhere (Ambrosone, Colafemmina, Palazzo, & Ceglie, 2000). The data were well described by a lognormal function " # 1 ð1nD 1nD0 Þ2 P ðDÞ ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð3Þ exp 2r2 2pr2 D0 which is dependent on two parameters, D0 an r. Many properties of the experimental distribution function can be derived by the moments: Z 1 Dn P ðDÞdD ð4Þ ln ¼ 0
where n is the moment order and l0 = 1. Particularly useful are D10, the average diameter, and D32 the volume/surface ratio (or Sauter average). In order to study the interphase role in polydispersed systems is noteworthy the specific surface area of dispersed phase: AD ¼
6l2 6 ¼ D32 l3
ð5Þ
Plots of D10 versus local concentration of ascorbic acid and ascorbate monoanion, respectively, reveal unexpectedly a direct relationship between the mean size of water droplets
Fig. 3. Size distribution function P(D) as a function of diameter D at different ascorbic acid concentration.
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between the formation rate of hydroperoxides and the specific AD. Thus the surface/volume ratio of the dispersed phase drives the antioxidant action of ascorbic acid. Even in the plot in Fig. 5C is evident the presence of a region where ROOH formation rate stays unchanged. We investigated this point by plotting both v0 and AD versus the local concentration of ascorbate monoanion. As one can see in Fig. 6 the region of constant rate coincides with the region of constant specific surface. Such results seem to indicate that the antioxidant action of vitamin C takes place only if the specific surface area of the dispersed phase reaches a threshold value. This is because the surface activity of a-tocopherols is mild so that for low values of AD the quantity of a-tocopherols in the interfacial region is very low and it is scarcely accessible to ascorbate monoanion molecules. When the specific surface reaches a value as high as to host a large a-tocopherols amount per water droplet the Fig. 4. Mean diameter of water droplet as a function of the (A) overall Lascorbic acid concentration and (B) local ascorbate monoanion concentration.
and the conjugate base concentration (Fig. 4A, B). Inasmuch as this value, at fixed temperature, is completely determined by the dissociation constant, one deduces that thermodynamic and kinetic stability are interrelated. The relation between the mean droplet size and the ascorbate anion concentration suggests a direct involvement of the water/oil interface in determining antioxidant activity. Then, if the antioxidant action occurs at the water-oil interface, a direct influence of emulsion texture on the oxidation reaction rates is expected. On the other hand, the system is polydisperse in size so that the mean diameter and standard deviation are not enough to detect the characteristics of the distribution function (Kenney, 1939). Therefore we calculated l2 and l3 directly from micrographs and related these values to the formation rate. As one can see in Fig. 5A, B, the plot of v0 versus l2 and l3 does not show any simple relation, on the contrary Fig. 5C shows a direct correlation
Fig. 6. Comparison between initial rate and specific surface area as a function of the local ascorbate monoanion concentration.
Fig. 5. Initial rate of hydroperoxides formation as a function of (A) the second moment of the droplet size distribution function, (B) the third moment of the droplet size distribution function and (C) the specific surface area.
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Table 2 AscH content per unit surface area and Tocopherol/Ascorbate anion molar ratio of W/Olive oil emulsions at different total ascorbic acid content Ascorbic acid emulsion content (ppm)
AscH (mg m2)
a-Tocopherol/AscH-molar ratio (mol/mol)
50 100 900 1200 2000 3500 5000 8000
0.26 0.42 0.40 1.8 2.2 2.2 2.8 2.0
62 44 14 13 10 8 6 5
Oil phase
Asc-
TOH
ROO
Aqueous phase AscHTO
ROOH
size distributions was monitored together with the changes with time of olive oil PV value. This allowed us to correlate the initial rate of hydroperoxides formation with the specific area of dispersed aqueous phase. The results show that vitamin C activity is concentration-dependent and that the antioxidant is effective only if the specific surface area of dispersed phase reaches a threshold value. So a link between water droplet surface area and vitamin C activity can be suggested. This could be explained by the setting in of synergistic interactions between the intra-droplet ascorbic acid and other natural antioxidants present in olive oil, such as tocopherols. In this regard, the W/O interphase appears to play a key role in modulating the effectiveness of antioxidants, when they are dispersed in the water phase of a W/O emulsion. Acknowledgements The authors acknowledge Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGIFirenze) and Italian MIUR for the financial support (PRIN 2003).
