.~.—-.. in . . I .1; . ~ - —. .v . . .. . p l . . _ . .4 . . . I. f r! . . _ r I . . .. Y z . e . .o . . . . . e > . - v _. .. .r VA .. . .. .. .. . , ._ . o 1.}: . ., .. . . . . .. . . .3 _ . I .. Jovv .. I . v _ . . . l I . . . ..¢ . . w I . . . . u u . . o . . . . .. . . _ I..l. . _ . . v o'- If .. . Q \3 . .ra. . ..... . . a. . t . E. v a D I! . a. -. .Q K . o. a .c v .~ 4.. > . I. . ..V(..Sa o . .lfiov.l‘1¢. u. . . oo .1 .n .I. p .. .. . . . .. . u .4 .uo...l'av A . - fit. . . e . a r 4. .3. . a A . .) a ‘ . I r 1....v . ~10 I - .1..- I o . . o .7 _. .. .r . .. v r 9’. \ . . . . ... . l...r.. . . .. 1’,- .4 . . u.¢lf.\. fl. I o .u _ . V . . . . _ . n. . . .. .f 1 p . .(L . . . u . . . ., I...I 9 . u . I , \ ‘... l . . . . . . . . . .— Io-Il . .. e. r .P ‘ . . .l . . . I “, I I ‘ . ,4 n c n I I O. A STABILITY OF VIRGIN OLIVE OIL Thesis for the Degree of M. “S. MICHIGAN STATE UNIVERSITY APOSTOLOS K. KIRITSAKIS 1977 II III III II II III" IIIIQIIIIII I f .71“. ’J .’ J ‘7 ’-.- '~ Q __. ’ I A- n A “I _. - ‘W ' ,~9~ ‘L I V J v" ABSTRACT EFFECT OF SELECTED ANTIOXIDANTS ON THE STABILITY OF VIRGIN OLIVE OIL By Apostolos K. Kiritsakis This is a study to assess the effect of the antioxidants butylated hydroxyanisole (BHA), butylated hydroxytolyene (BHT), ter- tiary butylhydroquinone (TBHQ) and propyl gallate (PG), on the stability of virgin olive oil obtained from Greece. Three different samples were used. The oil was treated with the above antioxidants which were used either alone or in different combinations, at different levels of concentration. Samples were then stored at room temperature in the dark for 22 weeks and at 50°C for 24 weeks, and were analyzed at regular intervals. In addition, samples containing antioxidants were stored at room temperature in light for a six month period. The peroxide value, diene conjugation and thiobarbituric acid (TBA) tests were conducted throughout the storage period to measure the oxidation of olive oil. The Schaal Oven Test was also employed. In this test the peroxide value was used as a measure of oxidation. The fatty acid composition of the olive oil was determined by gas liquid chromatography. Apostolos K. Kiritsakis Under accelerated conditions, it was found that antioxidants increased the stability of the oil and their effectiveness varied between the three different samples of olive oil used. In the oven test, where the sample of oil used was the same as that used for storage conditions (room temperature and 50°C), the relative inhibi- tion effect of the antioxidants used was in the following order TBHQ = BHA > BHT. The antioxidant combination which was found to be the most effective in the oven stability studies was 0.0l% BHA + 0.0l% TBHQ. Olive oil stored at room temperature in the dark did not undergo any oxidative deterioration during the 22 weeks storage period. Therefore the antioxidants used in this case had no effect. On the other hand, olive oil stored without antioxidant at room temperature in light underwent a high degree of oxidation. Results obtained from peroxide value, diene conjugation and TBA tests corre- lated well in this experiment. The presence of antioxidants in the samples stored at room temperature in light had no effect with respect to retarding the peroxide formation. The addition of antioxidants to olive oil stored at 50°C exhibited a beneficial effect. Citric acid used alone, however, showed a prooxidant effect. Potency of the antioxidants under these conditions was in the following order: TBHQ > BHT > BHA. Combina- tions of TBHQ with BHA and BHT provided good results, but they never exceeded the results obtained by using TBHQ alone. EFFECT OF SELECTED ANTIOXIDANTS ON THE STABILITY OF VIRGIN OLIVE OIL By ; . Apostolos KT kiritsakis A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science T977 To my parents, my wife and my son ii ACKNOWLEDGMENTS The author expresses sincere gratitude to Dr. C. M. Stine and Dr. L. R. Dugan, Jr. for their inspiration, counsel and patience during this study and for their assistance in the preparation of this thesis. Sincere appreciation is also extended to Dr. P. Markakis and Dr. D. Dilley for their time and effort in serving on my committee and critically reading this manuscript. Acknowledgment is also extended to my parents, my parents-in- law, and my brother Mimis, for their financial and moral support during the course of my undergraduate and graduate education. Appreciation is also expressed to my brother-in-law Steve for refining the English in my thesis. The author further thanks Karim Nafisi for the useful discussions. Finally, for the inspiration that has made the agony of this graduate study more than worthwhile, I thank my wife Ritsa. iii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. INTRODUCTION. REVIEW OF LITERATURE . Mechanism of Autoxidation. . . Flavor Compounds of Oxidized Olive Oil . Role of Chlorophyll on the Oxidation of Olive Oil. Bleaching of Chlorophyll . . . Effect of Free Fatty Acids (FFA) on Oxidation . Metal Catalysts . . Measurement of Oxidative Rancidity. Peroxide Vaue (PV) Diene Conjugation. . Thiobarbituric Acid (TBA) Test Role of Antioxidants in the Oxidation of Fats and Oils . Phenols and Toc0pherols as Natural Antioxidants in Olive Oil Phenols . Tocopherols. The Use of Synthetic Antioxidants in Olive Oil. MATERIAL AND METHODS . Raw Material. Virgin Olive Oil. . Dispersion of Antioxidants into the Samples. Oven Test. . Preparation of the Samples for the Storage Stability Studies . . . . . Analytical Techniques . Free Fatty Acid (FFA) Determination. Preparation of Methyl Esters for Gas Liquid Chromato-. graphy (GLC). iv Page vi viii —l HOKO LOCDNO‘U'l-Dw w ._a_.a._n N _a .b ._a_a h-b ”-1—! .—J -—I ooooo 00 01 NN NO NM NM Page Fatty Acid Composition of Olive Oil. . . . . . . . . 22 Measurement of Rancidity . . . . . . . . . . . . 23 Peroxide Value (PV) . . . . . . . . . . . . 23 Ultraviolet Absorption (UV). . . . . . . . . . . 24 Purification of 150- octane . . . . . . . . . . . 24 TBA Test . . . . . . . . . . . . . . . . . 24 Removal of Interfering Pigments. . . . . . . . . . . 25 RESULTS AND DISCUSSION . . . . . . . . . . . . . . 27 Gas Liquid Chromatography (GLC) Analysis. . . . . . . . 27 Oven Stability Studies. . . . . . 29 The Effect of Sample Size on the Rate. of Oxidation . . . . 36 The Effect of Antioxidants on the Storage Stability of Olive Oil . . . . . . . . . 37 Oxidation of Olive Oil at Room Temperature in the Dark . . 38 Removal of Interference Pigments by a Chromatographic Column. . . . . . . . . . . . 42 Oxidation of Olive Oil at 50° C . . . . . . . . . . 48 Photooxidation of Olive Oil . . . . . . . . . . . . 62 Discoloration of the Oil . . . . . . . . . . . . . 69 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 74 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . 77 Table TO. ll. 12. LIST OF TABLES Antioxidants and Synergist Used in Virgin Olive Oil Studies. . . Fatty Acid Composition of Greek Olive Oil . Stability Studies with Olive Oil No. l Treated with Antioxidants (Oven Test at 65°C) . . . Stability Studies with Olive Oil No. 2 Treated with Antioxidants (Oven Test at 65°C) Stability Studies with Olive Oil No. 3 Treated with Antioxidants (Oven Test at 65°C) . The Effect of Sample Size on the Stability of Olive Oil No. l Treated with Antioxidants (Oven Test at 65°C . . . . . . . . . . . . Peroxide Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at Room Temperature (20-28°C) . Peroxide Values Obtained from Olive Oil No. 2 Treated with a Combination of Antioxidants and Stored at Room Temperature (20-28°C) TBA Absorption Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at Room Temperature (20-28°C) . TBA Absorption Values Obtained from Olive 011 No. 2 Treated with a Combination of Antioxidants and Stored at Room Temperature (20-28°C) . Ultraviolet Absorption Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at Room Temperature (20-28°C) . . . Peroxide Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at 50°C . vi Page 19 28 3O 32 34 37 39 4O 44 45 47 49 Table 13. T4. 15. 16. 17. 18. Peroxide Values Obtained from Olive 011 No. 2 Treated with a Combination of Antioxidants and Stored at 50°C. TBA Absorption Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at 50°C TBA Absorption Values Obtained from Olive Oil No. 2 Treated with a Combination of Antioxidants and Stored at 50°C Ultraviolet Absorption Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at 50°C The Effect of Light on the Oxidation of 01ive Oi1 No. 2 Stored at Room Temperature (20-28°C) . The Effect of Light on Peroxide Formation in Olive Oil No. l Treated with Antioxidants and Stored at Room Temperature (20-28°C) . . . vii Page 50 55 56 60 65 67 Figure 10. 11. 12. 13. LIST OF FIGURES The Effect of Certain Antioxidants on Peroxide Formation in Olive Oil No. l (Oven Test at 65°C). The Effect of Certain Antioxidants on Peroxide Formation in Olive Oil No. 3 (Oven Test at 65°C). Peroxide Formation in Olive Oil No. 2 Containing Antioxidants. The Absorption Spectrum of Yellow Pigments Present in Olive Oil No. 2. Peroxide Formation in Olive Oil No. 2 Containing Antioxidants and Stored at 50°C Peroxide Formation in Olive Oil No. 2 Containing Antioxidants and Stored at 50°C TBA Absorption Values of Olive Oil No.2 Containing Antioxidants and Stored at 50° C . Ultraviolet Absorption in Olive Oil No. 2 Containing Antioxidants and Stored at 50°C Changes in Olive Oil No. 2 during Storage at 50°C . The Effect of Light on Peroxide Formation in Olive Oil No. 2(Room Temperature Storage). Changes in Olive Oil No. 2 during Storage at Room Temperature in the Presence of Light. The Effect of Light on Peroxide Formation in Olive Oil No.1 Containing Antioxidants (Room Temperature Storage). . . . . . . . . . . . Changes in the Color of Olive Oil Containing Antioxidants and Stored at Room Temperature in the Presence of Light. viii Page 31 35 41 43 52 53 58 61 63 66 68 7O 72 Figure page l4. Changes in the Color of Olive Oil during Storage at Room Temperature in the Presence of Light. . . . . 72 l5. Changes in the Color of Olive Oil Containing Anti- oxidants and Stored at 50°C . . . . . . . . . 