~11.” .. ..:;.~1. .‘ . ~.. ~~~uo 11...... 1““ ~ ‘uwx.;...l 3:3:-;.‘::.‘;_~o - II ‘ . IA -. . I A. Q . 8.15"“: 3.35.1 ”515%? I; &I§\ "'4 V ‘Fr'. as) 1 21:1». :1! :.-.»1_ 7 ”11.1., . ' " qt“: 3'1, no_1 1' I" 1.1 {1 1 v1: 572.6113"? $43.11.“ lI ;. . '-'1 :1 $111111. ONO). 50:35.3:{a' I i- Nc‘éa‘éck'l I. n ‘ g , . ' 13 ‘0 ‘1 13.1: {I‘Kl‘h’ 7:522“ ‘1 “h- ~ 1.212.; . ' Y" f,” 3% (1" 1‘: .‘ ‘ $5323.?iw :1 31‘5" ' a I'J 5.11% :Il\o'\“ -: V. '2 ....1, . ‘\ $.35. 4 It". lutan' ‘ ‘1‘ vv .5‘ wk?“ up. J“ .32; ‘ w. a»; VERY“: '“ " w '5' --~‘_‘ -.. .152?” 2.5.41;th ‘1“ ".415? .;’~‘V1‘€T..°‘°..~‘~‘}h“v ”M" xvi-78" v ‘l o s. . WV. .'.&-5" V .. {gawk ' "-9. '1. ~ 1“" a1aq~.~‘y*}".'( \‘J W 11 1.1 mm a 7' J". u 4 u . -€;:3:'§::1:;$IU. J ‘5“, 5": 31% ‘35 1,... 5%": 7'11 '91" tr ‘3': ”1" ' 4:}: {{- A\ ’1'. -;:'o:,;-,*.», 1‘ Y :“':‘ m. -. ..- ' W; i'.‘ $17.33,“ ._J’ :9. 1 1-.“ {(244751 Q ‘ 1 v3.3.1! ""rqiv: «’1' A v wvgc 2‘. i 8 -4 l ‘. 64"“ V,..._ 12. I 4 1 .‘ -1.".‘! ‘ ”1-H ' o ~,_v.;.. 10171-539 M :57: ‘v- 11“ ‘J..'..",|'.' ‘1?“ .1. . _‘ 1 1| 11:": n ‘ ‘ . '1}- ‘ 1%, '1'... ‘ 1 ‘1‘ :41. :1. NE?" _ -.-‘.1?)n‘-'_ \ p.12”. "Win/1‘ - 1 \I' ‘\ 1 1 V 1 . I 1 -\‘:-".'*}.“1-3.‘1" - . .. 1411. 1 |:-W1<1 (‘1;‘1 'n 1 .3 1: “r 1‘ .. I." v’ . : ‘. u |»J-:1"1"1l'l AW ”1“"; $1“ . 41‘3". . y 1.. 1 .412" 1""? S 11.1}- "vi? .1.|1,‘E, 'P.“ :1 1 '. xn.‘;y. 11%;; Vii) * 1 Vil~l> " k ' Ni”. .11 I, '1‘” . '¢ '._ ._, '_ a. :7: r7!" H 1”||"‘.l’ 11..- LIBRAJ: w' 1. a W W, Michigan Stew ' University This is to certify that the thesis entitled STUDIES WITH SELECTED ANTIOXIDANTS IN VEGETABLE OILS presented by Tranggono has been accepted towards fulfillment of the requirements for M. S . degree in Food Science WQM .974 Major profeiér {1:/ Date ? NM). x77? 0-7639 STUDIES WITH SELECTED ANTIOXIDANTS IN VEGETABLE OILS By Tranggono A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1978 ABSTRACT STUDIES WITH SELECTED ANTIOXIDANTS IN VEGETABLE OILS By Tranggono The effect of the antioxidant, 6-hydroxy-2,5,7,8- tetramethylchroman-Z-carboxylic acid (Trolox C) was evalu- ated and compared with currently used antioxidants in coconut, peanut and corn oils. Oxidative changes were determined by peroxide value, conjugated diene absorption and measurement of weight increase. Good correlations were obtained among these methods, particularly between peroxide values and conjugated diene absorption. Trolox C when used alone has been found to be the most effective among the antioxidants tested in both natural aging and accelerated (63°C) tests. The order of effective- ness in decreasing order was Trolox C, TBHQ, BHA or BHT and citric acid. Trolox C when used in combination with BHA or BHT displayed a negative synergism. However, a huge increase in stability of oils was obtained when treated by Trolox C in combination with BHA or BHT and citric acid. ACKNOWLEDGEMENTS The author would like to express his deepest appreci- ation to Dr. Leroy Dugan, Jr., his major advisor in selecting the author's course of study and for his guidance and advice during various phases of the research work. Appreciation and thanks are extended to the members of the committee, Drs. P. Markakis, H.A. Lillevik and T. Wish- netsky for their critical review of this manuscript. The author expresses gratitude to the Midwest University Consortium for International Activities, Inc. (MUCIA) — Indonesian Agricultural Higher Education Project for the financial support for his graduate program. Last, but not least, the author is especially grateful to his wife, Murdiasih and his daughter, Iwing for their sacrifices, patience and encouragement throughout his graduate program. ii TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . LITERATURE REVIEW . Mechanism of Oxidative Rancidity. Metal Catalysts . Measurement of Lipid Oxidation. Peroxide Value. . Conjugated Diene Absorption Method. Weighing Method . . Antioxidants. Mechanism of Antioxidant Action Synergists and Synergism. . . . . . . The Use of Synthetic Antioxidants in Coconut, Peanut and Corn Oils. MATERIAL AND METHODS. Oils. . . Antioxidants and Chemicals. Procedure . . Determination of Peroxides. Conjugated Diene Absorption Iso- octane Purification Weight Gain Technique . . Preparation of Methyl EsterL . Fatty Acid Composition of Oils. . . Free Fatty Acid (FFA) Determination Iodine Value. . . . RESULTS AND DISCUSSION. The Properties of Original Oils . . Gas Liquid Chromatography (GLC) Analysis. Correlations Among Peroxide Value (PV), Weight Gain and Conjugated Diene Absorption (CDA) Tests . . Page vi —l d —l 0000 KO 0‘ bN—‘dOQCDV-h & Nd—J—J The Effect of Type and Concentration of Anti- oxidants Upon Oil Stability. . . Effects of Combinations of Antioxidants Upon Oil Stability. . Room Temperature Storage SUMMARY. . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . . iv Page 37 43 69 71 Table 10 ll 12 LIST OF TABLES Properties of original oils. Fatty acid composition of original oils. Regression equations among PV, weight gain and CDA of oils during storage at 63°C . Correlation coefficients among PV, CDA and weight gain of oils during storate at 63°C . Mean peroxide values (meq/kg) of peanut oil during storage at 63° C . . . . . . . Mean weight gain (%) of corn oil during storage at 63°C. . . . . . . . . . . . . . . . . . Stabilization of peanut oil at 63°C. Stabilization of corn oil at 63°C. Mean conjugated dienS absorption of coconut oil during storage at 63 C for 8 weeks . Mean peroxide value of coconut oil stored at room temperature (20- 28°C) for 24 weeks. Mean conjugated diene absorption of peanut oil stored at room temperature (20— 28°C) Mean conjugated diene absorption of corn oil stored at room temperature (20-28°C) Page 26 28 32 36 39 41 44 49 53 60 62 65 Figure 10 ll 12 13 LIST OF FIGURES Peroxide value vs conjugated diene absorption of oils stored at 63°C. Peroxgde value vs weight gain of oils stored at 63 C . . . . . . . . . . . Conjugated diene absorption vs weight gain of oils stored at 63°C . . . . . . . . Conjugated diene absorption vs time for coconut oil during storate at 63°C. . Pesoxide