interfacial region Fig. 7. Schematic representation of the synergistic interaction between the ascorbate monoanion and a-tocopherols in the interfacial region.
synergistic effect of these two compounds starts. It is useful to calculate the AscH content per unit surface area and then to estimate the tocopherol/AscH molar ratio at the interface, as displayed in Table 2. As one can see, the surface content of AscH increases by increasing the initial ascorbic acid concentration while the a-tocopherol/AscH molar ratio decreases. This means that a more efficient synergistic interaction can take place at the interface between a-tocopherol and the ascorbate anion by increasing ascorbic acid content in the water phase. According to the mechanism proposed by Frankel et al. (1994) for oil-in-water emulsions and on the basis of the results discussed above, the synergistic interactions between a-tocopherol and ascorbic acid in emulsified olive oils, besides being explained by the recycling of the tocopheroxyl radical intermediate to the active tocopherol, might also be due to the ability of ascorbic acid to act as a metal chelator and thus inhibit the initiation of oxidation chain reactions (Fig. 7). 4. Conclusions In this paper olive oil samples were emulsified by adding aqueous solution of vitamin C in order to improve oil resistance to light-promoted oxidative damage. To test vitamin C effectiveness in protecting the oil in continuous phase, the time evolution of water droplet
References Alpaslan, M., & Gu¨ndu¨z, H. (2000). The effects of growing conditions on oil content, fatty acid composition and tocopherol content of some sunflower varieties produced in Turkey. Nahrung, 44(6), 434–437. Ambrosone, L., Mosca, M., & Ceglie, A. (2006a). Oxidation of water emulsified olive oils. Food Hydrocolloids, 20, 1080–1086. Ambrosone, L., Mosca, M., & Ceglie, A. (2007). Impact of edible surfactants on the oxidation of olive oil in water-in-oil emulsions. Food Hydrocolloids, 21, 1163–1171. Ambrosone, L., Colafemmina, G., Palazzo, G., & Ceglie, A. (2000). NMR studies of food emulsions: The dispersed phase self-diffusion coefficient calculated by the least variance method. Progress in Colloid and Polymer Science, 115, 161–165. Ambrosone, L., Cinelli, G., Mosca, M., & Ceglie, A. (2006b). Susceptibility of water emulsified extra virgin olive oils to oxidation. Journal of the American Oil Chemists’ Society, 83(2), 165–170. Ambrosone, L., Angelico, R., Cinelli, G., Di Lorenzo, V., & Ceglie, A. (2002). The role of water in the oxidation process of extra virgin olive oils. Journal of the American Oil Chemists’ Society, 79(6), 577–582. Decker, E. A., Warner, K., Richard, M. P., & Shahidi, F. (2005). Measuring antioxidant effectiveness in food. Journal of Agricultural and Food Chemistry, 53(10), 4303–4310. European Community, Regulation 2568/91 (1991). Off. J. European Community, L. 248. Frankel, E. N. (1993). In search of better methods to evaluate natural oxidants and oxidative stability in food lipids. Trends in Food Science and Technology, 4, 220–225. Frankel, E. N. (1998). Lipid Oxidation. Dundee Scotland: The Oily Press. Frankel, E. N., Huang, S. W., Kanner, J., & German, J. B. (1994). Interfacial phenomena in the evaluation of antioxidants: Bulk oils vs. emulsions. Journal of Agricultural and Food Chemistry, 42, 1054–1059. Hoffmann, G. (1989). The chemistry and technology of edible oils and fats and their high fat products. San Diego: Academic Press. Kenney, J. F. (1939). Mathematics of statistics. New York: Van Nostrand Company Inc (pp. 104–129).