73 ix c‘) _.. III (3 INTRODUCTION Olive oil is the oil extracted from the fruits of the tree Olea eurapaea and it is one of the very few, if not the only, plant 011 which can be consumed in its natural state without being chemi- cally treated. The olive tree probably originated in Mesopotamia and it has been cultivated for many centuries in the countries bordering the Mediterranean. For more than 3,000 years, the olive tree has been planted in Greece. While most of the world supply of olive oil is produced in the Mediterranean countries, some olive oil is also pro- duced in the United States (California). Before World War 1, Greece was the premier olive producing country in the entire Mediterranean region. Now Greece ranks third among the world's olive producing countries with Spain and Italy being number one and two, respectively. Many different cultivated varieties have been developed over the centuries and they differ from each other in various ways such as the size of the fruit, its color, and the percentage oil content. These characteristics determine what the fruits will be used for-- pickling or oil production. Olive oil is mainly used in its natural form in salads and in the preparation of foods and is considered to be the finest edible oil. Part of it is also used in the production of margarine in the olive producing countries. Officially reported production figures are usually concerned with edible oil. There is, however, a considerable amount of inedible oil , extracted by solvent methods from the residue remaining after pressing the olive paste. The oil which comes from the first pressing is called "virgin" olive oil and it is considered to be of highest qual ity. Virgin olive oil is the type of oil used in this study. Like the rest of the vegetable oils, 01ive oil undergoes oxida- ti ve deterioration as a result of several factors. The autoxidation of olive oil results in the modification of its organolepticicharac- The ter‘i stics and some of its physical prOperties such as viscosity. Prevention of autoxidation in olive oil is recognized as a problem of great importance from the standpoint of both health and economy. The devel0pment of synthetic antioxidants has played a vital ”01 e in the marketing of other vegetable oils by retarding oxidation. The present study was designed to gain knowledge on the effect 0‘: antioxidants in olive oil obtained from Greece. This work was based on storage stability tests at room temperature (both in the dark and 'i’ n light) and at 50°C. The Schaal oven test was also employed. pe"‘Oxide values, diene conjugation studies and thiobarbituric acid (TBA) determinations were conducted as chemical tests to evaluate the e1:‘Fect of antioxidants on the stability of the olive oil. The antioxidants used in this work were butylated hydroxy- an‘i Sole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydro- qui none (TBHQ), and WOW] gallate (PG)' REVIEW OF LITERATURE Mechanism of Autoxidation Autoxidation occurs when oxygen reacts with the double bonds in the unsaturated fatty acids. It is an autocatalytic mechanism in whi ch the rate of reaction increases with time due to the formation of Products acting as catalysts. The mechanism of autoxidation involves the following three steps (Dugan, l96l). 1. Initiation RH + 02 s R- + -00H (1) 2. Pr0pagation R. + 02 I ROO~ (II) 3. Termination R. + R- . RR (IV) R. + ROO- e ROOR (V) R00- + Roo- » ROOR + 02 (v1) RH refers to any unsaturated fatty acid in which the H is lab‘i 1e by reason of being on a carbon atom adjacent to a double bond. R’ Y‘efers to a free radical formed by removal of a labile hydrogen. The oxidation process becomes more complex after the development of a q“'ai'ltity of ROOH since the ROOH then decomposes either because of the"‘mal instability or through reaction with other materials to form More free radicals which in turn participate in the chain reactions (Dugan, 1951). The autoxidation of fats is affected by a number of factors. These factors are: (l) saturation of the fat, (2) heat, (3) light, (4) ionizing radiation, (5) enzymes, (6) catalysts, (7) presence of oxygen, and (8) use of antioxidants (Lea, 1962). In the autoxidation of fats, unsaturated fatty acids are oxidized to hydroperoxides which consequently undergo decomposition yie'l ding a number of volatile compounds including unsaturated esters, ketones, alcohols and hydrocarbons. These volatile compounds contri- bute to undesirable flavor in the autoxidized fats (Evans, 1961). The contribution of saturated and unsaturated aldehydes to off-flavor characteristics of the rancid fats has been reported by Hoffman (1962), Hammond and Hill (I964), and Horvat et al. (1965). Besides the oxidative rancidity formed during the autoxida- ti on of fats, there is another type of oxidative deterioration which is detectable in the very early stages of oxidation and is called 1:] avor reversion. This reversion is termed "grassy," "beany," "f'i Shy," etc. depending on the resemblance of the oil flavor. The ten“ reversion is a misnomer because the objectionable flavor and 0510" is not characteristically normal to the fresh fat (Spannuth, TSBAnggj). Although reversion occurs principally in vegetable oils con- tai '1 ‘ing polyunsaturated fatty acids (Lundberg, 1962), Gutierrez (1 96 3) observed the phenomenon of reversion in olive oil. flavor Compounds of Oxidized Olive Oil Studying the rancidity of olive oil Corrao (1966) demon— s t1'7ii1teed that the organoleptic threshold of this oil is independent of the peroxidation level. Swern (1964) suggested that oleic acid is probably responsible for the odor and the flavor of the true oxidative rancidity, while Badings (1970) was able to isolate seven aldehydes reSponsible for the off-flavor in autoxidized oleic acid at 20°C. Gutierrez and Romero (1960) observed that during the oxida- tion of olive oil different compounds are produced in different ways and 'in different proportion, and it is difficult to determine which groups are responsible for the off-flavor of this oil. Foresti (1964), however, found that the off-flavor in olive oil is caused by saturated aldehydes and dienals as well as a,B-unsaturated aldehydes. Role of ChlorOphyll on the Oxidation of Olive Oil Taufel et al. (1959) observed that chlorophyll in the pre- sen ce of light acts as a prooxidant for methyl oleate. This pigment, how ever, has no prooxidant effect in the dark; on the contrary it acts synergistically with phenolic antioxidants. In order to have OX‘idation in the presenCe of chlorOphyll in the dark, Tollin and Green (1960) showed that very strong prooxidants are necessary. Intewesse et al. (1971) found that, under the action of light, the fou7~ pigments chlorOphylls a and b and pheophytins a and b develop a" Oxidizing activity while in the dark they act as antioxidants. Due to the chlorophyll content, which according to Vitagliano (1 960) varies from O-9.7 ppm, virgin olive oil is easily oxidized and is Very sensitive to light. Studying the effect of light on the 0x1 dative stability of olive oil, Pretzch/(l970) observed that olive of 1 QXposed to light and air undergoes higher oxidation than that I s t0"‘ed in the dark. Valentinis and Romani (1960), however, PV‘OVEd that, in the absence of air, direct sunlight causes a decrease of peroxide and Kreis value during the storage of olive oil. It was also 1 observed by Gutierrez and Jimenez (1970) that virgin olive oil stored in polyethylene and exposed to light undergoes higher oxidation than that stored in dark. Radiations of wave length greater than 630 nm present a maxi- mum of absorption in virgin olive oils corresponding to the absorption of chlorophyll and are very active, while the zone between 530—630 nm ./ has; minimum activity (Borbolla et al., 1963). Francesco (1961) was more specific in finding that virgin olive oils have a chlorophyll absorption peak at 665 nm. A very important observation made by Vazquez et al. (1960)‘/is that, because of their chlorophyll content, 01 ‘i ve oils are very sensitive to radiation of wavelength between 320 and 720 nm whether in the presence or the absence of antioxidants. Bleaching of Chlorophyll The different types of chlorophyll have the ability to sensi- tize , in the existence of an excited state, the oxidation of organic S“1381:.ances by molecularoxygen (Seely, 1966). Rawls and Van Santen (1970) demonstrated that, in chlorophyll catalyzed photooxidation, a mechanism which would supply singlet O2 is necessary for the forma- ti on of the original hydroperoxides. These hydroperoxides, formed by 5“ ng‘let 02, are produced at a much faster rate (1450 times) than by “hi Plet 02. During photooxidation, bleaching of chlorophyll occurs. Sasthy et al. (1973) proposed the following mechanism for the b] eaching of chlorophyll as the effect of visible light. cm “V __.. am 5 3cm 3 3 1 13111 + 02 , 02 + cm 102 + RH 45 ROOH ROOH “V + ROO- + H- ROO- + cm _, ROOH + cm- ] O . c111. + 02 L, cm 02 cm. - 02 + H- , cm - o2 (bleached) Chlorophyll when exposed to visible light gets excited and is (:onverted to the triplet state of chlorophyll. This triplet state of“ «:hlorophyll reacts with triplet oxygen resulting in singlet oxygen, wh‘i ch in turn reacts with substrate to form hydroperoxides (Rawls and Van Santen, 1968). Hydroperoxides give rise to peroxy radicals on exposure to IT SJfrtg These peroxy radicals abstract hydrogen atoms from chlorOphyll, trilJSS disturbing its conjugated electron system. Abstraction of a P"<>1t<3n from chlorophyll causes a free radical. The resulting radical r'eélc‘ls with oxygen to give a peroxy free radical of chlorophyll. This corTllb'ined with a proton stabilizes itself to a stable peroxide. Effect of Free Fatty_Acids (FFA) on Oxidation Among the factors that might promote oxidation and influence the effectiveness of antioxidants used is the presence of free fatty E“lids. Olcott (1958) observed that the addition of oleic acid to refi ned olive oil and other vegetable oils decreased the stability of 1:"‘3 (Jils at 60°C. A similar observation was made by Chahine and El ‘Shobaki (1966) in unpurified shark liver oil at 100°C. 1) due / 1C5, Dunc Vega et al. (1960) and Catalano and Felice (1970) reported that the presence of FFA in concentrations as low as 0.5% have a strong prooxidant effect on olive oil and cause the stability of the oil to diminish. The later investigations pointed out that this prooxidant effect can be realized by means of a catalytic mechanism. They further demonstrated (1970) that the autoxidation of olive oil is related to the FFA concentration and that the chain length or the ' presence of a double bond plays a secondary role. Olcott (1958) noted that the antioxidant action can be modi- fied by the presence of free fatty acids in triglyceride systems and Cat alano and Felice (1970) found that the antioxidant effect of NDGA, BHA and ascorbyl palmitate was reduced in the presence of FFA. Metal Catalysts The presence of small quantities of prooxidant metals such as ‘i ron, copper and manganese which occur in oils naturally or as a I”esu‘l t of processing plays an important role in the stability of the Oi 1 S - These metals catalyze the rate of formation and destruction of Per‘oxides thus leading to the more rapid formation of substances with u"desirable odors and flavors (Swern, 1964). Some of the metals are such extremely powerful