value of peanut oil during storage at 63 C . . . . . . . . . Conjugated diene absgrption of peanut oil during storate at 63 C . . . . . Weight gain of corn oil during storage at 63°C. Conjugated diene absorption of corn oil during storage at 63°C . . . . . . . . Stabilization of coconut oil stored at 63°C for 6 weeks (measured by PV). Stabilization of coconut oil stored at 63°C for 6 weeks (measured by weight gain) . . Stabilization of coconut oil stored at room temperature for 24 weeks (as measured by conju- gated diene absorption) . . . . . . Stabilization of peanut oil stored at room temperature for 24 weeks (as measured by peroxide value) . . . . Stabilization of corn oil stored at room temperature for 24 weeks (as measured by peroxide value) . . . . . . . . vi Page 33 34 35 4O 47 48 51 52 54 55 61 63 66 INTRODUCTION Coconut, peanut and corn oils are vegetable oils currently used for preparing food in Indonesia. Coconut oil is the oil extracted from the coconut palm (Cocos_ nucifera, Linn.). With an oil yield up to 65 percent, c0pra, the main product of the palm, is perhaps the richest material for vegetable oil extraction. Copra and coconut oil are traditional commodities in the world markets for oilseeds, oils and fats. Total world exports of copra and coconut oil are estimated at about l.45 million tons of oil equivalent which represent a share of approximately l0 to ll percent of the world's total export trade in oils and fats (Thampan, 1975). About two-thirds of the world's peanut crop (Arachis hypogaea) is crushed for oil. Peanuts supply about a fifth of the world's edible oil production and they comprise a third of the world's trade in edible oils and oil bearing materials (Woodroof, l973). Now legumes including peanuts are the crops being recommended by the Indonesian government, therefore peanuts will have a more important role in Indo- nesia in the near future. Corn, known botanically as Zea mays Linnaeus is one of the world's most versatile seed crops. The production of corn only to obtain oil is uneconomical. Corn oil is a by-product of both wet and dry milling (Reiners 33 11., 1970) so that the amount of it available depends on the demand for the other corn products. Coconut oil has both food and non-food uses. It is used as cooking oil, margarine and confectionery. It is also employed in the manufacture of soaps and detergents. ”(Corn_and_psanut_gils are mainly used for ediblg_pgrpgses_such as cook“ '1 ' mar a ' . From a nutritional point of view, peanut and corn oil are important because these oils are rich in essential fatty acids. In recent years, there have been a dramatic increase in the use of corn and peanut oil due largely to the increasing awareness of the importance of polyunsaturated fatty acids in the diet. Al- though coconut oil has a low unsaturated fatty acid content, it has a greater digestibility coefficient and economically is cheaper than those other oils. Coconut, peanut and corn oils areWsusceptible to oxide: tive deterioration. The development of synthetic antioxi- dants has played a vital role in the marketing of vegetable oils by retarding oxidation. Several chemical compounds having antioxidant efficacy in fats and oils have been cleared for use by governmental regulatory agencies. Some new antioxidants have been developed recently, although these have not been released for human consumption. The recent trend toward increased use of vegetable oils in the human diet has emphasized the need for better antioxidant systems than those currently available. The present study was designed to gain knowledge in the effect of new and currently available antioxidants on the stability of coconut, peanut and corn oils. This work was based on the Schaal oven test at temperature of 63°C and storage stability tests at room temperature. Peroxide value, conju- gated diene absorption and weight gain were measured to follow the oxidative changes in the samples. The antioxi- dants used in this work were Trolox C (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), CA (citric acid) and TBHQ (tertiary butylhydroquinone). LITERATURE REVIEW Mechanism of Oxidative Rancidity. Lipid oxidation is very important and of much interest K to the food chemist because it results in the formation of off flavors and odors, destruction of essential fatty acids, formation of brown pigments, and alteration of_pigments and ‘ _jjavors: The common feature of oxidative rancidity is the reactivity of the unsaturated fatty acid moieties in lipids. In a refined fat or oil the oxidation usually proceeds through autocatalytic processes. The term "auto catalysis" refers to a reaction which increases in rate with time due to the formation of products which themselves catalyze the reaction (Dugan, 196l; Lundberg, T962). Oxidation of fatty substances is believed to take place in three stages (Dugan, l962; Sherwin, l974): (:) Initiation: This probably corresponds to the oxidation induction period of a fat or oil, and during this stage fat or oil molecules convert to unstable free radicals which can catalyze further free radical formation in the substrate. This may be depicted as: Initiaterisl, RH R' + H' (fat molecule) (free radical) [Various agents such as light (especially in the UV region) and_heavy metals (particularly copper and iron) are princi- pal initiators of autoxidation. (:) Propagation: Free radicals which have formed can combine with molecular (atmospheric) oxygen to form more radicals and hydroperoxides. This reaction may be depicted as: R' + 02 ------ 9 R00' (peroxy free radical) ROO‘ + RH ------ '> ROOH + R' (hydroperoxide) ROOH ------ ? R0' + 'OH 'OH + RH ------ > R' + H20 During propagation, especially in the presence of catalytic agents, decomposition of tthbverperoxides leads to forma: ktion of a wide varietyiof aldehyges, ketones, acids, etc., responsible for the obnoxious odor and flavor characteristics go: rancid food fats and oils. c) Termination: Termination of the oxidation chain reaction occurs when the free radicals (autocatalysts) are