M. Mosca et al. / Food Research International 41 (2008) 201–207 Kumler, W. D., & Daniels, T. C. (1935). Titration and dissociation constant of L-ascorbic acid (vitamin C) and diethyldihydroxymaleate. Journal of the American Chemical Society, 57, 1929–1930. Lambelet, P., & Lo¨liger, J. (1985). The fate of antioxidants radicals during lipid autoxidation I. The tocopheroxyl radicals. Chemistry and Physics of Lipids, 35, 185–198. Lercker, G., Frega, N., Bocci, F., & Servidio, G. (1994). Veiled extravirgin olive oils: Dispersion response related to oil quality. Journal of the American Oil Chemists’ Society, 71, 657–658. Menendez, J. A., Vellon, L., Colomer, R., & Lupu, R. (2005). Oleic acid, the main monounsaturated fatty acid of olive oil, suppresses Her-2/neu (erbB-2) expression and synergistically enhances the growth inhibitory effects of trastuzumab (Herceptin ) in breast cancer cells with Her-2/ neu oncogene amplification. Annals of Oncology, 16, 359–371. TM
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Niki, E. (1991a). Action of ascorbic acid as a scavenger of active and stable oxygen radicals. American Journal of Clinical Nutrition, 54, 1119–1124. Niki, E. (1991b). Vitamin C as an antioxidant. World Review of Nutrition and Dietetics, 64, 1–30. Official and Tentative Methods of the American Oil Chemist’s Society (1990). Method Cd 8-53. Champaign, IL: American Oil Chemist’s Society. Packer, L. (1997). Vitamin C and redox cycling antioxidants. In L. Packer & J. Fuchs (Eds.), Vitamin C in health and disease (pp. 95–107). New York: Marcel Dekker. Packer, J. E., Slater, T. F., & Wilson, R. L. (1979). Direct observation of a free radical interaction between vitamin E and vitamin C. Nature, 278, 737–738.
Food Research International 41 (2008) 201–207 www.elsevier.com/locate/foodres
Antioxidant dispersions in emulsified olive oils Monica Mosca, Andrea Ceglie, Luigi Ambrosone * Consorzio per lo sviluppo dei Sistemi a Grande Interfase (C.S.G.I.), c/o Department of Food Technology, DISTAAM, Universita` del Molise, via De Sanctis 86100 Campobasso, Italy Received 7 September 2007; accepted 25 November 2007
Abstract In this paper water droplets dispersed in olive oils were used as containers of L-ascorbic acid and its inhibitory effect on lipid oxidation was studied. In detail, olive oil samples were emulsified by adding a certain quantity of vitamin C aqueous solutions and then oxidized by UV light. Data collected from sample analysis by optical microscopy were used to calculate the average droplets parameters and to characterize the droplet size distribution function. The results revealed a strong correlation between the initial rate of hydroperoxide formation and the specific surface area of aqueous dispersed phase. Such correlation has been explained as the consequence of a synergistic interaction between the ascorbic acid contained in the droplets and the natural tocopherols present in olive oil. A key role is played by the water–olive oil interface which is the boundary where the synergisms take place. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Antioxidant; Emulsion; Olive oil; Oxidation; Specific surface area; Vitamin C
1. Introduction Olive oil, the main fat component of the Mediterranean diet, has received great attention since it has been found out its protective role against oxidative stress (Menendez, Vellon, Colomer, & Lupu, 2005). Evaluation and control of lipid oxidation are of concern in the storage of olive oils because it results in the loss of nutritional value and the formation of compounds that may be detrimental to health (Frankel, 1993, 1998). Lipid oxidation leads to the formation of several compounds of different molecular weight and polarity which makes it difficult to evaluate the degree of oxidation. The situation becomes even more complex in the case of veiled olive oils due to additional factors that can be decisive in determining the rate and pathway of lipid oxidation. Many of these factors arise from the presence of a large interfacial area between the dispersed and continuous phases. Veiled oils represent a particular class of olive oils obtained directly from the extraction process and not *
Corresponding author. Tel.: +39 0874 404715; fax: +39 0874 404652. E-mail addresses: [email protected] (M. Mosca), [email protected] (A. Ceglie), [email protected] (L. Ambrosone). 0963-9969/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2007.11.006