catalysts of ”<1. dation that their effect is high even in concentrations as low as °ne part in 100 million (Ingold, 1968). Fedeli et a1. (1973) found that the oxygen absorption rate in autOxidized olive oil was related to the amount of catalytic metals (Ca: Na, Co, Ni, Fe, Cu and Mg) present in the oil. It was also IF OUNd (Vioque et al., 1959 and Vioque, 1968) that upon removal of most IEIIC SIT) 311311 test 52" / Hair SUI) RFC of the catalytic metals present in olive oil by passing it through cat-i on exchange resins the stability of the oil was increased. He further found (1968) that there is a correlation between olive oil stability and iron concentration as is shown below: K = (Fe)aoS where K and a are constants (Fe) = ppm and S = stability (A.O.M.) Measurement of Oxidative Rancidity Many methods have been developed for measuring the oxidative rancidity or the stability of fats and oils. From these the most comonly used methods are: the active oxygen method (A.O.M.), Schaal Oven test, peroxide value, thiobarbituric (TBA) test, and carbonyl tes t (Dugan, 1955). PSY‘Oxide Value (Pfl For peroxide value determination a number of analytical pro- Cedures have been developed, such as Wheeler (1932), Lea (1939) and 511i he et a1. (1954). The peroxide value method involves the quanti- tat‘i ve measurement of the primary products of oxidation (peroxides), exp Pessed as milliequivalents of reactice oxygen per kilogram of fat. For determining the peroxide value of virgin olive oil Maur‘ikos et al. (1972) used a new polarographic method, where the SUPporting electrolyte was LiCl in methanol-benzene with a dropping m e ”(luv-y electrode. Burr tion 0i p) IIydES 1 . uIDer ACCDF In YT Virgil (1131') 10 The peroxide value is the most important method in evaluating the present condition of olive oil. Montedoto and Petruccioli (1972), however, observed that the peroxide values that are computed during the test are usually affected by the presence of quinones which are formed by the phenolic substances during storage. me ne Conj ugati on During the oxidation of polyunsaturated fatty acids, an inc rease in ultraviolet absorption occurs due to the formation of con- jug ated diene and triene hydr0peroxides. The change of ultraviolet absorption, however, is not easily related to the degree of oxidation, 51 r1<:e the effects upon the various unsaturated acids (oleic, linoleic, li rtcjlenic, etc.) are different in quality and magnitude (Holman and Burr, 1946). . Bartolomeo and Sergio (1969) observed that the primary oxida- ti c>r1 products of olive oil are hydroperoxides with an absorption peak at 232 nm and that the greater the absorbance the greater the degree 01: primary oxidation. For the secondary oxidation products--a1de- “Sides, ketones, etc.--they found an absorption peak at 270 nm. Jime Inez and Gutierrez (1970) and Bartolomeo and Sadini (1959) found f that the rancidity of olive oil causes the ratio A232/A270 to decrease according to the degree of alteration. This ratio remains constant in Virgin olive oils stored in the dark and decreases rapidly in Vi r‘Sl‘in oils stored in sunlight due to the rapid increase of oxidation (J 1‘ mi nez and Gutierrez, 1970). A quasilinear relation was found (Montefredine and Luciano, . 1 968) between the absorbance at 232 nm and the peroxide value and these a. .$ )I‘ebtl Iira‘il J '.’f-,T 11 between the absorbance at 270 nm and the acidity in virgin olive oil. 01 ive oil, like all oils free of conjugated double bonds, shows a slow i ncr'ease in absorptivity at 232 and 268 nm during the induction period whi ch is followed by a sharp, sudden increase. The absorbance at these wavelengths can be used to predict the thermal stability of the oi 1 (Ninnis and Ninni, 1968).“ Ninnis and Ninni also demonstrated (1966) that ultraviolet spectrophotometric analysis can be used to detect the adulteration of vi rgin olive oil. Th i obarbituric Acid (TBA) Test The 2-thiobarbituric acid (TBA) test has been used widely for measuring oxidative changes in foods containing unsaturated fatty acids. Kohn and Liversedge (1944) observed that animal tissues which had been incubated aerobically gave a red color when mixed with Z-thiobarbituric acid. Bernheim et al. (1947) f0und that the red (:01 or was formed from the oxidation products of unsaturated fatty acids and 2-thi0barbituric acid. Many researchers have reported that the red color is a con- de'I‘lsation product of one molecule of malonaldehyde with two molecules or 2-thiobarbituric acid. Sinnhuber et al. (1958) and Dahle et al. ‘ ( 1 969), found that the original material formed is predominantly a no'Tvolatile substance and therefore is not malonaldehyde. This non— VO] atile substance undergoes decomposition under the conditions of the TBA test and produces malonaldehyde which then reacts with TBA. A mechanism was proposed by Pryor et al. (1976) in which the "1&1 onaldehyde arises at least in part from the acid catalyzed, or 12 thermal decomposition of endoperoxides (2,3-dioxanorbornane compounds). They applied Dahle's et a1. (1969) theory to explain the formation of the thiobarbituric acid reactive material in a diene system and demon- strated that endoperoxides can be produced in a diene system but in a ‘lcyvvter'ratio than in a triene system. Gutierrez and Romero (1960), Franjo (1963), and Casillo (1968) used the thiobarbituric (TBA) test to measure oxidative rancidity in '<)'I‘i \ve oil. It was found by Casillo (1968) that TBA test detects the rancidity of olive oil at a lower level than other tests (peroxide va1 ue, Kreis test, acid number). Role of Antioxidants in the Oxidation of Fats and Oils The antioxidant role of various substances was known for a great number of years, but the technology of the antioxidants in re”! ation with foods, fats and oils did not start until the late 19405. Antioxidants are substances which in small quantities are able to Prevent or to retard the oxidation of easily oxidizable materials SUCh as fats (Chipault, 1962). Higgins and Black (1944) summarized the requirements of an T de an antioxidant as follows: 1. It should exert no harmful physiological effect. 2. It should contribute no objectionable flavor, odor or color to the fat. 3. It should carry through and effectively protect from rancidity the foods made with the fat. 4. It should be sufficiently fat soluble. 5. It should be effective in low concentrations. 1111 1:1 ('l‘h . “I Ca") 13 6. It should be readily available. 7. It should be reasonable in cost. Antioxidants may interfere with the process of autoxidation in two general ways, either as inhibitors of free radicals or as pe mxi de decomposers . Uri (1961) proposed the following mechanism for the antioxi- d an 1: function. ROO- + AH (antioxidant) b ROOH + A- The radical A- may be stabilized by recombination in two ways: A: + A- ’ A2 ROO- + A. w ROOA As peroxide decomposes, antioxidants act as catalysts in the decomposition of the peroxides which are initially present or formed during the oxidation. The decomposition function of antioxidants aDwears when they are used in high concentration exceeding 0.02% (Hi 1 ‘I et al., 1969). Dugan (1961) noted that the decomposition process r‘es u‘lts in the formation of products which are not free radicals. Synergism is a phenomenon which occurs when two or more com- poul"lds used together give a more pronounced antioxidant effect than the sum of their individual effects. Synergists may be organic or inc) Y‘ganic compounds and they are usually acidic in character. These aci ds are only active in conjunction with primary phenolic antioxi- dants. Synergistic action was also observed by Mahon and Chapman (1 953) and Dugan et al. (1954) when certain phenolic antioxidants ere used 1n comb1nat1on. 14 There are some theories on the action of the synergists. Synergists may work as metal scavengers, peroxide decomposers and sparing agents, as in the interaction of phenolic antioxidants or the interaction of other agents with phenolic antioxidants (Dugan, 1957). Phenols and Tocopherols as Natural Antioxidants in Olive Oil Phenols The phenolic concentration of olive oil depends on cultivation procedures and environmental factors and is correlated to the stability of the oil. Vazquez et al. (1973, 1975) demonstrated that the poly- phenol content of olive oil varies. They also found (1975) that the mai n polyphenols present in virgin olive oil are tyrosol and ‘ 3—hydroxytyrosol and observed some antioxidant effect in 3-hydroxyty- rosol. Polyphenols that were extracted from olive leaves were found to act as antioxidants in olive oil. These polyphenols in a concen- 13"‘<‘=ltion of more than 20 mg/100 g of oil inhibit the oxidative rancidity of the oil (Notte and Romito, 1971). Cantarelli and Montedoro ( 1 969, 1972) observed that phenols extracted from olive oil with 80% MeOH acted as antioxidants and inhibited rancidity. These phenols demonstrated a high stabilizing effect in other oils while the extrac- ti on of phenols from olive oil caused its rapid oxidation. \TO QOpherol s Tocopherols are natural antioxidants which are primarily rs es‘lbonsible for the stability of vegetable oils. It is known that an 0 pt ‘imum quantity of tocopherols must exist for stabilization. An 15 excess of this quantity will provide lesser stability through an apparent prooxidant effect (Oliver et al., 1944). Hove and Hove (‘l 944), in their experiment with carotene and ethyl oleate, found that gamma tocopherol was more effective than beta which in turn was more effective than alpha. Using dimensional paper chromatography, Grasian and Arevalo ( 1 965) identified only o-toc0pherol in olive oil. They noted that y—tocopherol must be considered as a product of o-tocopherol oxidation. Vi tagliano (1960) and Boatella (1975) both agreed that olive oil con- ta i ns a-tocopherol but they reported different quantities; the first from 12-162 ppm and the second from 70-150 ppm. Addition of Vitamin E at the level of 0.05% in the bottled 01 i ve oil retarded the oxidative rancidity of the oil (Fahmy and E1 Said, 1962). Vazquez et a1. (1973) found that olive oil was Stabilized against oxidative rancidity by adding tocopherols and other p0 1y phenol i c compounds . Ninnis et a1. (1969) demonstrated that the tocopherol content of Greek olive oil can be used for the detection of its adulteration ”‘5 th other vegetable oils. The Use of Synthetic Antioxidants in Olive Oil The addition of various antioxidants as a means of preventing the oxidation of olive oil has been reported by many researchers. Gutierrez (1961, 1962, 1963) studied the effect of the antioxidants BHA, BHT, NDGA, octyl gallate and dodecyl gallate as well as the s")"Tergistic effect of citric acid on the olive oil. 16 Astudillo et a1. (1968) found that