PaPer 90 Ital. J. Food Sci., vol. 28 - 2016 - Keywords: chlorophyll, kinetics, thermal degradation, virgin olive oil - Differential methoD to Determine thermal DegraDation kinetics of chlorophyll in virgin olive oil ahmet levent inanc Department of Food Engineering, Engineering and Architecture Faculty, Kahramanmaras Sutcu Imam University, 46100 Kahramanmaras, Turkey email: linanc@ksu.edu.tr AbstrAct Differential method is presented to study thermal degradation kinetics of chlorophyll in vir- gin olive oil. the oil samples, naturally containing 20.0 mg/kg chlorophyll were stored at 150°, 160°, 170°, 180°, 190° and 200°c until the time at which chlorophyll contents had reduced to the certain amounts. the concentration gradually decreased as heating time increased. A half or- der equation was found as the best model for the present experimental data. Differential meth- od with graphic and substitution methods was compared for the determination of the rate con- stant and the half-time. the rate constants and half life at 150°c were determined in the range of 0.20-0.22 and 12.14-13.12 for the thermal process of chlorophyll in virgin olive oil, respective- ly. the reaction rates increased approximately 1.1 times with increment of every 10°c from tem- perature of 150°c. conversely, the half lifes decreased 0.9 times for increment of every 10°c. the activation energies were approximately 24 J/kg for differential method, and 22 J/kg for graphic and substitution methods. mailto:linanc%40ksu.edu.tr?subject= Ital. J. Food Sci., vol. 28 - 2016 91 IntroDuctIon chlorophylls are responsible for the green col- or of all vegetables and fruits. Animal tissues can’t synthesis chlorophylls, though animal cells can chemically modify them for assimila- tion. these compounds should be supplied from food (GIuFFrIDA et al., 2007). chlorophyll and its various derivatives have been used in tradi- tional medicine and for therapeutic purposes for many years and perhaps have the potential role of these pigments in the prevention of human cancers that has drawn more recent attention (FErruZZI and bLAKEsLEE, 2007). the color of olive oil is principally related to its perceived quality, and therefore to its accept- ability. the economic importance of the appear- ance of the oils is unquestionable. the color of virgin olive oil is due to the natural pigments chlorophylls, and carotenoids (MInGuEZ Mos- QuErA et al., 1994). olive oil contains originally the chlorophylls a and b. chlorophyll a, pheophytin a, is typical- ly found in higher amounts than chlorophyll b. the distribution and content of chlorophyll in olive oil are dependent on a number of factors including species, agroclimactic conditions, pre- and postharvest treatment, and type and degree of food processing (MInGuEZ-MosQuErA et al., 1990, GAnDuL-roJAs et al., 1996, GIuFFrIDA et al., 2007, crIADo et al., 2008, cErrEtAnI et al., 2008, GIuFFrIDA et al., 2011). the grades of oil extracted from the olive fruit are classified as virgin, lampante, refined and ol- ive pomace oil. Virgin oil is produced by the use of mechanical means only, with no chemical treat- ment or heat. Virgin oil includes both virgin olive oil (Voo) and extra-virgin olive oil (EVoo) prod- ucts, depending on quality. therefore, virgin olive oil should be preferably added as the final sea- soning in fresh salads, soups, or more elaborated dishes (cArLA et al., 2013) but olive oil like oth- er vegetable oils is used in several cooking pro- cesses such as deep-frying, pan-frying, roasting, microwave cooking, etc. (WAtErMAn and LocK- WooD, 2007; bosKou, 2009). Each thermal pro- cessing type has particular characteristics as de- pending on process temperature and time. cAr- LA et al. (2013) summarized several works re- lated to olive oil that used as the cooking base, grouped the works by real and simulated cook- ing method and showed the analytical parame- ters chosen by the authors to evaluate olive oil performance. For example, in frying process the both methods were tested with several olive oil commercial grades, at temperatures ranging from 170°c to 180°c in real frying, and from 160°c to 190°c in simulated frying, i.e. being the olive oil heated without any food. some authors also compared the effects of adding fresh oil between frying sessions in the oil performance. In the pre- vious studies it was made on thermal