filtered. Therefore they are characterized by the presence of water and particulate suspensions. Recently, a great deal of studies on vegetable oil oxidation have shown that water and small particles dispersed in the oil can act as anti-oxidants, allowing longer preservation periods (Lercker, Frega, Bocci, & Servidio, 1994). In this context we have verified that in the absence of particles and in the presence of water only (emulsified oil), the antioxidant function takes place anyway (Ambrosone, Angelico, Cinelli, Di Lorenzo, & Ceglie, 2002; Ambrosone, Mosca, & Ceglie, 2006a; Ambrosone, Cinelli, Mosca, & Ceglie, 2006b) and the oil’s shelf-life depends on the characteristics of the water-in-oil emulsions (Ambrosone, Mosca, & Ceglie, 2007). In general, the inhibitory effect of antioxidants on lipid oxidation is affected by the physico-chemical state of the lipidic substrate and various evaluation systems using different physical conditions are required to provide a good understanding of antioxidant properties in different media (Decker, Warner, Richard, & Shahidi, 2005). Lipid hydroperoxides are more polar than the starting lipids due to the presence of oxygen. We suggested that the high polarity of lipid hydroperoxides would cause them to diffuse towards the water–oil interface
202
M. Mosca et al. / Food Research International 41 (2008) 201–207
of emulsions which acts as a ‘‘sink” for hydroperoxides molecules so affecting the course of the oxidation reaction (Ambrosone et al., 2006a; Ambrosone et al., 2006b). The results indicated that the contact time between water droplets and oil is an important parameter both for oxidative stability and for emulsion stability. Therefore, it is interesting to keep working on this topic to find out ways to improve the oxidative stability of water-in-oil emulsions, by acting on the characteristics of the water dispersed phase. The aim of the present study is to analyze the oxidative degradation of emulsified olive oils when L-ascorbic acid (vitamin C) is contained in the water droplets of dispersed phase. Up to now there is no substance which is cheaper, better tolerated by the body, and with less nuisance problems associated with orally administration than ascorbic acid. 2. Experimental procedure 2.1. Materials Chloroform, acetic acid and potassium iodine were reagents of analytical grade of 99.9% purity purchased from Carlo Erba Reagenti (Milan, Italy). L-ascorbic acid was kindly donated by Dr. P. Andrea Lo Nostro (University of Firenze) and used without further purification.
hydroxytyrosol, for instance) microemulsions might form. Since such systems are thermodynamically stable and not visible to the optical microscope, we verified that all the added water was dispersed in emulsion droplets by centrifuging an emulsified sample at 4000 rpm per 30 min. In this way the quantity of added water was totally recovered indicating that no appreciable amount of water was dispersed in microemulsion droplets. 2.2.3. Oxidizing conditions Aliquots of emulsified oil samples (2.5 g) were put into polyester vessels (40 mm 40 mm 8 mm) and exposed to UV light (wavelength k = 254 nm). The lamp power was 30 W and the direction of irradiation was perpendicular to oil surface. The distance between the lamp and the oil surface was 11.5 cm. This distance was the same for all samples so that such parameter is a constant of the oxidation process. All analyses were performed ‘‘simultaneously” on different samples. Each sample was exposed to oxidation for different time intervals, at 25 °C. The oxidative state of samples was monitored by measuring the peroxide value (PV). The peroxide value of samples was determined according to AOCS official method Cd 8-53 (American Oil Chemist’s Society, 1990). The solution was titrated against standard sodium thiosulfate (0.01 N). PV was calculated and expressed as meq peroxide per kg of oil sample:
2.2. Methods PVðmeqiv=kgÞ ¼ 2.2.1. Oil sampling Olives of mixed cultivars (Cima di Mola, Cima di Bitonto and others) were picked during the 2004/2005 oil campaign and pressed with a discontinuous process (Hoffmann, 1989). The oil samples were taken directly from the crusher and treated for 15 min with an N2 current and then stored at 4 °C in 50 ml flasks. In this way each sample, once it was slowly taken back to room temperature, was completely used for the analyses. The olive oil was characterized by measuring PV, total acidity, spectrophotometric indexes and refractive index, according to the official methods recommended by the European Community (European Community, 1991). The total tocopherols concentration was determined at 520 nm according to the methodology developed by Emmerie–Engel (Alpaslan & Gu¨ndu¨z, 2000). 2.2.2. Preparation of water-in-extra virgin olive oil emulsions Water in olive oil emulsions were prepared by adding distilled water or L-ascorbic acid aqueous solution directly to 15 g of oil. All emulsified samples were prepared so that the aqueous part was always the 3 wt%. As described elsewhere (Ambrosone et al., 2002) the texture of these emulsions, i.e. their stability, is a function of the excess of mechanical energy used for the preparation. For this reason all the samples were shaken for the same length of time (15 min) with Ultraturrax T8-S8NG, 100W, Ika Labortechnik (Janke & Kunkel, Gimbh, Stanfen, Germany). Due to the presence of minor constituents (tyrosol,
ðS BÞ N F 1000 W
ð1Þ