antioxidants such as BHA and a-tocopherol used in low concentration (0.05%) did not have any effect on the oxidation of irradiated olive oil. Tadaaki and Kazuhito ( ‘l 967) observed that citric acid added to olive oil stored at 50°C for 96 hours showed an antioxidant effect which was greater in con- current use with antioxidants. Studying the antioxidant action of ascorbyl palmitate in olive oi 'l , Cerutti (1956) found that addition of 0.02% ascorbyl palmitate i 5 sufficient to assure good preservation of olive oil. Miric et al. ( 1 964) observed that 150propylidene-L-ascorbic acid is more effective than ascorbyl palmitate and NDGA in preventing oxidation of olive oil. It was observed by Schorderet (1969) that the stabilizing effect of as corbyl palmitate was enhanced by 2-tert-butyl-4 hydroxyanisole and prepyl gallate while a mixture of ascorbyl palmitate and NDGA caused greater peroxide formation than that in the unstabilized oil. Vitag- 1 ‘5 ano and Vodret (1960) demonstrated that ascorbic acid was efficient ‘5 n controlling the rise of peroxide formation by inhibiting lipase activity in olive pulp. The addition of 0.01-0.l% p-aminosalicylic ac‘i d in solution to unrefined olive oil showed a good antioxidant e1°1=ect (Didenko et al., 1968). Urakami et a1. (1961) observed some antioxidant effect of egg yolk cephalin methyl oleate made from the oleic acid fraction of 0] ‘i ve oil. An antioxidant effect also was observed by Hirahara et al. ( 1 974) when alcohol or ether extract from clove was added to olive Di 1 . Natural inhibitors of oxidation that are present in olive 1 eaves were also found to favor the stability of olive oil (Notte a" cl Romito, 1971 ). 17 Gutierrez (1962) found that the proprietary blend antioxidant Tenox II added to olive oil kept the oil in better conditions than BHT during storage at room temperature. He also observed (1963) that some antioxidants used in a mixture of olive oil and soy bean oil in a 50/ 50 ratio showed a good effect on the conservation of this blend. MATE RI AL AND METHODS Raw Material: Virgin Olive Oil The oil used in this study was obtained from Greece. Three di fferent samples of virgin olive oil were shipped over, each coming from a different part of the island of Crete. In all cases the oil was extracted by hydraulic pressure from fruits of the variety The samples from the three different regions were found to They ts ounati. di ffer in fatty acid composition and initial peroxide value. were numbered as olive oil No. 1, No. 2, and No. 3 and had initial peroxide value of 16, 35, and 12 respectively. To prevent the peroxide va1ue from increasing, the oil was kept in a cool place (36.5°F) until the day it was used. Dispersion of Antioxidants into the Samples The antioxidants evaluated in these studies were butylated h.ydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ). and propyl gallate (PG) (Eastman Chemical CC’nuoany). These antioxidants were used alone and in combinations. The type and the concentration of the antioxidants used in this study are shown in Table 1. Citric acid at 0.005% was also used, without the presence of antioxidants. In order to insure complete solution and uniform dispersion 01: the antioxidants, the oil was placed in beakers and heated to 60°C be ‘Fore antioxidants were added. As for the citric acid it was first 18 19 Table 1.--Antioxidants and Synergist Used in Virgin Olive Oil Studies. Kn t ioxi dant Concentration Addeda % (w/w) cert/x. 0.01 Eifilflk 0.02 Esli‘f' 0.01 Eifi‘t’ 0.02 lFlBiliQ 0.005 'flESIiQ 0.01 '1'E3l—N1 0.02 F’IE; 0.02 BHA + TBHQ 0.01 + 0.005 BHA + TBHQ 0.01 + 0.01 BHT + TBHQ 0.01 + 0.005 BHT + TBHQ 0.01 + 0.01 CA 0.005 -———____. aBHA = Butylated Hydroxyanisole BHT = Butylated Hydroxytolyene TBHQ = Tertiary Butylhydroquinone PG CA Propyl gallate Citric Acid 20 dissolved in a mixture of ethanol and distilled water 1:1(v/v) and was then added to olive oil. All treated samples and the control were left at room temperature for 24 hours, being stirred occasionally for about 20 minutes. Oven Test For the oven test (usually referred to as the Schaal oven test) the oil was placed in petri dishes (5.5" in diameter and 3/4" in height) and kept in an electric oven, and tested periodically for peroxide formation. The petri dishes were arranged on two shelves, the bottom shelf being 4 1/4" from the bottom of the oven, and the top shelf 8 1/4" from the bottom. All the samples were rearranged shelfWise and positionwise after each testing to get more uniform results because, according to Eubank and Gould (1949), the arrangement of the samples inside the oven is a factor affecting the results. A total of four tests were run for the oven stability studies, using samples of the olive oil No. 1, No. 2 and No. 3. One of these tests was run at 100°C while the other three were run at 65°C. The sample size was uniform 1009 in all tests, except for one where samples of both 50 g and 100 g were used in order to study the effect of the sample on the rate of oxidation. The end point for the oven test was the day when the peroxide value of the oil reached 120 meq/kg oil. Preparation of the Samples for the Storage Stability Studies For the long term storage studies 100 g of olive oil No. 2 (with or without antioxidants) was placed in clear glass jars 2" in 21 diameter and 4.5" in height. A11 jars were loosely capped. The one hundred and five (105) jars used were separated into three groups. The first group of 50 jars was stored in an incubator maintained at 50°C. The second group of another 50 jars, containing samples identi- cal to those in the first group, was stored at room temperature ranging from (20-28°C) in darkness, while the third group of 5 jars, containing no antioxidants and used as control, was stored at room temperature but was exposed to daylight. The latter experiment was intended to study the effect of light on the photooxidation of virgin olive oil. For each oil sample, five jars were stored and each time a sample was taken from a different jar until all five were used and then sampling was repeated following the same order. This sampling technique was employed to reduce the number of samplings taken from the same jar in a short time span. At one week intervals during the first month, and two weeks for the rest of the period, a jar was removed and shaken in order to mix the oil well, and samples were taken and examined for peroxide value, ultraviolet absorption at 233 nm and TBA absorption values. To evaluate the effect of antioxidants in virgin olive oil in the presence of light, another experiment was conducted at room temperature using olive oil No. 1. Five jars of 150 g of oil treated with antioxidants and one containing no antioxidants were stored under diffused light for a period of six months. This oil was analyzed every two months for peroxide formation. 22 Analytical Techniques Free Fatty Acid (FFA) Determination The official AOCS (1974) method was used for free fatty acid determination of olive oil No. 1, No. 2 and No. 3. The content in FFA of the samples used was found to be 0.5%, 2.1% and 1% respectively expressed as oleic acid. Preparation of Methyl Esters for Gas Liquid Chromatography (GLC) Methyl esters of olive oil were prepared by a modified Morisson and Smith (1964) method. Boron Trifluoride-methanol (Sigma Chemical Company) was the reagent used. Two hundred to three hundred mg of oil were dissolved with 1 m1 benzene in a 30 ml test tube. A total of 2.5 ml of 14% (BF3 - MeOH) was added and the tube was sealed with a screw cap and was placed for 40 minutes in a steam bath. After cooling the sample at room temperature, the esters were extracted by adding two volumes of hexane and one volume of water to the tube. The tube was then shaken vigorously until two layers were formed. A portion from the upper layer was taken and dried with approximately 0.3 g of anhydrous sodium sulfate, and then different quantities of the dried samples were used for analysis by gas liquid chromatography. Fatty Acid Composition of Olive Oil The fatty acid of olive oil was identified using a Beckman- GC-4-Gas Chromatograph equipped with a hydrogen flame detector. The glass column (6 ft x 2 mm (i.d.)) was packed with 10% (w/w) 23 diethylene glycol succinate (DEGS) on 100/120 mesh Supelcoport (Supelco, Inc.). The column oven temperature was 100°C, the injection port was maintained at 210°C and the detector at 185°C. The helium carrier was adjusted to 40 ml/minute. The flow rate of hydrogen and oxygen was 30 ml/minute. Esters were identified by a comparison of their retention time to those of standard mixtures of known fatty acid methyl esters. Peak areas were calculated by multiplying peak height by the width at half-height and from this it was possible to determine the relative' percentage composition of the total fatty acids. Measurement of Rancidity Peroxide Value (PV). Peroxide values were determined by a modified Wheeler method (1932), and were reported as milliequivalents/ kg oil. A 4.4 g or 5 m1 of oil taken by weight from the stored samples or by pipetting from the oven test samples was dissolved in 30 m1 of glacial acetic acid-chloroform (3:2) solution, and 0.5 m1 of saturated KI solution was added. The mixture was shaken and allowed to stand for one minute. It was then diluted with 100 ml of distilled water, and titrated with 0.1 N sodium thiosulfate. The reason for using 100 ml of distilled water is that the olive oil used, had a dark green color and because Stansby (1941) reported that the emulsion which is formed when the iodine in a colored oil is titrated with thiosulfate makes the starch end point very difficult to distinguish. This can be avoided by using a larger ratio of water-solvent for the titration. 