stability of olive oil. the studies on thermal stability of olive include the thermal decomposition of commer- cial vegetable oils of some of their thermal prop- erties (DWEcK et al., 2004), the thermal degrada- tion study of four unsaturated or saturated ester- ified c18 fatty acids with glycerol (VEccHIo et al., 2008), stability of olive oil during heating (bErA- sAtEGI et al., 2012), the heat-oxidation stability of binary blends made with palm oil and several extra virgin olive oils (DE LEonArDIs and MAccI- oLA, 2012) and effects of the main virgin olive oil antioxidants under mild temperature conditions (MAncEbo-cAMPos et al., 2014) but, virgin olive oil contains minor constitutes together with tri- glycerides, the thermal effect on chlorophyll sta- bility and degradation in olive oil has not been studied extensively. Kinetic modelling recently gaining increas- ing interest in food science gives the possibility of controlling changes in foods such as to con- trol food quality during processing and shelf life (nIAMnuY et al., 2012; GouLA, 2013; GrAuWEt et al., 2014; rEMInI et al., 2015). Microbiologi- cal changes which are called as predictive mi- crobiology have been worked up to recent years but it can also be applied for biological, chemical and physical changes. the rate of a reaction and its temperature dependence, the occurrence of such a reaction can be predicted and controlled under specified conditions. the difficulties in ki- netic modelling are choosing the right model for a reaction. For example; one of the difficulties is that too few data points are available to de- cide for the correct order. In general, research- ers in food science have limited themselves of- ten to simple reaction kinetics. i.e. it is trying to fit a zero-, first- or second order model to their data (VAn boEKEL, 1996; VAn boEKEL, 1999). the present study focused on the determina- tion of rate order and characterization of the Ar- rhenius parameters governing the thermal deg- radation reactions of chlorophyll in virgin olive oil by using the differential method and was to compare it with other two rate order determina- tion methods. 2. MAtErIAL AnD MEtHoDs 2.1. Materials olive oil from olive fruits harvested in 20012 - 2013 season were obtained from a local olive oil plant (Demirkol Ltd., Kahramanmaras, turkey). Working principle of the plant is that olives are stored in the hopper of olive elevator and trans- ported to washing machine. First leaves of ol- ives are removed by leaf remover. then olives are washed without giving harms to its pulp in the ol- ive washing unit. olives are transported to crush- er by crusher elevator. olives are crushed and become semi paste in crusher. semi paste olives are mixed to obtain oil in malaxers. crushed olive is fed into the decanter without water through a 92 Ital. J. Food Sci., vol. 28 - 2016 pulp pump. Input product comes out of decant- er as oil and pomace with black water. the char- acteristic of the olive oil are as follows: free acid- ity, 0.49 % oleic acid; peroxide value, 5.22 meq o 2 /kg; K 232 and K 270 extinction coefficients, 1.89 and 0.15; respectively, according to the analyt- ical methods described in European regulation EEc 2568/91 (EEc, 1991) and chlorophyll con- tent, 20.0 mg/kg (PoKornY et al., 1998). the oil samples (25  ml each) were transferred into 50 mL glass bottles. the bottles were sealed with teflon-coated rubber seals and aluminum caps and stored at 150°, 160°, 170°, 180°, 190° and 200°c under dark condition in a forced air oven. chlorophyll content was measured with 2-h in- tervals from initial time until the time at which chlorophyll contents had reduced to 1 mg/kg All samples were prepared in duplicate. 2.2. Determination of chlorophyll content in olive oil the chlorophyll content of olive oil was ana- lyzed using the method described by PoKornY et al. (1998). the sample was measured at 630 nm, 670 nm and 710 nm in a 10 mm spectro- photometer cell against air, instead of a reference cell. the method is suitable for the determina- tion of quantities of chlorophyll pigments higher than 1 mg/kg. the following equation was used for determining the chlorophyll content Where: [c] = content of chlorophyll pigments in mg of pheophytin a in 1 kg of oil, A = absorbance at the respective wavelength (nm), L = thickness of the spectrophotometer cell (mm). 