where S is the titer for the sample, B is the titer for the blank, N is the normality of the sodium thiosulfate solution, F is the factor from standardization with potassium dichromate, and W the sample mass. The experimental uncertainty was estimated by calculating the standard deviation (SD) of repeated measurements (not less than three). 2.2.4. Optical microscopy Optical micrographs were obtained by means of an optical microscope Optech B5, at 25 °C. In order to get representative results from the system Video enhanced microscopy (VEM) was used. Such a technique combines the magnification power of the microscope with the digital image acquisition capability of a video camera (Panasonic, model GP-KR222). A series of images (9–10 pictures, 1000–1500 particles) were examined to determine the size polydispersity which was estimated by counting the ‘‘average number” of droplets at different radii in micrographs of a Thoma grating. Image analysis software (Sigma Scan Pro, SPSS Science Software Products) which provides a wide range of analytical features in addition to image enhancement was used to digitize the images. 2.2.5. H2O/vitamin C binary systems The weight fraction of dispersed phase in each emulsified oil sample was converted to volume fraction by means of density data measured by Densito30P (Mettler-Toledo
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GmbH-Switzeland). All ascorbic acid–water solutions were prepared by weight using distilled water as solvent. Density data for H2O/ascorbic acid at (22 ± 0.5) °C, expressed as a function of mass fraction of vitamin C, were fitted to the equation q ¼ 0:9960 þ 0:415W 0:01044W 2
ð2Þ
with standard deviation of 0.00005 gcm3. 3. Results and discussion 3.1. Peroxide measurements Before investigating the antioxidant effect of the aqueous dispersions, the olive oil was characterized analytically. The results are gathered in Table 1. Water-in-olive oil emulsions are complex multiphase systems in which different molecular species interact with each other. Oil oxidation in such systems is greatly affected by the nature of the interface. Because of the complexity of the oxidation process itself and of the difficulties in its accurate quantification, we decided to oxidize the system under controlled conditions. Therefore, natural olive oil samples were emulsified with aqueous solutions of vitamin C at different concentration and then were exposed to UV lamp. In Fig. 1 PV changes with oxidation time for emulsified oils without vitamin C or with different amounts of vitamin C are calculated as the ratio PV/PV0, where PV is the peroxide value at time t and PV0 is the peroxide value at t = 0. It can be seen that the larger the vitamin C amount the smaller the peroxide value of olive oil indicating that vitamin C succeeds in acting as an effective antioxidant even if it is confined into water droplets, out of the reaction site. However, to gather some information about the involvement of vitamin C in the oxidation process of emulsified oils, we analyzed the initial rates of hydroperoxides formation, defined by m ¼ ðdPV Þ . The plot of v0 versus the overdt t¼0 all concentration of vitamin C, displayed in Fig. 2A, exhibits a constant trend up to a value of 1200 mg/Kg of emulsified oil and then decreases for higher vitamin C concentration. Ascorbic acid is a weak diprotic acid (AscH2) with enediol group into a five membered heterocyclic lactone ring. Its dissociation constants in pure water at 22–23° are
Fig. 1. PV/PV0 versus UV oxidation time of extra virgin olive oil emulsified with aqueous solution of L-ascorbic acid. Oil emulsified with water only (s), is compared with dispersion containing an overall concentration of 50 ppm (d), 100 ppm (j), 900 ppm(), 3500 ppm (N), 5000 ppm (.), and 8000 ppm (L) of vitamin C.
Ka1 = 7.58106 Ka2 = 3.09 1012, respectively (Kumler and Daniels,1935). It is well known that ascorbate anion (AscH) is the species directly involved in the oxidation process through the formation of the ascorbyl radical anion. Assuming the inner of droplet to be pure water and neglecting the second acid dissociation reaction we calculated the local concentration of ascorbate anion as 2 a solution of the algebraic equation ½AscH þ k a1 ½AscH k a1 ½AscH2 0 ¼ 0; where [AscH2]0 is the initial concentration of ascorbic acid. The perfect linearity between the hydroperoxides (ROOH) formation rate and the local concentration of the ascorbate anion, displayed in Fig. 2B, demonstrates the direct involvement of this anion in the oxidative process also in water-in-olive oil emulsions. It is interesting the presence of a region in Fig. 2A where the formation rate of hydroperoxides is poorly sensitive to vitamin C concentration. This is an
Table 1 Chemical characteristics of olive oil samples. The results are expressed as mean of repeated measurements ± standard deviation (n P 3) Peroxide valuea (meq O2Kg1) 13.19 ± 0.02 a b c d e
Acidity valueb (% oleic acid)
Total tocopherolsc (mg Kg1 as a-tocopherol)
Refractive indexc
Spectrophotometric indexes k232d
k270d
Dke
0.850 ± 0.004
232 ± 9
1.4675 ± 0.0005
1.74 ± 0.01
0.27 ± 0.01
0.085 ± 0.02
n = 18. n = 3. n = 4. kk = specific extinction coefficient at wavelength k. Dk = k2700.5 (k266 + k274).