deacti- vated or destroyed. This may occur in various ways such as: R' + ROO' -------> ROOR Various extraneous influences may be present that affect the rate of oxidation. These factors are temperature, light radiation, various metals, metal salts, and organic compound of metals, oxidative enzymes such as lipoxidases and photochemical pigments which act as accelerators in the presence of light (Lundberg, l962). The reaction of RH + 02 ------ 9' ROOH requires a change in total spin, RH and ROOH being in singlet states while 02 is in triplet state, moreover the reaction is endo- thermic by about 64 kcal/mole (Waters, 1946). Both the energy and spin barriers could be overcome however if instead of ordinary triplet state 02, singlet state 02 was the active species. Since the plant pigments could serve as sensitizer, the photosensitized production of singlet 02 is believed to be the mechanism for the production of fatty acid hydro- peroxides. The two most likely mechanisms of photooxidation involve either a biradical moloxide (Schonberg, T935): hv '|S _______ ‘IS-k ______ 9 35* 3 35* + 02 ------ > .s-o-o. ,s-o-o, + RH ------ 9 ROOH + 1s or singlet 02 as the reactive intermediate (Rawls and van Santen, l970): 1s + hv ------ 9 15’“ ------ -> 35* 3 * 'I * I S + 302 """ ? 02 + S 'l * o + RH ------ a ROOH ROOH ------ ‘) free radical product. S is the sensitizer, the superscript refers to the spin multiplicity and the asterisk indicates electronic excita- tion. In the structure of chlorOphyll, a five membered ring condensed to the porphyrin system contains a carbonyl group. Rawls and van Santen (1970) reported that the chlorophyll reaction involved the non singlet O2 oxidation mechanism since electronically-excited carbonyl groups are known to be very effective proton abstractors and since the visible light is sufficient to excite the carbonyl n ------ > H * state. The following mechanism was proposed: chl + hv ------ 7 ch1* = n ------ > 11* excited state of the carbonyl group. chl* + RH ------ ; R"+ (chlorophyll product) R + 02 """" 9 R02 R02° + RH ------ 9 ROOH + R' CEEtal Catalysts:) Heavy metals, particularly those possessing two or more valency states with a suitable oxidation - reduction potential between them (e.g. Cu, Co, Fe, Mn, Ni, etc.), generally WW _:gts and oils. 4 Swern (l964) stated that,cgppgg_in particular, is a very strong prooxidant, being effective in a concentration of much less than one p.p.m. Tappel (1955) listed copper as a well known catalyst for the oxidation of unsaturated fats. Cooney _t 31. (1958) stated that both copper and iron were potent oxidative catalysts in cottonseed oil. Vioque gt 31. (1965) demetalized olive and soybean oils by passing them through columns packed with cation exchange resins. This lowered the trace metal content and increased the stability of the oils. Mertens gt 11. (1971) studied trace metals and the flavor stability of margarine. Levels of 0.1 p.p.m. copper led to rapid flavor deterioration. In order to expect good flavor stability, the maximum copper amounts which can be tolerated are about 0.02 p.p.m. Berger (1975) stated that variation in metal content accounts for stability and anti- oxidant activity variation in many fats and oils. Measurement of Lipid Oxidation. The acceptability of a food product depends on the extent to which the oxidative rancidity has occured. Thus some criterion for assessing the extent of oxidation is required. This can be followed by determining the total consumption of oxygen, by determining the amount of a product of lipid oxidation or by measuring the decrease in the concentration of unsaturated lipids. Many methods have been developed and among these are peroxide value, conjugated diene absorption and weight gain methods. Peroxide Value. The primary products of lipid oxidation are hydro- peroxides which are generally referred to as peroxides. Therefore it seems reasonable to determine the concentra:_ tion of peroxides as a measure of the extent of oxidation. The iodometric methods of Lea (1931) and Wheeler (1932) are widely used, and these are based on the measurement of the iodine produced from potassium iodide by the peroxides present in the oil. The iodine, liberated in a stochiometric ratio of two atoms of iodine for each atom of active oxygen, can be quantitated by titration with sodium thiosulfate. According to Mehlenbacher (1960), the two principal sources of error in these methods are (a) the absorption of iodine at unsaturated bonds of the fatty material, and (b) the liberation of iodine from potassium iodide by oxygen present in the solution to be titrated. Lea (1931) attempted to eliminate this error by filling the sample tube with nitrogen at the beginning of the test and assuming that the vaporization of chloroform thereafter would prevent the reentry of oxygen into the tube. Wheeler (1932) used a homogenous solution in an attempt to eliminate the need for shaking thereby minimizing the effect of oxygen. Conjugated Diene Absorption Method. Oxidation of polyunsaturated fatty acids is accom- panied by increased ultraviolet absorption due to the forma- tion of conjugated diene and triene hydroperoxides. Fatty 10 acids with conjugated unsaturation absorb strongly in the region 230 to 375 nm, diene unsaturation at 233 nm and triene unsaturation at 268 nm. The magnitude of change is not readily related to the degree of oxidation because the effects upon the various unsaturated fatty acids vary in quality and magnitude. However, the changes in the ultra- violet spectrum of a given substance can be used as a rela- tive measurement of oxidation (Gray, 1978). Oils containing linoleate or more highly unsaturated fatty acids are oxidized to conjugated diene systems that can be measured by ultraviolet absorption at 233 nm. Farmer and Sutton (1943) indicated that absorption increased propor- tionately to the uptake of oxygen and to the formation of peroxides in the early stages of oxidation. Angelo gt 31. (1975) studied the autoxidation of peanut butter by measuring the peroxide value and the increase in absorption at 234 nm due to diene conjugation. They conclu- ded that the conjugated diene hydroperoxide (COHP) method can be used as an index of progressive staling in place of, or in addition to, the peroxide value. The CDHP method is faster than the peroxide value method, is much simpler, requires no chemical reagents, does not depend upon chemical reaction, and can be conducted on smaller samples. This method is applicable for the analysis of peroxides in vege- table oils produced containing polyunsaturated fatty acids. 