2.3. Kinetic theory Differential method was used for determina- tion of the degradation rate order and the rate constant of chlorophylls in olive oil. It was expressed the concentration at any tem- perature as a function of time in a power series, with constants a, b, c by deriving from the ex- perimental concentration-time data [c] = at2 + bt + c Where concentration ([c]) and time (t) were expressed in mg/kg and in hour. rate of reaction in mg/kgh (v) was estimated from the following equation; the most simple general rate equation was used for a single reactant at concentration [c]: Where n = rate order, k n = rate constant at or- der n. by taking the logarithm of the above equation to base e it follows that: ln v = nln [c] + ln k n rate order and rate constants at different tem- peratures were determined by plotting graph ln v versus ln [c] the half-life value (t 1/2 ) of chlorophyll degrada- tion was calculated using the equation given be- low after founding rate order and rate constants: Lnk 1/2 was plotted versus 1/t to determine Arrhenius parameters (A and E a ) by taking the logarithm of Arrhenius equation; k 1/2 = Aexp(- E a /rt) to base e; where E a is the activation en- ergy (J/kg), A is the pre-exponential factor or Arrhenius constant, r* is the specific gas con- stant for pheophytin a (9.543 J/kg K), and t is the absolute temperature (K). Differential method using for determination of the rate constant was compared with substi- tution and graphic methods. In substitution method the k value at a temper- ature was calculated by substituting initial con- centration, concentration at any time and time values into the following half order rate equation: k 1/2 = 2/t×([c 0 ]1/2 - [c]1/2) for n = ½ In graphic method the above equation was rearranged as [c]1/2 = [c 0 ]1/2 - (k 1/2 /2)×t and [c]1/2was plotted versus t to determine k 1/2 val- ue (the plot not shown). 3. rEsuLts AnD DIscussIon Virgin olive oil is a food matrix contains tri- glyceride having a high percentage of monoun- saturated fatty acids and also other minor con- stituents such as the phenols, chlorophyll and carotenoids fundamental in contributing to spe- cific characteristics of virgin olive oil. therefore the kinetic study and characterization of the Arrhenius parameters related with the thermal degradation reactions of chlorophyll in Voo were performed in an oil matrix system to establish mathematical models enabling the prediction of the degradation of this pigment during Voo thermal processing. changes with respect to the time in chloro- phyll concentration in oil matrix during thermal Ital. J. Food Sci., vol. 28 - 2016 93 processing, expressed in mg/kg, were shown in Fig. 1. the chlorophyll concentrations gradual- ly decreased while heating times increased. the experimental data was transferred to sigmaplot (version 12.0) program and trial and error meth- od was applied to find the best fit curve equa- tion on the data. the chlorophyll concentration at any temperature was expressed as a function of time. the best fit mathematical equations for the changes in the experimental data with the reaction time were selected to verify the rates of reaction at any temperature. the equations and their constants are shown in table 1. the ini- tial concentration of chlorophyll was arbitrarily set at 20.0 units. the reaction mechanism for chlorophyll degradation kinetics was assumed as a simple reaction type; Pheophytin a → colorless products where k n = rate constant for n order the rates of reaction were obtained by taking derivatives of the concentrations with respect to time. so lnv versus ln[c] was plotted to esti- mate the rate order and rate constants at differ- ent temperatures (Fig. 2). table 2 shows the best fit equations for lnk - ln[c] data. After estimating rate order as half order reaction it was calculated coefficients of the best equations for it (table 3). An assump- tion had been made for order of reaction of ther- mal chlorophyll degradation in a lot of previous studies on the processes of different food ma- trices such as fermentation of pickles coleslaw and olives (MInGuEZ-MosQuErA et al., 1992, MInGuEZ-MosQuErA et al., 1994; HEAton et al., 1996) or thermal processing of spinach (cAn- JurA et al., 1991; YonGXI et al., 2000) and also such as the visual green color a degradation (stEEt and tonG, 1996; WEEMEAs et al., 1999; AHMED et