24 Ultraviolet Absorption (UV). The absorption of the oil at 233 nm was determined during storage using the following procedure. After four weeks of storage and every two weeks thereafter ten (10) mg of oil were weighed accurately into small petticups and placed into 30 m1 test tubes. Ten (10) ml of purified iso-octane (2,2, 4-trimethyl pentane) was added to the sample and shaken in a Fisher mini shaker to assure complete dilution of the oil. The mixture was then filtered through a Whatman No. 1 filter paper and the ultraviolet measurements were taken on a Beckman DU spectrophotometer using pure iso-octane as a blank. Purification of Iso-octane. This solvent was purified using silica gel, according to AOCS (1974) method. Silica gel was activated before use by being dried in an oven at 130°C for four hours. About 3 1/2" of glass wool was put in the bottom of a (24" x 2") filter tube and then 12" of silica gel was added. The tube was fastened verti- cally to a ring stand and a l-liter Erlenmeyer flask was put under the stopcock. Iso-octane was poured slowly into the tube, filling it almost to the top and the tube was then covered with aluminum foil. The iso-octane was filtered through the silica gel as many times as needed to get an absorbance lower than 0.070 at 233 nm wavelength. TBA Test. The 2-thiobarbituric acid (TBA) test employed in this study was a modification of the Dunkley and Jennings (1951) method. The first test was run after six weeks of storage and then repeated every two weeks for both the oil samples stored at room temperature and at 50°C. 25 Reggent§--The TBA reagent consisted of 0.025 M 2-thiobarbituric acid in M phosphoric acid. The TBA solution was prepared by dissolving 0.720 g of TBA with distilled water in a 100 ml volumetric flask and completing the volume to 100 ml. The solution was then poured into a 250 ml beaker where 19.6 g of phosphoric acid, diluted to exactly 100 m1 of distilled water in a volumetric flask, was added. This TBA mixture was then heated for several minutes and was continuously stirred as to assure complete dilution. The TBA solution was prepared fresh each week that it was used. Procedure--Four (4) m1 of oil was pipetted into a 55 m1 test tube and an equal volume of TBA solution was added. Mixing by shaking in a Fisher mini shaker followed and the tubes were put in a boiling water bath for 25 minutes. Two to three granules were added to each tube for smooth boiling. Tubes were cooled in cold water upon removal from the bath. For the color extraction, 8 m1 of chlorofonm, distilled in glass, was added in each tube. After they were vigorously shaken in the Fisher mini shaker, the tubes were left for a few minutes to settle and the aqueous layers were transferred to tubes which were centrifuged for five minutes in a centrifuge at a minimum of 3,000 rpm. Part of the clear layer was removed and its absorbance at 532 nm and 535 nm was determined with a Beckman DU spectr0photometer using TBA solution as a blank. Removal of Interfering Pigments Many researchers have reported the presence of yellow and orange colors with an absorption maxima at 450-460 in TBA reaction 26 solution. In this study, a separation of pigments by a chromatographic column was employed using the Yu and Sinnhuber method (1962). Pyrex chromatographic tubes 300 x 10 mm were packed with cellulose powder (Whatman standard grade) to a height of about 12 cm under a 9.0 psi nitrogen pressure. Five ml of the TBA reaction solution were added to the column. After the solution had passed through the cellulose column, five ml of phenol solution (phenol 5 9, ethanol 15 m1 and water 25 ml) were added, to wash the sides of the tubes. The column was then washed with 1-3 ml of 0.1 N HCl in order to remove all the yellow pigments which were collected in 15 ml test tubes. Finally about 20 m1 of 0.1 N NaOH were added to the column to elute the pink color. When the pink band was 2-3 cm above the bottom of the column, the eluate was collected in a total volume of 10 ml in a volumetric flask containing 1 ml of 1.2 N HCl and 2 ml 25% ethanol. Using a Beckman DU spectrophotometer the absorbance was read at 532 nm and 535 nm for both yellow and pink color against a TBA solution used as blank. RESULTS AND DISCUSSION The olive oil used in this study was in its natural form, that is there had been no refining of the oil. Virtually all olive oil is consumed in this state and it seemed appr0priate therefore to study the effect of antioxidants on such olive oil. The antioxidants used were evaluated under long term storage and accelerated conditions (Schaal Oven Test). The results presented in this study are the average of dupli- cate determination. Gas Liquid Chromatography_(GLC) Analysis The fatty acid composition of olive oil was determined by gas liquid chromatography. The data in Table 2 reveal the fatty acid composition of olive oil No. l and No. 2. Both samples were found to contain traces of linolenic (C18:3) and palmitoleic (C16:l) acid and they both contained a high percentage of oleic acid (c18:1)° The difference in quantitative composition between the two samples may be due to the difference in growth conditions, unsatura- tion of the fruits and parental trees, since these samples came from different areas and the fruits were harvested at different times. Swern (1964) reported that olive oil tends to become more unsaturated with advancing maturity of the fruits and this probably explains the higher percentage of unsaturation in olive oil No. 2 (Table 2). 27 28 Table 2.--Fatty Acid Composition of Greek Olive Oil. Percentage Sample No. l 2 Year of Production December 1975 March 1976 Palmitic, C16:0 13.57 7.57 Palmitoleic, Cl6:l Tr. Tr. Stearic, C18:0 3.03 , 3.25 Oleic, c18:1 75.61 83.82 Linoleic, C18:2 7.63 5.31 Linolenic, C18:3 Tr. Tr. Instrument: Beckman GC-4 chromatograph Detector: Hydrogen flame Column: Glass ( 6 ft x 2 mm i.d.) Carrier gas flow rate: 40 ml/minute Column temperature: 100°C Injection temperature: 210°C Oven temperature: 185°C Sensitivity (attenuation) of 5 x 103 29 Oven Stability Studies As indicated in Tables 3, 4, 5, and 6, all antioxidants used (BHA, BHT, TBHQ, and PG) had inhibitory effects to various degrees on the peroxide formation in all three samples of olive oil used. The oven test was terminated when the peroxide value had reached 120 meq/kg oil. The number of days required to reach this point varied between the control and the samples containing anti- oxidants and between the three samples of oil used. Data in Table 3 demonstrate that samples containing 0.005% TBHQ exhibited better stability than those containing 0.01% BHT which in turn exhibited better stability than those containing 0.01% BHA. When the phenolic antioxidants BHA and BHT were used in combina- tion with TBHQ, better stability of the samples was achieved (Figure 1). It is interesting to note that the order of effectiveness of the antioxidants was reversed when olive oil No. 2, which had a higher initial peroxide value (35) was used. In other words, samples containing 0.005% TBHQ showed the lowest stability while those con- taining 0.01% BHA showed the highest (Table 4). When TBHQ was used in 0.01%, it caused the oxidative stability of the sample to be enhanced and it gave the same results as did 0.01% BHA. These data also show that the antioxidants BHA and BHT used in combination with TBHQ gave better results than when used alone. BHA performed better than BHT, regardless of whether they were used alone or in combination with 0.005% or 0.01% TBHQ. When 0.01% BHA was combined with 0.01% TBHQ it increased the protective factor from 1 to 2 and this was the best combination of those evaluated. 30 Table 3.--Stability Studies with Olive Oil No. l Treated with Antioxidants (Oven Test at 65°C). Stability of Olive Oila Antzgx;dant 100 9 Sample 50 9 Sample Oven Protective Oven Protective Daysb Factorc Daysb Factorc None (control) 32 1.00 28 1.00 0.01 BHA 36 1.12 30 1.07 0.01 BHT 38 1.18 31 1.10 0.005 TBHQ 41 1.28 36 1.28 0.01 BHA + 0.01 TBHQ 51 1.59 41 1.46 0.01 BHT + 0.01 TBHQ 51 1.59 41 1.46 aInitial peroxide value 16 bTime in days required for olive oil to reach a peroxide value of 120. cProtective Factor is expressed as: stability of the sample containinggantioxidant stability offithe control sample 31 _ .oz Poo e>e_o e? eoeerLOa meexoeoa ea moeeeexoeee< eeaocoo to oooeem wee Ao>oov «a on .AUOmc ea once ea>ov mes-b 9. 1 up w v d u - COD-<4 AR50 arm... + fired him $5.0 01m... .7 wowed (In ARmood 073... £906 #10 .RPOAV (yam AOKFZOO .P wczmwe ON own SOIXOHSd 301VA 32 Table 4.--Stability Studies with Olive Oil No. 2 Treated with Antioxidants (Oven Test at 65°C). Stability of Olive Oila Antioxidant (Wt %) Oven Daysb Protective FactorC ' None (control) 20 1.00 0.01 BHA 31 1.55 0.01 BHT 25 1.25 0.005 TBHQ 24 1.20 0.01 TBHQ 31 1.55 0.01 BHA + 0.005 TBHQ 34 1.70 0.01 BHT + 0.005 TBHQ 28 1.40 0.01 BHA + 0.01 TBHQ 40 2.00 0.01 BHT + 0.01 TBHQ 33 1.65 0.02 PG 60 3.00 0.005 Citric Acid 20 1.00 aInitial Peroxide value 35. bTime in days required for olive oil to reach a peroxide value of 120. cProtective Factor is expressed as: stability of the sample Containing antioxidant stability of the control sample 33 Good results were also obtained in the oven test when 0.02% PG was added to the sample. The addition of 0.005% citric acid had absolutely no effect on the results (Table 4). Since olive oil No. l and No. 2 had relatively high initial peroxide value, (16) and (35) respectively, a third quantity of olive oil was obtained from Greece which had a lower peroxide value (12) than the other two, but still somewhat higher than anticipated. This olive oil (No. 3) was used only for oven studies. In the oven test which was run with this oil, antioxidants were used at the maximum permitted level (0.02%). Results obtained from this test are shown in Table 5 and Figure 2. These results indicate that olive oil No. 3 which had the lowest degree of oxidative degradation (as determined by the initial peroxide value), showed lower stability than No. 1 and No. 2 (Tables 3, 4 and 5). It was found that BHA and PG were quite effec- tive in increasing the stability of olive oil No. 3, while TBHQ and BHT were only slightly effective. The difference in the oven stability of the three samples of olive oil might be due to the fact that the amount of natural anti- oxidants present in olive oil (toc0pherols and phenols) differed from one sample of oil to another. It might also be due to the percentage variations of unsaturated fatty acid of the oil (Catalano, 1971), which variations in turn depend on the area where the trees are grown (Petruccioli, 1966) and or on the difference in the degree of matura- tion of the fruits (Swern, 1964). 34 Table 5.--Stability Studies with Olive 011 No. 3 Treated with Antioxidants (Oven Test at 65°C). Stability of Olive Oila Antioxidant (wt %) Oven Protective Daysb Factorc None (control) 16 1.00 0.02 BHA 27 1.68 0.02 BHT 20 1.25 0.02 TBHQ 20 1.25 0.02 PG 26 1.62 aInitial Peroxide value 12. b Time in days required for olive oil to reach a peroxide value of 120. cProtective Factor is expressed as: stability of the sample containipg antioxidant stability of the controT'sample 35 3501— CONTROL 0 a H A 0.02 % 0 30°“ BHT 0.02 % o TBHQ 002% A PG 0.02 % I . 2501. m a ...l '< > 200.. 11.1 D 150'- x (3 a: I" 100.. o. 50 : -e—o av—r’ffzggggir . ' i“" O . l I I o 1 2 TIME (Weeks) Figure 2. The Effect of Certain Antioxidants on Peroxide Formation in Olive Oil No. 3 (Oven Test at 65°C). 