1. INTRODUCTION
⌅Olive oils (OO), as other vegetable oils, are mainly made up of triglycerides (between 98% and 99%). Their content in free fatty acids is well known (Barjol, 2013Barjol JL. 2013. Introduction, in Aparicio R, Harwood J (Eds.) Handbook of Olive Oil. Analysis and Properties, second ed., Springer, New York, pp. 1-17. https://doi.org/10.1007/978-1-4614-7777-8_1 ). The proportion of free fatty acids of OO is variable, depending on the extent of the hydrolysis of triglycerides. Thus, the free fatty acids in vegetable oils are generally considered as those that do not form part of the triglycerides. Olive oil free acidity is one of the major quality features of virgin olive oils. In fact, according to the standards in force of the European Commission (1991)European Commission, 1991. Commission Regulation (EEC) Ner 2568/91 of 11 July 1991, on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis. 128 pp., the classification into Extra Virgin Olive Oil class (EVOO) requires the free acidity to be lower than 0.8%. The class EVOO also requires the fruity sensory attribute to be perceptible, and for it to be free of organoleptic defects. The product without these features but with free acidity up to 2%, belongs to the class VOO. An acidity value higher than 2% implies that the product is to be classified as lampante olive oil, which should be refined. The official method of analysis of olive oil’s free acidity (European Commission, 1991European Commission, 1991. Commission Regulation (EEC) Ner 2568/91 of 11 July 1991, on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis. 128 pp.) is a volumetric titration. Briefly, 4 to 6 g of olive oil are weighed into 250 mL wide-mouth Erlenmeyer flasks, and 50-mL ethyl alcohol: ethyl ether at 1:1 and a few drops phenolphthalein are added, and it is then neutralized with NaOH 0.1 N until it turns a pink color, and expressed as a percentage of oleic acid. Thus, olive oil acidity is considered to be caused by the fatty acids liberated from triglycerides by hydrolysis processes, and expressed as the analyzed value according to the method explained above.
It is interesting to highlight that the physical state of cloudy virgin olive oil (VOO) is due to a phase formed by microscopic droplets of water in meta-stable dispersion (Lercker et al., 1994Lercker G, Frega N, Bocci F, Servidio G. 1994. “Veiled” extra-virgin olive oils: Dispersion response related to oil quality. J. Am. Oil Chem. Soc. 71, 657-658. https://doi.org/10.1007/s11746-999-0239-4 ). Moisture spreads homogeneously in the major mass of triglyceride liquid, making it an emulsion colloid. Besides, VOO has another phase formed by microscopic solid particles, thus it has been described as a sol-emulsion colloid (†Gómez-Herrera, 2007†Gómez- Herrera C. 2007. Matter transfer during virgin olive oil elaboration. Grasas Aceites 58 (2), 194-205. https://doi.org/10.3989/gya.2007.v58.i2.85 ). In this article, we explain how the fatty acids in veiled olive oils could not be free in the triglyceride mass, and that the functional free acidity of these olive oils could be lower than the acidity determined through official analysis.
2. CAPTIVE FATTY ACIDS IN VEILED OLIVE OILS?
⌅A study on the influence of the scattered matter in veiled olive oils on their shelf-life reported that the free fatty acids precipitated with the residue or sediment in the oil (Lercker et al., 1994Lercker G, Frega N, Bocci F, Servidio G. 1994. “Veiled” extra-virgin olive oils: Dispersion response related to oil quality. J. Am. Oil Chem. Soc. 71, 657-658. https://doi.org/10.1007/s11746-999-0239-4 ). The cited study related the precipitate of the solid particles contained in the olive oils to the attraction of opposite electrical charges. The same study pointed out that the scattered matter dampened the acidity, at the same time exerting an antioxidant effect.