al., 2002; tHron et al., 2001; AHMED et al., 2004; APArIcIo-ruIZ et al., 2011, AHMED et al., 2013; MErcALI et al., 2014; DonG et al., 2014), and kinetics studies had been gone on assuming an order of 1. but VAn boEKEL (2009) reported that the best model for the decomposi- tion of chlorophyll is not only first-order equa- Fig 1 - changes with respect to the time in chlorophyll con- centration in oil matrix during thermal processing. table 1 - best fit equations for the concentration-time data. T (oC) [C]=at2-bt+c R² a b c 150 0.012 0.970 20.0 0.999 160 0.014 1.066 0.998 170 0.018 1.183 0.993 180 0.028 1.489 0.993 190 0.030 1.558 0.991 200 0.035 1.678 0.987 [C]: Chlorophyll concentration; t: time; a, b and c: function coefficients. Fig 2 - lnv - ln[c] plot to estimate the rate order and rate constants at different temperatures. table 2 - best fit equations for lnk - ln[c] data. T (oC) lnv=n×ln[C]+lnkn R² n lnkn 150 0.53 -1.59 0.998 160 0.51 -1.45 0.999 170 0.49 -1.32 0.999 180 0.47 -1.04 0.997 190 0.53 -1.11 0.998 200 0.50 -0.97 0.999 [C]: Chlorophyll concentration; v: reaction rate; k: reaction constant; n: reaction order. table 3 - best fit equations for half-order rate. T (oC) [C]1/2=[C0]1/2 - k1/2/2×t R² k1/2 [C0]1/2 150 0.21 4.47 0.998 160 0.24 170 0.26 180 0.33 190 0.34 200 0.38 94 Ital. J. Food Sci., vol. 28 - 2016 tion, but also could be half-order equation; for example, applying nonlinear regression to the data of scHWArtZ and Von ELbE (1983), the best order n is 0.5 ± 0.5 for chlorophyll a and 0.6 ± 0.4 for chlorophyll b (± 95% confidence interval). thus, the present data similar to the data of VAn boEKEL (2009). It was compared the differential method with the other two methods; substitu- tion and graphic method. the results obtained from the three different methods are shown in table 4. It was found that the rate constants and half-lifes at each temperature determined by three methods were close together. the re- action rates increased approximately 1.1 times with increment of every 10°c from temperature of 150°c. but, in general the reaction rate dou- bles for each 10°c increase in temperature (APA- rIcIo-ruIZ et al., 2010). However, cLArK (2009) reports that this approximation (about the rate of a reaction doubling for a 10 degree rise in tem- perature) only works for reactions with activa- tion energies of about 50 kJ/mol fairly close to room temperature, and the rate constant goes on increasing as the temperature rise up, but the rate of increase falls off quite rapidly at higher temperatures. the half-life of a reaction is de- fined as the time at which the concentration of component A is at half its initial value. It pro- vides a highly detailed description of how fast a reaction is occurring. In the present work, the half-life decreased 0.9 times for each 10 °c in- crease in temperature. the activation parameters were determined for the thermal process of chlorophyll in vir- gin olive oil in the range between 150°c and Fig 3 - lnk versus 1/t plot for the thermal process of chlo- rophyll in virgin olive oil. table 4 - comparison of methods used for determination of the rate constant and the half-life. T (oC) rate constant (k1/2) half-life (t1/2) D G S D G S 150 0.20 0.21 0.22 13.12 12.44 12.14 160 0.24 0.24 0.24 11.36 11.22 11.12 170 0.27 0.26 0.26 9.96 10.09 10.27 180 0.35 0.33 0.33 7.55 8.12 8.09 190 0.33 0.34 0.35 8.12 7.79 7.63 200 0.38 0.38 0.38 7.04 7.11 7.03 D: differential method. G: graphic method. S: substitution method. table 5 - Arrhenius constant, and activation energy for chlorophyll. Method k=Aexp(-Ea/R*T) Best fit equations for lnk1/2 vs 1/T data A Ea (J/kg) R*(J/kg.K) R² D 79.84 24.05 9.543 lnk1/2= -2.52x103(1/T) + 4.38 0.93 G 55.70 22.43 lnk1/2= -2.35x103(1/T) + 4.02 0.97 S 55.15 22.43 lnk1/2= -2.35x103(1/T) + 4.01 0.97 D: differential method. G: graphic method. S: substitution method. 