36 Advanced maturation of the fruits also affects the free fatty acid content which in turn, as Suarez (1975) reported, affects the sensory characteristics of the oil. The Effect of Sample Size on the Rate of Oxidation An experiment was run in order to determine the effect of sample size on the rate of oxidation. Two oil samples of different sizes, 50 g and 100 g, were used. Table 3 shows that at 65°C a decrease in the sample size from 100 g to 50 g resulted in apparent lower stability for the control samples and those containing anti- oxidants. This may be due to the difference in surface-volume ratio between the two samples. This observation is in agreement with the finding of Ewbank and Gould (1942). Studying the effect of the sample size under accelerating conditions, they found that a decrease of the size of the sample markedly affected the rate of oxidation. The data in Table 6 also show that in the oven test run at 100°C, the 50 9 samples reached higher peroxide value than the 100 9 samples after one week. Further examination of peroxide formation indicated a decrease in peroxide value. This may be due to the fact that hydroperoxides were decomposed at a higher rate than they were fomred at 100°C. Privett and Quackenbush (1954) studied the destruction of hydroperoxides of lard at 100°C under vacuum and found a 50% loss after about 14 hours. Cooney et al. (1958) demonstrated that hydroperoxides are considered unstable products, especially at temperatures higher than 100°C. Table 6.--The Effect of Sample Size on the Stability of Olive Oil No. 1 Treated with Antioxidants (Oven Test at 100°C). Peroxide Values After 1 Weeka Antioxidant (Sample Size in 9) (wt %) 100 50 None (control) 214 263 0.01 BHA 211 275 0.01 BHT 216 287 0.005 TBHQ 204 257 0.01 BHA + 0.01 TBHQ 182 232 0.01 BHT + 0.01 TBHQ 190 241 aInitial peroxide value 16. Results obtained from the oven studies indicate that the effect of antioxidants differed from one sample of oil to another and their effectiveness depended on the concentration. In other words, equal amounts (0.01%) of BHA and TBHQ used in the 011 No. 2 resulted in equal stability while, whereas if double the quantity (0.02%) of the forementioned antioxidants was used in olive oil No. 3, different levels of stability were noted with BHA being superior (Tables 4 and 5). ' The Effect of Antioxidants on the Storage Stability of Olive Oil In this study the effect of antioxidants BHA, BHT and TBHQ used alone and in combinations in olive oil No. 2 was examined for two sets of samples, one stored at room temperature (20-28°C) and the other at 50°C. 38 The results were based on the measurements of peroxide value (PV), absorbance at 233 nm, and thiobarbituric (TBA) acid determina- tions at 532 nm, and 535 nm. Measurements of the peroxide value were taken once a week for the first month, but since the changes in this value were not considerable it was decided to make the analysis once every other week for the rest of the period. The ultraviolet absorp- tion measurements and the thiobarbituric (TBA) acid test, were begun four and six weeks respectively after this study was initiated and were repeated every two weeks. Oxidation of Olive Oil at Room Temperature in the Dark_ The peroxide values obtained as a result of storage at room temperature are presented in Tables 7 and 8. The data indicate that all samples stored at room temperature ranging from (20-28°C) had almost the same peroxide value at the end of the storage period as they did at the beginning, even though they varied widely during the course of the study. The reason that the final peroxide value of these samples was almost the same as that of the intial, might be due to the fact that the oil used was virgin containing chlorophyll, which according to Interesse et a1. (1971) acts as an antioxidant in the dark. Gut- finger et a1. (1975) found that olive oil stored in the dark at room temperature showed a reduction in peroxide value after a year of storage while a slight increase occurred in absorbance at 232 nm. Results obtained from this study make it obvious that anti- oxidants used in samples stored at room temperature in the dark had no effect on the outcome since no difference in peroxide value 39 Table 7.--Peroxide Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at Room Temperature (20-28°C). Antioxidant Treatment Storage (Lgflfis) Control BHA BHT TBHQ TBHQ CA 0.01% 0.01% 0.01% 0.005% 0.005% 0 35 35 35 35 35 35 1 41 42 41 41 40 40 2 55 55 55 54 57 42 3 41 40 39 39 39 40 4 40 39 39 39 38 38 5 52 45 52 45 51 51 6 41 42 41 40 40 40 8 51 51 48 50 53 51 10 51 51 51 50 49 51 12 44 45 44 41 42 41 14 47 45 45 43 43 45 15 41 42 40 38 39 41 18 37 35 35 34 34 35 20 38 38 35 34 34 35 22 37 37 36 34 34 36 40 Table 8.--Peroxide Values Obtained from Olive Oil No. 2 Treated with a Combination of Antioxidants and Stored at Room Temperature (20-28°C). Storage Comb1nat1on of Ant1ox1dants (alflfis) Control 0.01% BHA 0.01% BHT 0.01% BHA 0.01% BHT +0.005% TBHQ +0.00S% TBHQ +0.01% TBHQ +0.01% TBHQ O 35 35 35 35 35 1 41 41 42 42 4] 2 56 66 67 67 55 3 41 39 40 39 4O 4 4o 38 38 39 39 5 52 49 50 5o 49 5 41 41 41 41 40 8 51 49 53 50 51 10 51 49 50 48 49 12 44 42 40 40 39 14 47 44 44 42 44 15 41 40 39 38 39 18 37 34 35 34 34 20 38 34 34 34 34 22 37 34 34 34 34 VALUE PERO x105 41 140{ ROOM TEMP. 50‘ C CONTROL I V 120 811T oxnix .A (3 TBHQ 0.01% O D 100% 801- 551. /\ 40' Id,.s:iiiLlIIIIIIIIii577“i~.--~r‘---' .' 20. o ,. >1 . .. l .A .l . I. O 6 12 18 24 STORAGE TIME (Weeks) 'Figure 3. Peroxide Formation in Olive Oil No. 2 Containing Antioxidants. 42 between the control and the antioxidant treated samples was observed (Figure 3). The addition of 0.005% of citric acid alone brought about no changes. Removal of Interference Pigments py a Chromatogrgphic Column Caldwell and Grogg (1955) observed the presence of yellow interfering pigments in the TBA reaction solution and Yu and Sinn- huber (1962) demonstrated two methods that could be used for the separation of these pigments. One of these methods, the chromato- graphic column separation, was used in this study in order to determine any interference due to the presence of yellow pigments. This method showed that although yellow pigments were present, no significant difference in the absorption values of these pigments was observed between the control and the samples containing antioxidants, during the first month. All other TBA determinations were therefore per- formed without passing the solution through the column. The presence of the yellow pigments in the TBA reaction mix- ture, may be attributed to the fact that olive oil contains carote- noids and carotenes. Vitagliano (1960) reported that the olive oil content of the above pigments is 38-956 and 33-310 v/lOO 9 oil respectively. Figure 4 illustrates the absorption spectrum of yellow pigments present in olive oil No. 2. Tables 9 and 10 present the TBA absorption values at 532 nm and 535 nm, obtained from samples stored at room temperature in the dark. In all cases the absorbance at 532 nm exceeded the absorbance at 535 nm and generally all TBA absorption values were lower than in the case of samples stored at 50°C (Tables 9, 10, 14, and 15). 43 E :: CM (0 u) *- ¢l III C) 2: ‘l m C) a) on ( 0.04- 04x2- aooL 1 I 1 l I 420 440 460 480 500 520 540 . WAVE LENGTH (001) Figure 4. The Absorption Spectrum of Yellow Pigments Present in Olive Oil No. 2. 44 Table 9.--TBA Absorption Values Obtained from Olive 011 No. 2 Treated with Antioxidants and Stored at Room Temperature (20-28°C). Absorbance at 532 nm Storage 432:4 .831. .831. iii. .1332. .33.. 6 0.217 0.210 0.186 0.119 0.121 0.179 8 0.248 0.219 0.157 0.122 0.130 0.236 10 0.150 0.144 0.123 0.074 0.075 0.205 12 0.216 0.203 0.161 0.093 0.097 0.191 14 0.209 0.192 0.161 0.087 0.104 0.310 16 0.190 0.220 0.150 0.078 0.096 0.220 18 0.187 0.185 0.118 0.063 0.069 0.176 20 0.200 0.225 0.179 0.102 0.110 0.238 22 0.274 0.215 0.212 0.124 0.125 0.274 Aborbance at 535 nm 6 0.211 0.202 0.177 0.117 0.120 0.175 8 0.236 0.210 0.150 0.118 0.125 0.224 10 0.143 0.139 0.117 0.070 0.069 0.200 12 0.206 ‘0.194 0.154 0.090 0.094 0.182 14 0.199 0.185 0.152 0.083 0.100 0.295 16 0.180 0.210 0.143 0.075 0.094 0.211 18 0.180 0.176 0.114 0.060 0.063 0.168 20 0.191 0.216 0.171 0.098 0.105 0.227 22 0.259 0.206 0.204 0.121 0.122 0.254 45 Table 10.--TBA Absorption Values Obtained from Olive Oil No. 2 Treated with a Combination of Antioxidants and Stored at Room Temperature (20-28°C). Storage Absorbance at 532 nm Time ) Control 0.01% BHA 0.01% BHT 0.01% BHA 0.01% BHT (Weeks +0.005% TBHQ +0.005% TBHQ +0.01% TBHQ +0.01% TBHQ 6 0.217 0.128 0.116 0.115 0.113 8 0.248 0.144 0.140 0.127 0.144 10 0.150 0.082 0.075 0.070 0.080 12 0.216 0.095 0.090 0.092 0.083 14 0.209 0.091. 0.085 0.090 0.092 16 0.190 0.094 0.103 0.098 0.099 18 0.187 0.073 0.083 0.067 0.069 20 0.200 0.117 0.092 0.084 0.087 22 0.274 0.152 0.121 0.109 0.112 Absorbance at 535 nm 6 0.211 0.125 0.112 0.111 0.108 8 0.236 0.140 0.132 0.122 0.140 10 0.143 0.080 0.073 0.066 0.077 12 0.206 0.091 0.086 0.088 0.079 14 0.199 0.087 0.080. 0.087 0.088 16 0.180 0.090 0.100 0.094 0.092 18 0.180 0.069 0.079 0.063 0.066 20 0.191 0.112 0.089 0.081 0.082 22 0.259 0.146 0.116 0.104 0.107 46 Although no significant difference in peroxide values was observed during the 22 weeks of storage period between the control and the samples containing antioxidants and stored at room tempera- ture, TBA absorption values varied widely. The difference in TBA absorption values between the control and the antioxidant treated samples might be due to the fact that small variation in the peroxide values between the samples was enough to causé‘considerable differences in TBA absorption values. Samples containing 0.01% TBHQ exhibited lower TBA absorption values than those containing 0.005% TBHQ, while peroxide values obtained from these two samples throughout the storage period were almost identical. The fluctuation of TBA absorption values throughout the' storage period is probably due to the fact that the malonaldehyde precursor is not a stable product (Tarladgis and Watts, 1960). This also might be attributed to errors made during the analysis. An attempt was made to measure the absorbance of the oil samples at 233 nm during the storage period, and to learn if there is any correlation between peroxide, TBA and ultraviolet absorption values. Absorption at 233 nm is due to the presence of conjugated double bonds. Angelo et a1. (1975) noted that the mechanism causing the peroxidation of polyunsaturated fatty acids produces conjugated diene hydr0peroxides (CDHP). Table 11 shows the results from diene studies on samples stored at room temperature in the dark. Control samples (containing no antioxidant) gave slightly higher ultraviolet absorption values than the samples treated with antioxidants. 47 Table ll.--Ultraviolet Absorption Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at Room Temperature (20-28°C). Storage Absorbance at 233 nm (Ligfis) Control BHA BHT TBHQ TBHQ CA 0.01% 0.01% 0.01% 0.005% 0.005% 4 0.410 0.415 0.397 0.380 0.379 0.377 5 0.402 0.410 0.407 0.382 0.359 0.372 5 0.383 0.384 0.380 0.370 0.360 0.375 8 0.332 0 332 0.327 0.301 0.295 0.300 10 0.330 0 330 0.315 0.298. 