b
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itoring the texture measurements.
of
emulsions
through
VEM
3.2. Video enhanced microscopy measurements
Fig. 2. Initial rate of hydroperoxides formation as a function of the (A) overall L-ascorbic acid concentration and (B) local ascorbate monoanion concentration.
indication of some particular mechanism of action for scavenging free radicals. To understand this aspect it is worthwhile remarking that in olive oils there are different substances with antioxidant properties, like tocopherols and poliphenols, which play a key role in determining oil resistance to oxidation. Although the content of phenolic derivates is dependent on olive cultivar, climatic conditions, oil extraction process and storage conditions, the average amount of total tocopherols is quite constant and specific for olive oils and it is around 200–300 mg per kg of oil. Among tocopherols, a-tocopherol is the most abundant in olive oils and it is the molecule with the highest antioxidant activity (Hoffmann, 1989). This datum is particularly noteworthy since it has been demonstrated the synergic interaction between tocopherols and ascorbic acid in homogeneous systems (Lambelet & Lo¨liger, 1985; Packer, Slater, & Wilson, 1979). However, there have been some controversly on whether such an interaction can occur in heterogeneous systems in which tocopherols are located in the lipidic environment whereas ascorbic acid is located in aqueous environment (Niki, 1991a; Packer, 1997). Ascorbic acid, due to its high lipophobicity, is not able to exert its antioxidant function when the radicals are primarily formed within the oil phase where tocopherols are active. On the other hand a-tocopherol molecules are slightly surface active and therefore they might be located at the water–oil interphase especially when present in oxidized form. Indeed, once at the interface the tocopheroxyl radicals would be able to interact with vitamin C and to be regenerated back to the reduced form. This surface–mediated interaction between vitamin C and vitamin E was studied in oil-in-water emulsions by Frankel et al. and in liposomal membranes by Niki (Frankel, Huang, Kanner, & German, 1994; Niki, 1991b). Herein we have studied the role of the water–oil interface in the antioxidant action of vitamin C with mon-
Emulsion structure of freshly prepared samples is clearly identified through optical microscopy which shows a large size polydispersity of dispersed phase. Droplet size distributions of samples containing increasing amounts of vitamin C are shown in Fig. 3. The experimental VEM results were processed according to the technique we described in detail elsewhere (Ambrosone, Colafemmina, Palazzo, & Ceglie, 2000). The data were well described by a lognormal function " # 1 ð1nD 1nD0 Þ2 P ðDÞ ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð3Þ exp 2r2 2pr2 D0 which is dependent on two parameters, D0 an r. Many properties of the experimental distribution function can be derived by the moments: Z 1 Dn P ðDÞdD ð4Þ ln ¼ 0
where n is the moment order and l0 = 1. Particularly useful are D10, the average diameter, and D32 the volume/surface ratio (or Sauter average). In order to study the interphase role in polydispersed systems is noteworthy the specific surface area of dispersed phase: AD ¼
6l2 6 ¼ D32 l3
ð5Þ
Plots of D10 versus local concentration of ascorbic acid and ascorbate monoanion, respectively, reveal unexpectedly a direct relationship between the mean size of water droplets
Fig. 3. Size distribution function P(D) as a function of diameter D at different ascorbic acid concentration.