Together with this, there are several studies on the conservation of alternatively cloudy or filtered olive oils. The literature agrees that changes in the acidity during the period of olive oil storage are greater in veiled olive oils than in the filtered ones (Tsdimidou et al., 2005Tsdimidou MZ, Georgiou A, Koidis A, Boskou D. 2005. Loss of stability of ‘‘veiled’’ (cloudy) virgin olive oils in storage. Food Chem. 93, 377-383. https://doi.org/10.1016/j.foodchem.2004.09.033 ; Fregapane et al., 2006Fregapane G, Lavelli V, Leon S, Kapuralin J, Salvador MD. (2006). Effect of filtration on virgin olive oil stability during storage. Europ. J. Lipid Sci. Tech. 108, 134-142. https://doi.org/10.1002/ejlt.200501175 ; Stefanoudaki et al., 2010Stefanoudaki E, Williams M, Harwood J. 2010. Changes in virgin olive oil characteristics during different storage conditions. Europ. J. Lipid Sci. Tech. 112, 906-914. https://doi.org/10.1002/ejlt.201000066 ). The literature establishes that free fatty acids have a prooxidant effect (Frega et al., 1999Frega N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil. J. Am. Oil Chem. Soc. 76, 325. https://doi.org/10.1007/s11746-999-0239-4 ), and that high acidity exerts a negative influence on the stability of olive oil (Ciafardini, et al., 2006Ciafardini G, Zullo BA, Iride A. 2006. Lipase production by yeasts from extra virgin olive oil. Food Microbiol. 23 (1), 60-67. https://doi.org/10.1016/j.fm.2005.01.009 ; Frega et al., 1999Frega N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil. J. Am. Oil Chem. Soc. 76, 325. https://doi.org/10.1007/s11746-999-0239-4 ). The referred studies concur that a slightly better oxidative stability index (OSI) is determined for cloudy olive oils. Frega et al. (1999)Frega N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil. J. Am. Oil Chem. Soc. 76, 325. https://doi.org/10.1007/s11746-999-0239-4 reported that the suspended-dispersed material of cloudy virgin oil exerted a positive effect on oxidative stability. Moreover, the same authors found a higher antioxidant effect of the dispersed particles compared to the suspended particles. This last study proved the olive oil quality decay was significantly delayed by removing the scattered matter. The authors pointed out the possibility of an effect from the higher surface to dimension ratio of the dispersed particles compared to the suspended particles. It is possible to think the authors referred to the ‘suspended’ particles as those with little stability among all participating in the dispersion. Nevertheless, the study cited lacks a clear description of what particles are referred to by the concepts ‘suspended’ and ‘dispersed’. It is also interesting to consider that, according to Chaiyasit et al., (2007Chaiyasit W, Elias RJ, McClements DJ, Decker EA. 2007. Role of physical structures in bulk oils on lipid oxidation. Crit Rev Food Sci Nutr. 47, 299-317. https://doi.org/10.1080/10408390600754248 ), the interface of association colloids is a likely site of oxidation reactions since many prooxidants and antioxidants are also surface active and thus would concentrate at the water-lipid interface.
A recent microscopy study (Cayuela-Sánchez and Caballero-Guerrero, 2019Cayuela-Sánchez JA, Caballero-Guerrero B. 2019. Fresh extra virgin olive oil, with or without veil. Trends Food Science Tech. 83, 78-85. https://doi.org/10.1016/j.tifs.2018.11.014 ) revealed that the major part of the spread matter in cloudy virgin olive oil is formed by microscopic droplets of water, while solid particles seem to be contained within an aqueous medium inside colloidal globules (Figure 1). The name of reverse micelles does not seem appropriate for these globules, because their size, even larger than 15 μm, is too large for it. The diameter of reverse micelles ranges from 7 10-4 μm to 25 10-4 μm (Amararene et al., 1997Amararene A, Gindre M, Le Huérou JY, Nicot C, Urbach W, Waks M. 1997. Water Confined in Reverse Micelles: Acoustic and Densimetric Studies. J. Phys. Chem. B 101, 10751-10756. https://doi.org/10.1021/jp972718f ), thus quite smaller. The referred solid particles were reported to show a wide diversity both in shape and size. Besides, they were observed in motion. The peculiar movement of said solid particles inside the colloidal globules was recorded in various video sequences (Cayuela-Sánchez and Caballero-Guerrero, 2019Cayuela-Sánchez JA, Caballero-Guerrero B. 2019. Fresh extra virgin olive oil, with or without veil. Trends Food Science Tech. 83, 78-85. https://doi.org/10.1016/j.tifs.2018.11.014 ). The authors first related the observed movement to the possible physical influence of electrostatic charges. However, the observed particles’ agitation clearly matches the Brownian movement according to the literature (Einstein, 1905Einstein A. 1905. Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen. Ann. Phys. 17, 549. https://doi.org/10.1002/andp.19053220806 ; Smoluchowski, 1906Smoluchowski M. 1906. Zur kinetischen Theorie der Brownschen Molekularbewegung und der Suspensionen. Ann. Phys. 21 (14), 756-780. https://doi.org/10.1002/andp.19063261405 ; Einstein, 1956Einstein A. 1956. Investigations of the Theory of Brownian Movement. Dover Publications, 119 pp.). This physiochemical effect is observed particularly in emulsions. The major interest of this movement of the solid particles in olive oil colloidal globules is that it confirms the hypothesis the internal content of the colloidal globules is aqueous. This movement of particles is not observed in the mass of veiled olive oil, formed largely by triglycerides, outside the globules. Albert Einstein explained in detail (Einstein, 1905Einstein A. 1905. Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen. Ann. Phys. 17, 549. https://doi.org/10.1002/andp.19053220806 ) how the movement Brown had observed in pollen was the result of the particles being pushed by individual water molecules. Briefly explained, the notorious scientist attributed the movement to the atomic bombing force, whose direction is constantly changing. Therefore, the observed movement could exist independently from the microscopy illumination. However, artificial light should provide additional excitation because this physical phenomenon is temperature-dependent (Seddig, 1908Seddig M. 1908. The measurement of temperature dependency of Brown’s molecular movement. Physik. Zeitschrift 9, 465-468.). Thus, the heat provided by the light used for microscopy observation could be responsible for most of the observed movement.
The suspended matter identified by Frega et al., (1999)Frega N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil. J. Am. Oil Chem. Soc. 76, 325. https://doi.org/10.1007/s11746-999-0239-4 probably relates to these colloidal globules, despite such globular structures containing water was not described. Moreover, Frega et al., (1999)Frega N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil. J. Am. Oil Chem. Soc. 76, 325. https://doi.org/10.1007/s11746-999-0239-4 related the amino groups of lignin, derived from the olive nut, as primarily accountable for the cloudy material. However, the study did not consider the possibility that microscopic droplets of water are directly responsible for the opalescence of veiled olive oils. The referred authors hypothesized that after reacting with FFA, the amino groups may liberate the phenolic groups that were previously bonded to hydrogen, a state in which they were not able to display any antioxidant activity. Frega et al., (1999)Frega N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil. J. Am. Oil Chem. Soc. 76, 325. https://doi.org/10.1007/s11746-999-0239-4 related this to the observation that FFA bonded to the disperse particles and precipitated with them as a brown colored residue, as reported in a previous study (Lercker et al., 1994Lercker G, Frega N, Bocci F, Servidio G. 1994. “Veiled” extra-virgin olive oils: Dispersion response related to oil quality. J. Am. Oil Chem. Soc. 71, 657-658. https://doi.org/10.1007/s11746-999-0239-4 ). However, these studies did not consider that FFA may participate in the colloidal globules reported (Cayuela-Sánchez and Caballero-Guerrero, 2019Cayuela-Sánchez JA, Caballero-Guerrero B. 2019. Fresh extra virgin olive oil, with or without veil. Trends Food Science Tech. 83, 78-85. https://doi.org/10.1016/j.tifs.2018.11.014 ), among the structural units forming their pseudo-wall (perceptible in Figure 1), as the present study proposes. This last hypothesis agrees that FFA precipitate with the solid residue noted by Lerker et al., (1994)Lercker G, Frega N, Bocci F, Servidio G. 1994. “Veiled” extra-virgin olive oils: Dispersion response related to oil quality. J. Am. Oil Chem. Soc. 71, 657-658. https://doi.org/10.1007/s11746-999-0239-4 ; although a different scenario for the modulation of the acidity of veiled virgin olive oils can be shown. Frega et al., (1999)Frega N, Mozzon M, Lercker G. 1999. Effects of free fatty acids on oxidative stability of vegetable oil. J. Am. Oil Chem. Soc. 76, 325. https://doi.org/10.1007/s11746-999-0239-4 inferred that the effects from spread matter are so positive that avoidance of oil filtration is desirable in order to extend the olive oil’s shelf-life. However, this conclusion should be assessed considering that olive oil filtration implies a drastic removal of moisture, and the elimination of water soluble phenolics along with it. (Bakhouche et al., 2014Bakhouche A, Lozano-Sánchez J, Ballus CA, Martínez-García M, González Velasco, M, Olavarría-Govantes A. 2014. Monitoring the moisture reduction and status of bioactive compounds in extra-virgin olive oil over the industrial filtration process. Food Control 40, 292-299. https://doi.org/10.1016/j.foodcont.2013.12.012 ). The phenolic compounds which provide the highest antioxidant activity in olive oil, such as hydroxytyrosol and tyrosol, are the most water-soluble ones (Capasso et al., 1992Capasso R, Evidente A, Scognamiglio F. 1992. A simple thin layer chromatographic method to detect the main polyphenols occurring in olive oil vegetation waters. Phytochem. Analysis 3 (6), 270 -275. https://doi.org/10.1002/pca.2800030607 ; Bakhouche et al., 2014Bakhouche A, Lozano-Sánchez J, Ballus CA, Martínez-García M, González Velasco, M, Olavarría-Govantes A. 2014. Monitoring the moisture reduction and status of bioactive compounds in extra-virgin olive oil over the industrial filtration process. Food Control 40, 292-299. https://doi.org/10.1016/j.foodcont.2013.12.012 ). Therefore, the filtration of virgin olive oil implies a drastic loss in its antioxidant power, with negative influence both on its oxidative stability and nutritional benefits. It is advisable to consider all of the above to decide the most suitable handling of olive oil according to its destination.