11 Weighing Method. The technique of following the rate of oxidation of oils by weighing small samples at intervals during storage has been used from time to time for at least 75 years (Olcott gt gl., 1958). They found the weighing procedure to be a convenient method for estimating the relative effectiveness of antioxidants in marine and other oils and purified fatty esters. The procedure involved weighing the oil into beakers and the additives are then put in as ali- quots of solutions in volatile solvents. In constant draft oven at 50 or 60°C the solvents are removed quantitatively as judged by constant weight in subsequent weighing. Fuku- zumi and Ikeda (1969) used a vacuum desiccator at constant temperature (30 i 1°C) for 1 hour, at 1 mm Hg pressure to remove the solvents. Fukuzumi gt g1. (1976) studied the effect of new anti- oxidants, such as phenothiazine derivatives on the autoxi— dation of methyl linoleate and used the weighing method to estimate the induction period. They found that the induction period evaluated from the weighing method gives almost the same value as that from the peroxide value. Antioxidants. Antioxidants are substances capahlp of slguiflg_thg_niig‘ _9f oxidation in autoxidizable material, The choice of an antioxidant for a given purpose is governed by the require- ment of the system and the characteristics of the antioxidants 12 available. Desirable features of an antioxidant include the 'following: it must be effective at low concentrations, non- toxic, convenientlv and safely handled. and low in cggtl and it must not impart undesirable characteristics to the N system in which it is used (Dugan, 1963). #— All antioxidants are structurally similar in that they contain unsaturated benzene rings plus either hydroxy or amino groups. Stuckey (1962) divided antioxidants into phenols, amines and aminophenol groups. Most natural and synthetic food grade antioxidants belong to the phenolic class of compounds. Although the presence of hydroxy or amino groups on the aromatic ring is necessary for antioxidant activity, the potency of a given compound can be greatly enhanced by the introduction of certain substituents into the proper position on the aromatic nucleus. Morawetz (1949) and Thompson and Symon (1956) showed, after evaluating many phenolic compounds, that alkyl substitution in the ortho and para positions greatly enhance the potency of a given compound. The addi- tion of the tertiary butyl group in the ortho position seems to be particularly effective in this respect. Mechanism of Antioxidant Action. Observations from a number of studies have indicated that more than one type of action may occur depending upon the conditions of the reaction and the type of system being studied. Dugan (1963) noted that antioxidants might be 13 considered to function in two ways, either as inhibitors of free radical formation or as peroxide decomposers. _Antioxidants that function as free radical inhibitg:s LLSECt with free ragigals to_£nrm inert products as_jg_gfl termination stepgjg_the chain reaction mechanism. Studies by Bolland and Ten Have (1947) led to a proposal of a simple mechanism in which the antioxidants acted as hydrogen donors ‘ or free radical acceptors. From the kinetics of the reac- tion, it appeared that the free radical acceptors (AHz) react primarily with R02' and not with R', as follows: ROZ' + AH2 ------ #1 ROOH + AH° AH' + AH' ------ a A + AH2 Boozer gt g1. (1955) proposed a different mechanism, invol- ving complex formation, as follows: R0 ' + AH ------ > (R0 2 2 “‘2" 2 (ROZAH2)' + R02‘ ------ 9 stable product. The peroxide decomposers act as catalysts to decompose peroxides initially present as well as those that are formed during further oxidation. An important feature of this decomposition process is that the primary stable products are not free radicals. This naturally rules out the decom- position of peroxides by metals such as copper, cobalt and iron (Dugan, 1963). 14 Synergists and Synergism. The term synergism refers to the cooperative action of two or more agents in such a way that the total effect is greater than the sum of the individual effects taken inde- pendently (Dugan, 1963). The original description of anti- oxidants and synergists (Olcott and Matill, 1936) differen- tiated between substances which were effective alone in . relatively low concentration (tocopherols, phenols) and those which had little activity by themselves, but were effective in combination with the phenolic inhibitors. They were called synergists (citric and ascorbic acids, phospho- lipids, etc.). It has been well demonstrated that the chelation of metals is one of the principal mechanisms involved, where one of the antioxidants is a metal chelating agent. Kray- bill _t _l. (1949) showed that BHA exhibits synergism with certain acids, including citric, as well as with hydro- quinone, methionine, lecithin, and thiodipropionic acid. Citric acid was more effective against iron and nickel than against copper, and ascorbic acid was effective against copper but ineffective against iron (Morris gt gl., 1950). Cowan gt gt. (1962) with soybean oil and lard, showed that both sorbitol and citric acid were acting as a metal inacti- vator and did not have a true synergistic effect. Citric acid readily forms stable complex salts with many metallic ions (Lockwood and Irwin, 1963), thus it is able to retard 15 the increasing rate of free radical formation. Zeldes and Livingston (1971) identified three radicals while studying