0.279 0.295 12 0.255 0.254 0.252 0.248 0.250 0.251 14 0.324 0.300 0.293 0.257 0.259 0.252 15 0.252 0.250 0.249 0.215 0.230 0 259 18 0.427 0.425 0.395 0.374 0.391 0.401 20 0.430 0.430 0.400 0.378 0.380 0.411 22 0.435 0.432 0.410 0.390 0.398 - 0.420 Sigggge Control 0.01% BHA + 0.01% BHT + 0.01 BHA + 0.01% BHT + (Weeks) 0.005% TBHQ 0.005% THBQ 0.01% TBHQ 0.01% TBHQ 4 0.410 0 390 .0385 0.388 0 390 5 0.402 0.355 0.371 0 372 0.354 5 0.383 0.374 0.389 0.358 0.375 8 0 332 0.305 0 293 0.314 0.345 10 0.330 0.292 0.285 0.281 0.310 12 0.255 0.258 0 250 0.259 0.252 14 0.324 0.281 0.278 0 250 0.268 15 .0.252 0.229 0.239 0.235 0.240 18 0.427 0.374 0 370 0.375 0.375 20 0 430 0.378 0 375 0 374 0 378 22 0.435 0.397 0.395 0.398 0.394 48 Somewhat unexpectedly in this case, UV absorption values showed a decrease at some points during the storage period while, for the last few weeks, they showed a continuous increase, an indication of the formation of conjugated linoleate hydroperoxides (Table 11). Oxidation of Olive Oil at 50°C The effect of elevated temperature on peroxide formation in olive oil No. 2 is shown in Table 12 and 13. Data in these tables indicate that all samples stored at 50°C developed essentially the same hydroperoxide values during the first two weeks of storage. Up to the tenth week of storage, the samples containing no antioxidants (control) and those containing either 0.01% BHA or 0.01% BHT showed similar changes. From then on the control samples showed a continuous and rapid increase in peroxide formation while the samples with 0.01% BHA and those with 0.01% BHT showed a decrease in peroxide value, which continued until this study was terminated (Figure 5). The effect of 0.01% BHT was found to be slightly superior to that of 0.01% BHA in reducing the peroxide content of the oil. TBHQ did not prevent peroxide formation during the first two weeks, but it reduced the peroxide content of the oil for the rest of the storage period. The beneficial effect of this antioxidant increased when the quantity used was doubled from 0.005% to 0.01% (Table 12, Figure 5). When BHA and BHT were used in combination with TBHQ, better results were obtained than when BHA and BHT were used alone. Samples containing 0.01% BHT + 0.005% TBHQ and those containing 0.01% BHA + 0.005% TBHQ showed a decrease in peroxide value which started the 49 Table 12.--Peroxide Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at 50°C. Antioxidant Treatment Storage «1223» .331. .811. 138?. $333. .33.. 0 35 35 35 35 35 35 1 42 42 42 41 42 43 2 46 46 45 42 42 46 3 41 42 41 35 39 41 4 40 41 36 34 34 39 6 38 38 35 30 32 36 8 45 45 44 35 39 44 10 4O 40 38 31 33 40 12 38 38 33 27 32 39 14 42 i 31 30 22 26 44 16 48 24 23 18 24 84 18 80 25 22 18 22 99 20 99 24 22 17 20 121 22 136 24 20 16 18 172 24 164 22 18 13 16 190 50 Table l3.--Peroxide Values Obtained from Olive Oil No. 2 Treated with a Combination of Antioxidants and Stored at 50°C. Storage Comb1nat1ons of Ant1ox1dants (EZEES) Control 0.01% BHA 0.01% BHT 0.01% BHA 0.01% BHT +0.005% TBHQ +0.005% TBHQ +0.01% TBHQ +0.01% TBHQ 0 35 35 35 35 35 1 42 41 41 41 41 2 45 43 44 43 44 3 41 38 38 39 38 4 40 35 35 35 34 5 38 34 32 32 . 31 8 45 40 40 39 37 10 40 40 35 32 32 12 38 . 32 30 27 25 14 41 28 24 21 22 15 48 25 23 20 21 18 80 23 20 19 13 20 99 22 19 - 17 17 22 135 19 18 15 15 24 164 17 15 14 14 51 eighth and tenth week respectively and continued to the end of the storage period (Table 13, Figure 6). When the quantity of TBHQ com- bined with BHT or BHA was increased from 0.005% to 0.01% the anti- oxidant effect was even more pronounced. In other words, samples containing 0.01% BHA + 0.005% TBHQ showed always a higher peroxide value than those containing 0.01% BHA + 0.01% TBHQ and at the end of this study the two samples were found to have a peroxide value of 17 and 14 respectively. When 0.01% TBHQ was used, the results were almost the same throughout the storage period whether it was combined with BHA or BHT. When TBHQ was used at 0.005%, however, the combination of TBHQ with BHT was more effective than the combination of TBHQ with BHA (Table 13); therefore BHT was more effective than BHA whether it was used alone or combined with 0.005% TBHQ. It is interesting to note that the best results were obtained when 0.01% TBHQ was used alone (Figures 5 and 6). This superiority of TBHQ over BHT or BHA in various fats and oils agrees with the findings of Cort et al. (1975), and also with the findings of Sherwin and Luckadoo (1970) and Chahine and MacNeill (1974) who observed a higher antioxidant effect with TBHQ than with BHA. The results obtained when citric acid was used alone were unexpected. This acidic compound seemed to be without significance in the system until the twelfth week of storage; after which, samples treated with 0.005% citric acid developed a rapid peroxide formation at a much faster rate than the control samples (Figure 6). Our results are similar to those obtained by Lea (1944) who observed 52 1807f CONTROL 0 . 160— BHA 0.01% A BHT 0.01% U Ill TBHQ 0.005% . TBHQ 0.01% A D 140)- _l ‘1 -L > 80 o I.” C3 _ 60H- X C) m in 40 //5\ - .. 0. ._ 20 o .41 J . - .1 l .. a . l . 1. l 1 0 6 12 18 24 STORAGE TIME (Weeks) Figure 5. Peroxide Formation in Olive Oil No. 2 Containing Anti- oxidants and Stored at 50°C. 53 000 ° (24 “$351901 m+ In :1 80.. (24 wks,164) .J -L 1r 5! > 4°— . m l c: .. 30.. x (D o: 20— u: CONTROL A a CA 00057. 0 BHA 0.01%+TBHQ 0.005% O 10- BHT QO17€+TBHO 0.0057. I TBHQ 0.01% A o J I I |.. - O 6 12 18 24 STORAGE TIME (Weeks) Figure 6. Peroxide Formation in Olive Oil No. 2 Containing Anti- oxidants and Stored at 50°C. 54 a prooxidant effect of citric acid when used alone in milk fat. Lemon et a1. (1950) also pointed out that the citric acid in substrates free of primary antioxidants does not prevent the oxidation of fatty acids. In view of the findings in the present study, one may consider that peroxide value is a useful tool for measuring the extent of lipid oxidation and for evaluating the effect of different antioxidants. It is obvious that at room temperature (20-28°C) the use of antioxidants was not found to be of benefit during the time of the tests, while at 50°C the effectiveness of antioxidants was very clear. The peroxide value of all samples containing antioxidants and stored at 50°C was decreased during the storage period and this indicated that the anti- oxidants probably acted as peroxide decomposers (Dugan, 1961). The results obtained when the thiobarbituric acid (TBA) test was employed for the samples stored at 50°C, are presented in Tables 14 and 15. The data show that citric acid added to the oil resulted in TBA absorption values higher than the control samples and those con- taining antioxidants (Figure 7). This was also true for the peroxide values (Table 12). Citric acid resulted in a gradual increase in TBA values during the first 14 weeks of storage while a rapid increase in these values appeared at the sixteenth week. In the case of the control samples a gradual increase was observed until the sixteenth week with the exception that on the eighth week there was a decrease, while a rapid increase in TBA absorption values started on the eighteenth week of storage. A similar rapid increase was observed in peroxide values at the corresponding weeks (Table 12). Table l4.--TBA Absorption Values Obtained from Olive Oil No. 2 Treated with Antioxidants and Stored at 50°C. 55 Absorbance at 532 nm Storage (Eggfis) Control BHA BHT TBHQ TBHQ CA 0.01% 0.01% 0.01% 0.005% 0.005% 6 0.339 0.291 0.250 0.150 0.160 0.390 8 0.257 0.281 0.206 0.115 0.230 0.410 10 0.312 0.310 0.220 0.144 0.204 0.500 12 0.333 0.279 0.250 0.118 0.224 0.546 14 0.402 0.194 0.174 0.102 0.171 0.738 16 0.622 0.204 0.162 0.085 0.170 1.080 18 1.214 0.280 0.243 0.125 0.208 1.260 20 1.290 0.299 0.250 0.159 0.214 1.420 22 0.690 0.327 0.271 0.148 0.229 0.895 24 0.723 0.268 0.238 0.122 0.212 0.792 Absorbance at 535 nm 6 0.327 0.270 0.237 0.171 0.148 0.372 8 0.243 0.265 0.195 0.109 0.218 0.386 10 0.305 0.297 0.210 0.138 0.197 0.475 12 0.314 0.262 0.237 0.113 0.214 0.506 14 0.378 0.184 0.166 0.097 0.162 0.682 16 0.575 0.196 0.154 0.074 0.156 0.890 18 1.123 0.266 0.231 0.120 0.201 1.236 20 1.210 0.273 0.240 0.151 0.203 1.360 22 0.634 0.310 0.259 0.141 0.219 0.809 24 0.658 0.251 0.225 0.117 0.203 0.725 56 Table 15.--TBA Absorption Values Obtained from Olive Oil No. 2 Treated with a Combination of Antioxidants and Stored at 50°C. Storage Absorbance at 532 nm (Llflfis) Control 0.01% BHA 0.01% BHT 0.01% BHA 0.01% BHT +0.005% TBHQ +0.005% TBHQ +0.01% TBHQ +0.01% TBHQ 6 0.339 0.201 0.168 0.162 0.144 8 0.257 0.190 0.193 0.119 0.114 10 0.312 0.246 0.214 0.134 0.139 12 0.333 0.205 0.166 0.117 0.123 14 0.402 0.165 0.148 0.107 0.114 16 0.622 0.174 0.149 0.093 0.107 18 1.214 0.218 0.153 0.126 0.112 20 1.290 0.206 0.158 0.136 0.134 22 0.690 0.220 0.194 0.138 0.135 24 0.723 0.222 0.186 0.130 0.131 Absorbance at 535 nm 6 0.327 0.184 0.150 0.152 0.141 8 0.243 0.181 0.185 0.114 0.110 10 0.305 0.235 0.187 0.125 0.131 12 0.314 0.195 0.158 0.112 0.116 14 0.378 0.157 0.141 0.101 0.112 16 0.575 0.162 0.140 0.089 0.098 18 1.123 0.209 0.147 0.122 0.103 20 1.210 0.195 0.149 0.128 0.127 22 0.634 0.209 0.186 0.131 0.128 24 0.658 0.211 0.177 0.124 0.129 57 Both the control and the citric acid treated samples reached their maximum level after 20 weeks of storage and then began declining until the end of this study, as evidenced by the data plotted in Figure 7. This may be due to the fact that malonaldehyde production declines after reaching a peak as Tarladgis and Watts (1960) concluded that the malonaldehyde precursor is not a stable end product. The variations in TBA absorption values in samples containing antioxidants were probably due to the fact that when the decrease in these values was observed, the destruction of malonaldehyde was greater than its formation. Data in Table 14 show, as was the case with samples stored at room temperature, TBHQ at 0.005% resulted in . higher TBA absorption values than when it was used in 0.01%. As can be observed from Figure 7, BHA resulted in higher TBA absorption values than did BHT, which in turn gave higher values than TBHQ, when those three antioxidants were used in the same concentra- tions. The results obtained when the combination 0.01% BHA + 0.01% TBHQ was used were very similar to these obtained when 0.01% BHT + 0.01% TBHQ was used (Table 15). This also held true in the case of peroxide value. The pink color formation in olive oil, despite the fact that Gas Liquid Chromatography (GLC) analysis showed that there were only traces of linolenic acid (Table 2), can be explained if it is con- sidered that olive oil contains linoleic acid and, according to Pryor et al. (1976), malonaldehyde arises from the decomposition of endo- peroxides which can be formed in a diene system. Wilbur et a1. (1949) and Kenaston et a1. (1955) observed formation of TBA color in oxidized linoleate. 