The structure and dynamics of colloidal dispersions in veiled virgin olive oil (VOO) have been the subject of a study (Papadimitriou et al., 2013Papadimitriou V, Dulle M, Wachter W, Sotiroudis TG, Glater O, Xenakis A. 2013. Structure and dynamics of veiled virgin olive oil: Influence of production conditions and relation to its antioxidant capacity. Food Biophys. 8 (2), 112-121. https://doi.org/10.1007/s11483-013-9286-3 ). The authors used confocal microscopy and three different dispersive techniques for characterizing samples of turbid VOO, among which the best was the “Small Angle Light Scattering Apparatus” (SALSA). The SALSA data showed a colloid diameter range from 1.5 to 14 μm. The authors indicated that the resolution limit for the method used was 15 μm, with higher colloids not being detected. Some new details concerning colloidal globules found in olive oil have been reported recently (Cayuela and Caballero-Guerrero, 2019Cayuela-Sánchez JA, Caballero-Guerrero B. 2019. Fresh extra virgin olive oil, with or without veil. Trends Food Science Tech. 83, 78-85. https://doi.org/10.1016/j.tifs.2018.11.014 ). These colloidal globules are formed by amphiphilic molecules, among which proteins, sugar glycosides, and glycoproteins are found, according to the contents reported for fresh olive oils (Koidis and Boskou, 2006Koidis A, Boskou D. 2006. The contents of proteins and phospholipids in cloudy (veiled) virgin olive oils. Europ. J. Lipid Sci. Tech. 108, 323-328. https://doi.org/10.1002/ejlt.200500319 ; Lercker et al., 1994Lercker G, Frega N, Bocci F, Servidio G. 1994. “Veiled” extra-virgin olive oils: Dispersion response related to oil quality. J. Am. Oil Chem. Soc. 71, 657-658. https://doi.org/10.1007/s11746-999-0239-4 ), and to organic fragments of diverse complexity. It is important to note that free fatty acids, which determine olive oil acidity, are also amphiphilic. The shorter their chain and the greater their unsaturation, the greater their affinity for water (Nelson and Cox, 2017Nelson DL, Cox MM. 2017. Lehninger principles of biochemistry. 7th. ed. W. H. Freeman and Co. New York. 1340 pp.). Total unsaturated fatty acids in olive oils range from 77% to 88% and monounsaturated fatty acids averaging around 75% (García-González et al., 2013García-González DL, Infante-Domínguez C, Aparicio R. 2013. Tables of olive oil chemical data, in Aparicio R, and Harwood J (Eds.) Handbook of olive oil: Analysis and properties, Springer, New York, pp. 739-768.). However, up to now, the relation of fatty acids to the colloidal globules of olive oil has not been characterized. Here is a proposal on the hypothesis to a link of free fatty acids to the pseudo-wall of the colloidal globules, suggesting that adsorption of free fatty acids in the interface of water globules may result in a decrease in their chemical reactivity.