paramagnetic resonance spectra of radicals present during photolysis of aqueous solutions of citric acid. Smith and Dunkley (1962) showed ferrous ion was more effective than ferric ion in the peroxidation of linoleate. It was proposed that a perhydroxyl radical was produced by the reduced metal ion. Strouse gt gt. (1977) reported that triionized citrate formed a tridentate chelate with Fe (II) in which the protonated hydroxyl group, the central carboxyl group, and one terminal carboxyl group are coordinated to a single Fe (II) ion. Both oxygen atoms of the other terminal carboxyl group were coordinated to two other symetry-related Fe (II) ions. Hexaquoiron (II) was the counter ion. Cort gt g1. (1975) stated that it was not the metals per se, but their oxidation state which were important. Ascorbate converted Fe3+ to Fe2+ and Cu2+ to the lower oxi- dation states. It was the higher oxidation which reacted with tocopherol, Trolox C and ascorbic acid. Berger (1975) reported that ascorbic acid had some antioxidant activity and possibly it acted as a somewhat inefficient free radical scavenger. On the other hand, citric acid was particularly valuable as a chelating agent. Synergism has been produced also in fat by a combina- tion of two antioxidants (Mahon and Chapman, 1953; Dugan t 1., 1954). There are some theories regarding to syner- gism. These may be as metal scavenger, peroxide decomposer, 16 and sparing agents, as in the interaction of phenolic anti- oxidants or the interaction of other agents with phenolic antioxidants (Dugan, 1963). Ikeda and Fukuzumi (1977) with methyl linoleate showed that nucleic acid acted as a syner- gist with tocopherol through H bonding which protected tocopherol from direct air oxidation. The Use of Synthetic Antioxidants in Coconut, Peanut and Corn Oils. The effect of various antioxidants on the stability of coconut, peanut and corn oils has been reported by many researchers. Dugan gt gt. (1950) reported that BHA when used alone and in combination with propyl gallate and citric acid had been found useful in preventing rancidity in frying oils such as corn oil and peanut oil and in the food prepared in these oils. The addition of citric acid during the cooling stage of deodorization or of monoisopropyl citrate (Gooding gt gl., 1950) or monoglyceride citrate (Brown and Gooding, 1955) following deodorization negates the prooxidant effect of trace metal to a large degree, principally that of traces of iron and copper, which occur in all vegetable oils. The effect is to reestablish the inherent stability of vegetable oils owing to the naturally occuring antioxidants contained in vegetable oils. Tollenar and V05 (1958) in an extended study found a small protective factor with octyl gallate in various 17 vegetable oils tested at 100°C and 34°C. Their room tem- perature storage test with BHT and gallates in various com- binations in corn oil showed that these antioxidants under these conditions had no effect. They also mentioned the reason why animal fats yield considerably better results with antioxidants than vegetable oils as follows: a) Vegetable oils generally have a higher iodine value than animal fats. Antioxidants however exercise a stronger influence on the oxidation of oleic esters than on linoleic and linolenic esters. Although oxidative rancidity does not constitute a major problem for coconut oil, the positive action of phenolic antioxi- dants on this vegetable oil with relatively low unsatu— ration has been demonstrated in storage tests. b) Vegetable oils contain many more natural antioxidants than animal fats. The supplementary addition of antioxidants has less effect in the vegetable oil group. c) Fats as a rule are packed entirely differently from oils, packed fat frequently has unlimited quantities of oxygen available. d) The natural flavor constituents in vegetable oils and fats are entirely different from those present in animal fats and react differently to the addition of antioxi- dants. Fritsch gt _1. (1971) demonstrated that the addition of BHA, BHT and citrate to coconut oil increased the ADM 18 stability to about 350 hr even though the initial stability was 30 or 250 hr. These results suggested that the high resistence to oxidation of coconut oil might not be entirely due to its low level of unsaturation but due to the presence of natural antioxidants in varying amounts. Semiquantitative GLC showed no difference in the amount of tocopherols in coconut oils with low and high stabilities. Thewalt gt g1, (1969) reported detecting phenols other than tocopherols in amounts up to 100 p.p.m. in coconut oil samples. MATERIAL AND METHODS Oils. Coconut, peanut and corn oil were used for this study. Coconut oil was obtained from PVO Internationallnc. Boonton .‘—_..» N.J. 07005, {drimfijlhffdm'CPCMInternatiohal Inc. Argo Ill. 60501 while Shedd's pure peanut oil was purchased from a local store. All of the oils were refined and had no added antioxidants. Antioxidants and Chemicals. Five antioxidants were used for this study. These were Trolox C, a trivial name for 6-hydroxy-2,5,7,8-tetramethyl- chroman-Z-carboxylic acid (Roche Inc); BHA for butylated hydroxyanisole: a mixture of 2- and 3-tert-buty1-4-hydroxy- 1-methoxy benzene (Eastman Kodak Company); BHT for butylated hydroxytoluene: 3,5-ditert-butyl-4-hydroxy toluene (Ashland Chemical Company); Citric acid monohydrate (Fisher Scientific CompanY); and TBHQ for 2-tert-butyl hydroquinone (Eastman Kodak Company). All the reagents used during analysis were analytical grade. Procedure. In each series, 50 g of the oils were weighed into beakers. Some served as control and accurately measured 19 20 amounts of the antioxidant solution in absolute alcohol were added to others. The oil was stirred by magnetic stirrer for ten minutes at room temperature to assure the homogenous distribution of the antioxidants, then filled into weighed petticups. Solvent was carefully removed in a vacuum oven at a constant temperature of 30°C and pressure of 1 mm Hg, as judged by constant weight in subsequent