58 .uoom on eoeoom eee moeee_xoeee< aeee_eoeou N .52 P_o m>epo to mm=~e> eoeoacomc< ./O l v iii T\ 11.] .. 1596 S . nu o l u . , 8 JAXXWO uv ,. N 1 nu 3 L83 0 «.66 0:2. .. V I e. 3.0 t; 1. q 4.56 (In 9 . L58. 8 o °emcee <0 7. o 42:23 .. m 59 TBA absorption values were more meaningful for changes occur- ring in the samples stored at 50°C than th0se stored at roam tempera- ture (20-28°C). When the TBA is the only test used to determine the level of oxidation, its significance is variable because the products it measures do not increase at all stages in proportion to the degree of oxidation. This is shown in Table 12 and Figure 6 where, after the twentieth week, the degree of oxidation of the control and the citric acid treated samples appeared to increase by the peroxide test while the TBA test showed a decrease in values (Table 14, Figure 7). It seems that TBA color reactants are oxidized further and produce more unstable products or disappear by reacting with themselves or . with other components of the system. Ultraviolet absorption values at 233 nm for the samples stored at 50°C, are presented in Table 16. These samples exhibited better results than those stored at room temperature. Samples without antioxidant and those containing 0.005% citric acid followed an identical pattern. These samples showed a rapid increase in absorbance at 233 nm from the twelfth week on, due to diene conjugation (Figure 8). A continuous increase in peroxide value was also observed after the twelfth week and this is in accor- dance with the findings of Ninnis and Ninni (1966, 1968), Montefredine and Luciano (1968) and Bartolomeo and Sergio (1969), who repor ‘that the increase in ultraviolet absorption in olive oilxis_well/correlatedi with the change in peroxide value. It is evidenced from Figure 8 J that the addition of 0.005% citric acid resulted in higher ultraviolet absorption values and this correlated with the PV and TBA absorption values to indicate that Citric acid had a prooxidant effect. 60 Table l6.--Ultraviolet Absorption Values Obtained from Olive Oil N0. 2 Treated with Antioxidants and Stored at 50°C. Absorbance at 233 nm Storage (Lizfis) Control BHA BHT TBHQ TBHQ CA 0.01% 0.01% 0.01% 0.005% 0.005% 4 0 425 0 425 0.420 0.405 0.405 0.434 5 0.450 0.450 0.445 0.434 0.441 0.492 0.479 0.480 0.440 0.387 0 420 0.485 10 0.425 0.420 0.375 0.278 0.325 0.352 12 0.420 0.402 0.381 0.301 0.340 0 394 ‘14 0.505 0.452 0 395 0.304 0.358 0.550 15 0.524 0.455 0 407 0.350 0.435 0.945 18 1.120 0.524 0.505 0.382 0.471 1.200 20 1.400 0.559 ‘ 0.550 0.470 0.550 1.500 22 1.700 0.555 0.590 0.486 0.559 1.900 24 1.950 0.570 0.500 0.490 0.572 2.200 5593299 Control 0.01% BHA + 0.01% BHT + 0.01% BHA + 0.01% BHT + (Weeks) 0.005% TBHQ 0.005% TBHQ 0.01% TBHQ 0.01% TBHQ 4 0.425 0 415 0.403 0 359 0.385 5 0 450 0.440 0.443 0.440 0.420 8 0.479 0.450 0.448 0.424 0.435 10 0.425 0.348 0.314 0.281 0.290 12 0.420 0.355 0.330 0.290 0.280 14 0.505 0.448 0.382 0.305 0.323 15 0.524 0.412 0 390 0.315 0.328 18 1.120 0.545 0.445 0.413 0.420 20 1.400 0.554 0.450 0.433 0.435 22 1.720 0.579 0.507 0.485 0.489 24 1.950 0.585 0.510 0.488 0.492 61 doom um UGLopm Ucm mucouwxowuc’x OCwamucou N .02 :.O m>wpo :w cowHQLOmn—gx vm~0w>mLHP= .w mLzmwn. 3x062: m2... m0epo e2 memeeeo .m mesmea 80.002: ms..._. u0<¢o._.m mm .24. 8 2 a. w e o o - 4 q H A . fioood 8 t L8.3 r 8. 488 L on? +83 o .. I emu» as: Nmm pm we: mmm pm m:_m> mocmngoma< muwxocma mucmncomn< muwxocmm Anxmmzv unoppzma ms“ op ummoaxm scan mgp cw umcoum mmwwwwm .wo m>wpo co »o__wnmpm .Au°m~-omv wcsumaoasmk zoom on umtopm N .02 __o m>w_o do cowomuwxo web go o;m_4 co sumac“ m:e--.~p m_nmh 66 STORED IN DARK O EXPOSED TO THE LIGHT O 300m. I“ D __J _ < . ' :’ zoo). ° I“ c: x _ . (D a: Ill 0. wot, p. O ,1 1 n 1 , ll 0 4 8 12 16 20 24 STORAGE TIME (Weeks) Figure 10. The Effect of Light on Peroxide Formation in Olive Oil No. 2 (Room Temperature Storage). 67 Table l8.--The Effect of Light on Peroxide Formation in Olive Oil No. l Treated with Antioxidants and Stored at Room Temperature (20-28°C). . .Storage Antiox1dant Treatment (agflihs) Control BHA BHT TBHQ 0.01% BHA 0.01% BHT 0.01% 0.01% 0.005% +0.01% TBHQ +0.01% TBHQ 0 16 16 16 16 16 - 16 2 103 101 99 98 99 102 4 165 160 116 163 164 157 6 191 189 123 190 163 161 transfer of excitation energy from chromophoric impurities to oxygen, as is shown by the reaction: 3 Ch “V 4 |cn|* -—-—02———-->cn+‘o2 This singlet oxygen, which is more active than triplet, reacts rapidly with unsaturated fatty acids to give hydroperoxides which can decom- pose at room temperature, initiating the free radical mechanism of autoxidation (Foote, l968 and Kaplan, 1971). Skinner (1976) reported that singlet oxygen reacts in an electrophilic rather than in a free radical fashion. Figure ll illustrates that samples containing no antioxidants and exposed to the daylight followed a similar pattern in all three tests employed. The continuous increase in absorbance at 233 nm for the samples exposed to light indicates that conjugated linoleate hydroperoxides were formed during the photooxidation of olive oil. According to Rawls and Van Santen (1970) conjugated and non- conjugated hydroperoxides are formed as primary oxidation products 68 BOIXOHBd 301VA .wcmwb mo mucmmmcm mzp cw mcspocmaamp zoom um mmmcoum mcwcau N .02 FPO m>wpo cw mmmcmzu .~F mczmwd onoogv m2_h m0 mo.x0mwa 8 O ham... 1001. m a x C) m / CONTROL 0 I" so F B H A 0.01 % v n' e H T 0.01 % o 1' 8H 0 0.005% A BHA 0,01% “I'TBHO 0.005% - BHT 001% +TBHO 0.005% 0 0 I l. . . o 2 4 6 STORAGE TIME (Months) Figure 12. The Effect of Light on Peroxide Formation in Olive Oil No. l Containing Antioxidants (Room Temperature Storage). 71 carotene present, occurred. Sastry et al. (l973) postulated a bleaching mechanism of chlorOphyll during oxidation in visible light. Pictures of the samples used in this study, taken at the end of the storage period are shown in Figures 13, l4 and 15. The sample containing 0.0l% BHT maintained its color more than other samples (Figure 13). This sample had the lowest peroxide value when this study was terminated. The sample containing 0.005% TBHQ was able to maintain its color even though it showed almost the same degree of oxidation with the control sample. The presence of 0.0l% BHA, however, did not prevent the loss of the oil color. Figure l4 illustrates the difference in color between untreated samples, one stored in the dark and the other in the presence of light. The color difference between five of the samples which were stored at 50°C and underwent different degree of oxidation can be observed from Figure l5. It is shown in this figure that TBHQ, which provided the best stability, was able to maintain the original color of the oil. The control sample and the one containing citric acid, however, underwent the highest degree of oxidation and lost most of their original color (Figure l5). 72 I. _ $739.“ ’1 Figure l3. Changes in the Color of Olive Oil Containing Antioxidants and Stored at Room Temperature in the Presence of Light. Figure l4. Changes in the Color of Olive Oil During Storage at Room Temperature in the Presence of Light. 73 Figure l5. Changes in the Color of Olive Oil Containing Antioxidants and Stored at 50°C. SUMMARY AND CONCLUSIONS Three different samples of virgin olive oil (No. l, No. 2, and No. 3) with initial peroxide values of 15, 35, and 12, respectively, were used in this study. The effectiveness of the antioxidants BHA, BHT, TBHQ, and PG was evaluated under accelerated conditions (Schaal Oven Test) and other storage conditions. The development of rancidity in the olive oil under accelerated conditions was measured by the method of peroxide value only, while in the case of other storage conditions, results obtained from peroxide value, diene conjugation and TBA test were compared. The best results were obtained from peroxide values. The usefulness of the TBA test in olive oil is questionable, since the TBA absorption values of the control and the citric acid treated samples, after reaching a peak, fell to lower levels, while the same samples showed a continuous increase of oxidation when the other two tests were used. Gas Liquid Chromatography (GLC) analysis for oil No. l and No. 2 showed that each had a high percentage of oleic (C18:]), low percentage of linoleic (c18:2) and traces of linolenic (c18z3) acid. Sample size was found to affect the rate of oxidation; the smaller the size, the higher the rate of oxidation. 74 75 The use of antioxidants had a considerable effect on the oven stability of the oil. The degree of effectiveness, however, was found to be different in the three samples of olive oil used. When the antioxidants were used at 0.02% in the oven test the effectiveness was in the following order: BHA > PG > TBHQ and BHT. The most effective combination in the oven stability studies was found to be 0.01% BHA + 0.0l% TBHQ. The use of citric acid alone had no effect on the stability of the oil in the oven test. Results related to the oil stored at room temperature in the dark indicated that the use of antioxidants under these conditions had practically no significance over the period of the test. The peroxide values, ultraviolet absorption at 233 nm and TBA absorption values, were poorly related in this case. Samples containing antioxidants and stored at 50°C showed a considerable decrease in peroxide value during the storage period, in contrast with the control and samples containing citric acid which showed a continuous increase. A good correlation between the peroxide value and diene conjugation was obtained in the case of the control and the samples containing citric acid, especially after the four- teenth week of storage. In the storage conditions at 50°C, in contrast with the oven test, TBHQ was found to be the most effective antioxidant with the order of effectiveness being TBHQ > BHT > BHA. This held true even when the amount of TBHQ used was half as much as that of BHT or BHA. BHA and BHT were more potent when they were used in combination with TBHQ, but still not as effective as TBHQ used alone. 76 Citric acid did not affect the oxidative stability of the oil stored at room temperature but it did exhibit a negative effect at 50°C. Exposure of the samples to daylight resulted in rapid oxida- tion, probably due to the catalytic action of chlorophyll. The useful- ness of antioxidants in this case was not apparent. 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