200°c. the resulting logarithmic plot is shown in Fig. 3. the estimated values used in the Ar- rhenius Equation for chlorophyll degradation re- action during heating by using three methods is shown in table 5. the Ea determined by graphic method (22.43 J/kg) was the same value found in substitution method whilst the value in dif- ferential method was 24.05 J/kg. Average acti- vation energies for chlorophyll with respect to first order reaction were reported to be in range of 14.8 and 15.3 kcal/mol in the different tem- peratures and pH range (rYAn-stonEHAM and tonG, 2000; KocA et al., 2006). If a compound has low activation energies it is highly sensitive to temperature (JAIsWAL et al., 2012) 4. concLusIons thermal processing played an important role for degradation of chlorophyll in virgin olive oil during heating in high temperature range. the kinetics of degradations of chlorophyll in oily food matrices was studied by using differential meth- od. the rate order of chlorophyll degradation re- action was determined as half order reaction that are not yet reported in literature from the experi- mental results. the degradation reaction of chlo- rophyll in many of previous studies have been fit- ted first-order kinetic model by assumption. use of half-order reaction model for chlorophyll degra- dation should be encouraged by further studies. Ital. J. Food Sci., vol. 28 - 2016 95 rEFErEncEs Ahmed J, Al-salman F. and Almusallam A.s. 2013. Effect of blanching on thermal color degradation kinetics and rheo- logical behavior of rocket (Eruca sativa) puree. Journal of Food Engineering 119: 660-667. Ahmed J., Kaur A. and shivhare u.s. 2002. color degradation kinetics of spinach, mustard leaves and mixed puree. Jour- nal of Food science 67: 1088-1091. Ahmed J., shivhare u.s. and singh P. 2004. color kinetics and rheology of coriander leaf puree and storage characteristics of the paste. Food chemistry 84: 605-611. Aparicio-ruiz r., Minguez-Mosquera M.I. and Gandul-rojas b. 2010. thermal degradation kinetics of chlorophyll pigments in virgin olive oils. J. Agric. Food chem 58: 6200-6208. Aparicio-ruiz r., Minguez-Mosquera M.I. and Gandul-rojas b. 2011. thermal degradation kinetics of lutein, b-carotene and b-cryptoxanthin in virgin olive oils. J Food composition and Analysis 24: 811-820. berasategi I., barriuso b., Ansorena D. and Astiasaran I. 2012. stability of avocado oil during heating: comparative study to olive oil. Food chemistry 132: 439-446. boskou D. 2009. culinary applications of olive oil. Minor con- stituents and cooking. In D. boskou (Ed.), olive oil: Minor constituents and health. usA: crc Press, pp.1-4. canjura F.L., schwartz s.J. and nunes r.V. 1991. Degrada- tion kinetics of chlorophylls and chlorophyllides. J. Food sci., 56: 1639-1643. carla s.P., santos r.c., sara c. and cunha s.c. 2013. Effect of cooking on olive oil quality attributes Food research In- ternational 54: 2016-2024. cerretani L., Motilva M. J., romero M. P., bendini A. and Ler- cker G. (2008). Pigment profile and chromatic parameters of monovarietal virgin olive oils from different Italian culti- vars. European Journal of Food research and technology 226: 1251-1258. clark J. 2009. Edexcel IGcsE chemistry (student book). In: chapter 6. (Edexcel International GcsE) Publisher: usA: Edexcel pp. 41 criado M.n., romero M.P., casanovas M. and Motilva M.J. 2008. Pigment profile and colour of monovarietal virgin olive oils from Arbequina cultivar obtained during two consecutive crop seasons. Food chemistry 110: 873-880. De Leonardis A. and Macciola V. 2012. Heat-oxidation stability of palm oil blended with extra virgin olive oil. Food chem- istry 135: 1769-1776. Dong Z., sang-Min K., cheol-Ho P. and Donghwa c. 2014. Ef- fects of heating, aerial exposure and illumination on stability of fucoxanthin in canola oil. J. Food chemistry 145: 505-513. Dweck J. and sampaio c.M.s. 2004. Analysis of the thermal de-composition of commercial vegetable oils in air by simul- taneous tG/DtA. Journal of thermal Analysis and calo- rimetry 75: 627-630. EEc. 1991. European Economic community; commission reg- ulation (EEc) no. 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analy- sis. official Journal L 248, pp. 1-83. Ferruzzi M.G. and blakeslee J. 2007. Digestion, absorption, and cancer preventative activity of dietary chlorophyll de- rivatives. nutrition research 27: 1- 12. Gandul-rojas b. and Minguez-Mosquera, M.I. 1996. chlorophyll and carotenoid composition in virgin olive oils from various spanish olive varieties. Journal of the science of Food and Agriculture 72: 31-39. Giuffrida D., salvo F., salvo A., cossignani L. and Dugo G. 2011. Pigments profile in monovarietal virgin olive oils from vari- ous Italian olive varieties. Food chemistry 124: 1119-1123. Giuffrida D., salvo F., salvo A., La Pera L. and Dugo G. 2007. Pigments composition in