weighing. After they were weighed accurately, the samples were placed in a constant temperature oven at 63°C. Other samples were stored at room temperature for long term storage. Peroxide value, conjugated diene absorption and weight gain determinations were made to follow oxidative changes in the samples. Determination of Peroxides. Peroxides in the oils were measured by the iodometric method of Wheeler. The petticups containing oil were removed from the oven, cooled in a desiccator, weighed and transferred into a 200-300 ml Erlenmeyer flask where the samples were dissolved in 30 ml of glacial acetic acid - chloroform solution (3/2, v/v). Then 0.5 ml of saturated potassium iodide solution was added and swirled to provide mixing. After two minutes, 30 m1 of distilled water was added and the sample was titrated with standardized sodium thiosulfate solution. Starch indicator (1 percent, w/v) was added when near the end point. The solution was titra- ted to disappearance of the blue color. 21 The peroxide content was determined as meq/kg of sample. Conjugated Diene Absorption. About ten mg of oil were weighed accurately into small petticups and placed into test tubes. Ten ml of pure iso- octane (2,2,4-trimethyl pentane) was added to the sample and the oil was shaken in a Fishernfini shaker to assure complete dilution. Conjugated diene absorption was measured at 233 nm by a Beckman DU spectrophotometer using pure iso- octane as a blank. If the absorption was too large, dilu- tion was accomplished by taking 1 m1 of sample solution and mixing with 9 ml of pure iso-octane in order to bring the absorbance within the proper limits. Conjugated diene ab- sorption was calculated at a dilution of 1:1000 (w/v). Iso-octane Purification. The iso-octane used in the spectral determination of conjugated diene absorption should have an absorbance at 233 nm of not more than 0.07 when compared with distilled water. In order to conform to this requirement, the iso- octane was purified by passage through a 50 cm column of silica gel. Weight Gain Technique. This simple method is based on the concept that oxy- gen absorption is accompanied by a finite gain in weight of the oil undergoing the oxidative process. In this 22 technique, the petticups containing oil were removed from the oven, allowed to cool in a desiccator for 10 minutes, weighed and then placed in the oven. Preparation of Methyl Esters. Methyl esters were prepared by a rapid procedure des- cribed by Metcalfe gt gt. (1966). A total of 4 ml of 0.5 N methanolic NaOH was added to approximately 150 mg of oil in a 50 m1 volumetric flask. This mixture was heated in a steam bath for about 5 minutes until the oil went into solution. A total of 5 ml BF3-methanol was added to the flask and the mixture was boiled for 2 minutes. Enough saturated NaCl solution was added to the flask to float the methyl esters up, then the entire mixture was transferred into a separatory funnel. About 20 ml of petroleum ether (b.p 30-60°C) was added to the separatory funnel and the funnel was shaken vigorously for 1 minute and the layers were allowed to separate. The lower aqueous layer was drained off and discarded. The petroleum ether layer was drained through filter paper into a 50 ml beaker. The sol- vent was then evaporated on a 60°C water bath or removed by a gentle stream of air at room temperature. The esters were then ready for GLC analysis. Fatty Acid Composition of Oils. Chromatographic analysis of methyl esters was performed using a Beckman GC-5 equipped with a hydrogen flame detector. 23 A coiled stainless steel column 1/8 in x 6 ft packed with 10% DEGS-PS on 80/100 Supelcoport (Supelco Inc.) was used for methyl ester separation. The column oven temperature was 160°C, the injection temperature was maintained at 185°C and the detector at 205°C. The nitrogen carrier was adjusted to 27 m1/minute. The flow rate of hydrogen and oxygen were 26 ml/minute and 250 m1/minute respectively. The emerging peaks were identified by comparing retention time to those of standard mixtures of known fatty acid methyl esters. These standard were Supelco F&OR mix #1 for corn oil, F&OR mix #3 for peanut oil and F&0R mix #5 for coconut oil. Peak areas were calculated by multiplying peak height time peak width at half height and the percentage of total fatty acids were determined. Free Fatty Acid (FFA) Determination. The Official AOCS method (1974) was used for free fatty acid determination. In this method, 50 g of sample was weighed into an Erlenmeyer flask. Fifty ml of hot neutralized alcohol and 2 m1 of phenolphthalein (1% in 95% alcohol) were added to the solution. The solution was titrated with 0.1 N standard NaOH to the appearance of the first permanent color which must persist for 30 seconds. The percentages of free fatty acids in corn and peanut oils were calculated as oleic acid while in coconut oil it was expressed as lauric acid. 24 Iodine Value. The Hanus method was used for the iodine value deter- mination. The iodine value is a measure of unsaturation and is expressed in terms of the number of centigrams of iodine absorbed per gram of sample. The Hanus solution was prepared by dissolving 13.2 g of iodine in 1 liter of glacial acetic acid (99.5%) and enough bromine was then added to almost double the halogen content. In this method 0.1 - 0.2 gram corn or peanut oil or 0.5 gram coconut oil was placed in a dry 500 ml glass stoppered flask containing 10 m1 of chloroform. Twenty five ml of Hanus solution was added and the solution was allowed to stand for 30 minutes in the dark with occasional shaking. Ten ml of 15% potassium iodide was added, shaken and followed by the addition of 100 ml of freshly boiled and cooled water washing down any free iodine that might be on the stopper. The solution was titrated with 0.1 N Na2S203 until the yellow color had almost disappeared. The starch indicator was then added and the titration continued until the blue color had disappeared entirely. A blank determina- tion was conducted simultaneously with that for the sample. RESULTS