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between the formation rate of hydroperoxides and the specific AD. Thus the surface/volume ratio of the dispersed phase drives the antioxidant action of ascorbic acid. Even in the plot in Fig. 5C is evident the presence of a region where ROOH formation rate stays unchanged. We investigated this point by plotting both v0 and AD versus the local concentration of ascorbate monoanion. As one can see in Fig. 6 the region of constant rate coincides with the region of constant specific surface. Such results seem to indicate that the antioxidant action of vitamin C takes place only if the specific surface area of the dispersed phase reaches a threshold value. This is because the surface activity of a-tocopherols is mild so that for low values of AD the quantity of a-tocopherols in the interfacial region is very low and it is scarcely accessible to ascorbate monoanion molecules. When the specific surface reaches a value as high as to host a large a-tocopherols amount per water droplet the Fig. 4. Mean diameter of water droplet as a function of the (A) overall Lascorbic acid concentration and (B) local ascorbate monoanion concentration.
and the conjugate base concentration (Fig. 4A, B). Inasmuch as this value, at fixed temperature, is completely determined by the dissociation constant, one deduces that thermodynamic and kinetic stability are interrelated. The relation between the mean droplet size and the ascorbate anion concentration suggests a direct involvement of the water/oil interface in determining antioxidant activity. Then, if the antioxidant action occurs at the water-oil interface, a direct influence of emulsion texture on the oxidation reaction rates is expected. On the other hand, the system is polydisperse in size so that the mean diameter and standard deviation are not enough to detect the characteristics of the distribution function (Kenney, 1939). Therefore we calculated l2 and l3 directly from micrographs and related these values to the formation rate. As one can see in Fig. 5A, B, the plot of v0 versus l2 and l3 does not show any simple relation, on the contrary Fig. 5C shows a direct correlation
Fig. 6. Comparison between initial rate and specific surface area as a function of the local ascorbate monoanion concentration.
Fig. 5. Initial rate of hydroperoxides formation as a function of (A) the second moment of the droplet size distribution function, (B) the third moment of the droplet size distribution function and (C) the specific surface area.
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Table 2 AscH content per unit surface area and Tocopherol/Ascorbate anion molar ratio of W/Olive oil emulsions at different total ascorbic acid content Ascorbic acid emulsion content (ppm)
AscH (mg m2)
a-Tocopherol/AscH-molar ratio (mol/mol)
50 100 900 1200 2000 3500 5000 8000
0.26 0.42 0.40 1.8 2.2 2.2 2.8 2.0
62 44 14 13 10 8 6 5
Oil phase
Asc-
TOH
ROO
Aqueous phase AscHTO
ROOH
size distributions was monitored together with the changes with time of olive oil PV value. This allowed us to correlate the initial rate of hydroperoxides formation with the specific area of dispersed aqueous phase. The results show that vitamin C activity is concentration-dependent and that the antioxidant is effective only if the specific surface area of dispersed phase reaches a threshold value. So a link between water droplet surface area and vitamin C activity can be suggested. This could be explained by the setting in of synergistic interactions between the intra-droplet ascorbic acid and other natural antioxidants present in olive oil, such as tocopherols. In this regard, the W/O interphase appears to play a key role in modulating the effectiveness of antioxidants, when they are dispersed in the water phase of a W/O emulsion. Acknowledgements The authors acknowledge Consorzio Interuniversitario per lo Sviluppo dei Sistemi a Grande Interfase (CSGIFirenze) and Italian MIUR for the financial support (PRIN 2003).
interfacial region Fig. 7. Schematic representation of the synergistic interaction between the ascorbate monoanion and a-tocopherols in the interfacial region.
synergistic effect of these two compounds starts. It is useful to calculate the AscH content per unit surface area and then to estimate the tocopherol/AscH molar ratio at the interface, as displayed in Table 2. As one can see, the surface content of AscH increases by increasing the initial ascorbic acid concentration while the a-tocopherol/AscH molar ratio decreases. This means that a more efficient synergistic interaction can take place at the interface between a-tocopherol and the ascorbate anion by increasing ascorbic acid content in the water phase. According to the mechanism proposed by Frankel et al. (1994) for oil-in-water emulsions and on the basis of the results discussed above, the synergistic interactions between a-tocopherol and ascorbic acid in emulsified olive oils, besides being explained by the recycling of the tocopheroxyl radical intermediate to the active tocopherol, might also be due to the ability of ascorbic acid to act as a metal chelator and thus inhibit the initiation of oxidation chain reactions (Fig. 7). 4. Conclusions In this paper olive oil samples were emulsified by adding aqueous solution of vitamin C in order to improve oil resistance to light-promoted oxidative damage. To test vitamin C effectiveness in protecting the oil in continuous phase, the time evolution of water droplet
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