3. EVOLUTION OF THE ACIDITY OF VEILED OLIVE OILS
⌅The literature on the evolution of acidity in the conservation of veiled and filtered olive oils (Tsdimidou et al., 2005Tsdimidou MZ, Georgiou A, Koidis A, Boskou D. 2005. Loss of stability of ‘‘veiled’’ (cloudy) virgin olive oils in storage. Food Chem. 93, 377-383. https://doi.org/10.1016/j.foodchem.2004.09.033 ; Fregapane et al., 2006Fregapane G, Lavelli V, Leon S, Kapuralin J, Salvador MD. (2006). Effect of filtration on virgin olive oil stability during storage. Europ. J. Lipid Sci. Tech. 108, 134-142. https://doi.org/10.1002/ejlt.200501175 ; Stefanoudaki et al., 2010Stefanoudaki E, Williams M, Harwood J. 2010. Changes in virgin olive oil characteristics during different storage conditions. Europ. J. Lipid Sci. Tech. 112, 906-914. https://doi.org/10.1002/ejlt.201000066 ) is consistent with the possible link of ‘free’ fatty acids to the globules as amphiphilic molecules, as explained below. Their binding to the pseudo-walls of such globules may involve some restraint of their chemical reactivity. This hypothesis is consistent with a brief report on the idea that the scattered matter of veiled oil drops its acidity level (Lercker et al., 1994Lercker G, Frega N, Bocci F, Servidio G. 1994. “Veiled” extra-virgin olive oils: Dispersion response related to oil quality. J. Am. Oil Chem. Soc. 71, 657-658. https://doi.org/10.1007/s11746-999-0239-4 ), although these referred authors did not give reasons.
The segregation of the molecules framing the pseudo-wall of the colloidal globules may explain the subsequent decantation of water and sedimentation of solids. This is consistent with the slight increase in acidity noted during the storage of cloudy olive oils (Tsdimidou et al., 2005Tsdimidou MZ, Georgiou A, Koidis A, Boskou D. 2005. Loss of stability of ‘‘veiled’’ (cloudy) virgin olive oils in storage. Food Chem. 93, 377-383. https://doi.org/10.1016/j.foodchem.2004.09.033 ; Fregapane et al., 2006Fregapane G, Lavelli V, Leon S, Kapuralin J, Salvador MD. (2006). Effect of filtration on virgin olive oil stability during storage. Europ. J. Lipid Sci. Tech. 108, 134-142. https://doi.org/10.1002/ejlt.200501175 ; Stefanoudaki et al., 2010Stefanoudaki E, Williams M, Harwood J. 2010. Changes in virgin olive oil characteristics during different storage conditions. Europ. J. Lipid Sci. Tech. 112, 906-914. https://doi.org/10.1002/ejlt.201000066 ). Also, according to our microscopy study, the solid particles seem to lodge inside the colloidal globules, but not spread into the triglyceride matrix. Under microscopy observation, the repulsion exerted by the triglyceride mass seems to force the droplets of water through the amphiphilic compounds of the pseudo-wall, towards the inside of the globules. Therefore, the colloidal globules collapse, due to the coalescence of microscopic droplets of water is the logical necessary outcome. Thus, the globules collapse implies the disassembly of their amphiphilic compounds. The solid particles previously suspended in a watery medium inside the colloidal globules must also be released during the proves. Consequently, the solid matter may begin the process of precipitation. From these results, a logical hypothesis is that the free fatty acids released from the pseudo-walls from the collapse of the globules, then begin exerting their characteristic chemical activities.
4. CONCLUSIONS
⌅Colloidal globules have been reported in forming part of the sol-emulsion colloid characteristic of unfiltered virgin olive oils, as well as in the product before a rest period in tanks. In this study it is hypothesized that the adsorption of free fatty acids on the pseudo-wall of these globules may reduce the functional olive oil acidity. Therefore, the fatty acids resulting from the triglyceride hydrolysis, are not free in the veiled olive oil but ‘captive’ in the pseudo-walls. It is reasonable that this fact can reduce its chemical activity, as may be the case with its prooxidant activity. Thusly, it is logical to believe that when the fatty acids adhere to the globules pseudo-walls, they may have less influence on the perception of acidity by the consumer of these cloudy virgin olive oils. The official analysis method of olive oil acidity can’t detect this last effect since it is a titration test. If this is true, veiled fresh virgin olive oils can offer natural modulation of their acidity, while preserving said physical state. To the best of our knowledge, these ideas have yet to be considered in scientific literature.