AND DISCUSSION The oils used in this study were refined and had no added antioxidants. The majority of oils are usually marketed in refined condition thus it seemed appropriate to study the effect of antioxidants on refined oils. The results presented in this study were performed at least duplicate determinations. The Properties of Original Oils. The properties of original oils in terms of free fatty acids content, iodine value, peroxide value and conjugated diene absorption are shown in Table 1. All samples exhibi- ted 1ow free fatty acids content. Among these samples, corn oil exhibited the highest iodine value while peanut and coconut oils had the medium and the lowest iodine values respectively. The data agree with the literature values reported by Swern (1964). The susceptibility of fats and fatty acids to oxidation is asso- ciated with the presence of unsaturated bonds. This reaction leads to the formation of primary, secondary and tertiary oxidation products which may make the oils unsuitable for consumption. Thus corn oil was more susceptible to oxida- tive deterioration than peanut oil which in turn was more susceptible than coconut oil. 25 26 .A>\zv ooo_ F L0 =o_p=_Fn a “a umpapau_oo. ¢N_.o m.m om.op eeo.o aseooou NFN.o ¢.¢_ m¢.~m «No.o uzcmma wmm.o ~.~_ .m.o__ Nmo.o eaou .5: mmm pa compaaomna Am¥\amsv Au\muv Aav wcmwo umummzncou m=Pm> mcwxogma m=Fm> mcwvom muwum qumm omen mpwo mpwo _m:wmwgo mo mmwugmaoga .F mpamh 27 The degree of oxidation that has taken place in fats and oils can be expressed in terms of peroxide value and conjugated diene absorption. As shown in Table 1, all samples exhibited substantial amounts of peroxides. These indicated that the oxidative process had been started. It was noted that although corn oil had a lower peroxide value than peanut oil it exhibited higher conjugated diene absorption. Gas Liquid Chromatography (GLC) Analysis. The original oils were also analyzed for fatty acid composition by gas liquid chromatography as shown in Table 2. The data show that the coconut oil contained appreci- able quantities of saturated °8:O to °14:0 fatty acids, whereas the corn and peanut oils contained low levels of saturated fatty acids but high levels of C18 unsaturated fatty acids. In the coconut oil, lauric and myristic made up over 75% of the total acids. It contained also caproic, capric, caprylic, palmitic, oleic and linoleic. The fatty acids detected were in agreement with the data reported by Sreenivasan (1968). The small differences in the percen- tages of each acid may be due to the difference in growth conditions or varieties of coconut trees. Worthington and Hammons (1977) studied variability in fatty acid composition among peanut genotypes. They repor- ted that the two major fatty acids, oleic and linoleic 28 Table 2. Fatty acid composition of original oils Fatty acid (%) Fatty acid* Coconut oil Peanut oil Corn oil 8:0 2.9 - - 10:0 3.3 - - 12:0 53.6 - - 14:0 21.9 - - 16:0 8.1 10.9 12.1 18:0 2.3 1.5 1.8 18:1 6.3 55.1 28.8 18:2 1.6 27.7 56.2 18:3 - 0.8 0.8 20:0 - 0.8 0 3 22:0 - 1.9 - 24:0 - 1.4 - *The notation used to describe fatty acids is number of carbon atomznumber of double bonds. 29 ranged between 36-69% and l4-40%, respectively, and together made up 75-85% of the total fatty acids. The very long chain (°20'°24) fatty acids made up 4-9%, palmitic acid 7-13%, and stearic acid 2-5% of the total fatty acids. The fatty acid composition of peanut oil as shown in Table 2, agrees with the values reported by Worthington and Hammons (1977). As shown in Table 2, the corn oil contained a high percentage of linoleic acid. It contained also palmitic, stearic, oleic, linolenic and arachidic acids. Linoleic and oleic acid made up 85% of total acids. These data were in agreement with the range of values tentatively adopted by the Food and Agriculture Organization/World Health Organi- zation Codex Alimentarius Committee on Fats and Oils (Spencer and Herb, 1976). Correlations Amogg_Peroxide Value (PV), Weight Gain and Conjugated Diene Absorption (CDA) Tests. Peroxide value (PV) determination is the most widely used chemical method to measure oxidative rancidity of fats and oils. Oxidation of polyunsaturated fatty acids produces peroxides and the position of the double bonds shifts to a conjugated form. Conjugated linkages absorb light in the ultraviolet region of the spectrum and have absorption maxima at the wave length of 233 nm. Although the conjuga- ted diene absorption (CDA) test has not been adopted forda'y to day quality control in the food industry, its usefulness for 30 oil products has been indicated. Some researchers use the weight gain procedure as a convenient method for estimating the extent of oxidative rancidity. Fukuzumi and Ikeda (1969) and Ikeda and Fukuzumi (1977) used the weighing method, UV and IR spectra to follow the extent of autoxidation. They found that the induction period evaluated from the weighing procedure gave almost the same value as that from the peroxide method. Ke _t _l. (1977) used weight gain, PV, TBA and free fatty acid tests to follow the oxidation rate while studying the potency of antioxidants on mackerel skin lipid as the tested model system. Angelo _t _t. (1975) plotted PV against the correspon- ding conjugated diene hydroperoxide (COHP) values to test for possible correlation between the two methods in deter- minations of shelf life stability of peanut butters. They found a linear relationship between CDHP and PV values with a high correlation coefficient which indicated that the results from the two methods were highly correlative. The linear regression equation had a negative intercept and positive slope. To test for possible correlation among PV, weight gain and CDA methods in this study, the results from one method were plotted against the corresponding values of the other methods measured on the same sample of oil stored at 63°C (Figure 1, Figure 2 and Figure 3). The linear regression 31 equations for the three methods are shown in Table 3. It was noted that the regression equation