monovarietal virgin olive oils from various sicilian olive varieties Food chemistry 101: 833-837. Goula A.M. 2013. ultrasound-assisted extraction of pome- granate seed oil - Kinetic modeling. J. Food Engineering 117: 492-498. Grauwet t., Vervoort L., colle I., Van Loey A. and Hendrickx M. 2014. From fingerprinting to kinetics in evaluating food qual- ity changes. trend in biotechnology 32:125-131. Heaton J.W., Lencki r.W. and Marangoni A.G. 1996. Kinetic model for chlorophyll degradation in green tissue. J. Agric. Food chem. 44: 399-402. Jaiswal A.K. Gupta s. and Abu-Ghannam n. 2012. Kinetic eval- uation of colour, texture, polyphenols and antioxidant ca- pacity of Irish York cabbage after blanching treatment Food chemistry 131: 63-72. Koca n., Karadeniz F. and burdurlu H.s. 2006. Effect of pH on chlorophyll degradation and colour loss in blanched green peas. Food chemistry 100: 609-615. Mancebo-campos V., salvador M.D. and Fregapane G. 2014. Antioxidant capacity of individual and combined virgin ol- ive oil minor compounds evaluated at mild temperature (25 and 40 °c) as compared to accelerated and antiradical as- says. Food chemistry 150: 374-381. Mercali G.D., schwartz s., Damasceno L., Marczak F., tessaro I.c. and sastry s. 2014. Ascorbic acid degradation and color changes in acerola pulp during ohmic heating: Effect of elec- tric field frequency. J. Food Engineering 123:1-7. Minguez-Mosquera M.I., Gandul-rojas b. and Gallardo-Guer- rero M.L. 1994. Mechanism and kinetics of the degradation of chlorophylls during the processing of green table olives. J. Agric. Food chem 42: 1089-1095. Minguez-Mosquera M.I., Mosquera b., rojas G. and Gallar- do-Guerrero M.L. 1992. rapid method of quantification of chlorophylls and carotenoids in virgin olive oil by high-per- formance liquid chromatography. J Agricultural and Food chemistry 40: 60-63. Minguez-Mosquera M.I., Gandul-rojas b., Garrido-Fernandez J. and Gallardo-Guerrero M.L. 1990. Pigments present in virgin olive oil. J. Am. oil chem. soc. 67: 192-196. niamnuy c., nachaisin M., Poomsa n. and Devahastin s. 2012 Kinetic modelling of drying and conversion/degradation of isoflavones during infrared drying of soybean. Food chem- istry 133: 946-952. Pokorny J., Kalinova L. and Dysseler P. 1998. Determination of chlorophyll pigments in crude vegetable oils. Pure Appl. chem., 67(10): 1781- 1787. remini H., Mertz c., belbahi A., Achir n., Dornier M. and Mada- ni K. 2015. Degradation kinetic modelling of ascorbic acid and colour intensity in pasteurised blood orange juice dur- ing storage. Food chemistry 173: 665-673. ryan-stoneham t. and tong c.H. 2000. Degradation kinetics of chlorophyll in peas as a function of pH. Journal of Food science 65: 1296-1302. schwartz s.J. and Von Elbe J.H. 1983. Kinetics of chlorophyll degradation to pyropheophytin in vegetables. J Food sci 48:1303-1306. steet J.A. and tong c.H. 1996. Degradation kinetics of green color and chlorophyll in peas by colorimetry and HPLc. Jour- nal Food science 61: 924-931. thron M., Eichner K. and Ziegleder G. 2001. the influence of light of different wavelengths on chlorophyll-containing foods. Lebensm.-Wiss.technol 34: 542-548. Van boekel M.A. 1996. statistical aspects of kinetic modeling for food science problems Journal of Food science 61: 477-485. Van boekel M.A. 1999. testing of kinetic models: usefulness of the multiresponse approach as applied to chlorophyll degra- dation in foods. Food research International 32: 261-269. Van boekel M.A. 2009. Kinetic modeling of reactions in foods. crc Press taylor & Francis Group 6000 broken sound Parkway usA nW. Vecchio s., campanella L., nuccilli A. and tomassetti M. 2008. Kinetic study of thermal breakdown of triglycerides con- tained in extra-virgin olive oil. J. thermal Analysis and cal- orimetry 91: 51-56. Waterman E, Lockwood b. 2007. Active components and clin- ical applications of olive oil. Alternative Medicine review 12: 331-342. Weemeas c.A., ooms V., Loey A.M. and Hendrickx M.E. 1999. Kinetics of chlorophyll degradation and color loss in heat- ed broccoli juice. Journal of Agricultural Food chemistry 47: 2404-2409. Yongxi t., Jian-Hui J., Hai-Long W., Hui c. and ru-Qin Y. 2000. resolution of kinetic system of simultaneous degradations of chlorophyll a and b by PArAFAc. Analytica chimica Acta 412: 195-202. Paper Received November 5, 2014 Accepted April 18, 2015