between PV and CDA had a negative intercept and positive slope thus it was consis- tent with the finding reported by Angelo gt gt. (1975). Table 4 shows the correlation coefficients among PV, weight gain and CDA measured on coconut, peanut and corn oils stored at a temperature of 63°C. High correlations were demonstrated between PV and CDA of all samples observed. The correlation between PV and weight gain as well as CDA and weight gain were high enough but weaker than the corre- lation between PV and CDA. These weaker correlations may be due to the vaporization of volatile oxidation products which affected the measurements made by the weight gain method. As mentioned, the present study has demonstrated that the CDA test is more sensitive and correlates better with peroxide development than that of the weight gain method in vegetable oils. The high correlations observed between PV and CDA may be explained, in part, by the suitability and accuracy of the CDA method for monitoring the oxidatiVe changes in place of or in addition to PV. The advantages of CDA over PV are that it is faster and simpler, it does not depend on chemical reaction or color development and can be carried out on much smaller samples. It was interesting to note that the correlation coef- ficient between PV and CDA of corn oil was higher than that of peanut oil which in turn was greater than that of coconut 32 x _¢.m + mo.p u > x mo.mom + mm.mv u > x mm.mm + ¢~.m~u u > csoo x mm.~ + ¢¢.o u > x m_.¢¢~ + pm.mF u > x vw.- + P¢.o_1 u > panama x mm.~ + -.o u > x mm.ope + mu.“ u > x Fv.F~F + mm.o—u u > uncooou =_am u;m_mz m) >a <98 m> >a .Po game so mmmgoum mcpgau mPPo #0 (cu use even pgmwmz .>m mcosm mcowumacm :owmmmgmmm .m mpnmh 33 .uomo um umcoum meo Lo cowuaLOmnm mcwmu umummahcou m> mapm> muwxocma E: mmm um :owuagomnm m:mwu umpmmzncou m m o m w m N P - 1 q q q q d - on .wo uncoooo .u . 4 A .Fo.o v a .0de «meme mpacppaz mcmmz mo goggm usmucmum HmoNNP thmoNN m%0m.©m flmcpN flN.¢P NN0.0 HIM m¢.mom xnwm.om gmm~.~m nm~.¢~ ao.mp xpo.o hxm L¢.mmp P~.mm gmmm.mo me.p~ u~.¢_ xmo.o (:m cuan.nm mm.- m<.om mm.mp am.¢_ xmo.o u xopoge a~.-~ wcmp.no mm.m~ mo.mp mo.mp xpo.o u xoposp om.o~¢ :¢.mm~ E¢.FNP mmvm.m¢ mm.¢~ Fogpcoo e m N F o 1 -, Mlmwzv as P - acmuwxowpc< mx w .uomm pm mmagoum acmgsc Pwo vacuum mo Amx\cmsv mmzpm> muwxogma :mm: .m mpamp 40 .uomm um mmmgopm mcwcau Fwo pzcouou com memo m> :o_pagomnm mcmwu umummsncou .e mgamwu 3:003. NSC. e N o m . . o 3.. Q VIN 898 1' BONVSUOSBV d I 8.0 FIG I pod #20 fl «0.0 (In x pod (In R «0.0 0 ~29... fl rod 0 x295. 2.02.3.2: 02 ‘ o elooo '1 41 .Po.o v a Agate magma m_acu_=z 3oz m.:mo::o ADV acmcmmwww xpgcmowepcmwm we: «gm: mgmppmr cossou mcwgmgm mmspm> u uam .Nmmo.o u mcmmz mo Logsu cgmvcaum epqo._ Lamkm.o numow.o memo.o amo.o sz sepa.~ m¢-.o au¢_m.o unammp.o Npo.o Hzm zomo._ Lam¢.o uumpm.o m_¢o.o amo.o «In Pmmm.P mmkk.o akmm.o ammmF.o spo.o <=m xm~¢.o Lampa.o ammo.o aomo.o NNo.o u xopoah comm.F come.o uaomp.o naomo.o Npo.o u xo_oa» neme.m .mmm.~ LmNo¢.o oammp.o Scauwxo.u=a oz 1 a .1 1m , , N P “cauwxo.p=< Amxmmzv mswh .uomo um mamgoum mcwcsu Pwo cgoo Lo ARV cwmm pgmwmz cum: .0 mpnmp 42 The difference between samples containing Trolox C at 0.01% and 0.02% was not significant for the first three weeks. There was also no significant difference in inhibi- tory effects between BHA 0.01% and 0.02% or BHT 0.01% and 0.02% for the first three weeks. It was noted that the concentration of 0.01% for these antioxidants seemed to be optimum in retarding the peroxidation of peanut oil. As measured by peroxide method, Trolox C had greater efficacy than either BHA or BHT, which confirmed the results obtained by Cort ££.il- (1975). Figure 4 displays the development of conjugated diene absorption measured on coconut oil during storage at 63°C over a period of 8 weeks. As can be seen, the control sample had the highest rate of conjugated diene absorption development. The sample containing Trolox C had higher stability than that containing either BHA or BHT. The effects of antioxidant concentration were clearly noted for all antioxidants used after a six weeks storage period. Table 6 shows the mean weight gain of corn oil during storage at 63°C. for four weeks. Analysis of variance indi- cated a highly significant difference between treatments. As can be seen, the antioxidants used had inhibitory effects to various degree upon oxygen uptake as indicated by lower weight gains than that of the control sample. As in pea- nut oil, Trolox C had better efficacy than either BHA or BHT in corn oil. As the concentration increased, the in« hibitory effect also increased, It was noted that the 43 effect of antioxidant concentration was more significant in corn oil than in peanut oil. As mentioned, all antioxidants used exhibited higher effectiveness as the concentration increased. The use of antioxidants in concentration as small as possible is an important consideration in applying the antioxidants to vegetable oils. BHA and BHT are very soluble in oils. The solubility of Trolox C in peanut and corn oils is 0.18 g/100 9 while its toxicity as measured by L050 is greater than 1,000 mg/kg of body weight in mice, rats and rabbits (Cort et al., 1975). The maximum concentration of anti- oxidants permitted in vegetable oils are defined by regu- lations. BHA and BHT are food approved antioxidants, while Trolox C has not been released for human consumption. This study established that the concentration of 0.01% for Trolox C in vegetable oils gives good results. Effects of Combinations of Antioxidants Ugon Oil Stability Trolox C, BHA, BHT and citric acid were used for this study and evaluated at a temperature of 63°C. Peroxide value, conjugated diene absorption muiweight gain were mea- sured on samples during the storage period. Table 7 shows the stabilization of peanut oil at 63°C. as measured by the weight gain method. Figure 5 exhibits the development of peroxide value in peanut oil over a per- iod of four weeks. Figure 6 displays the change of conjuga- ted diene absorption observed in the same samples. As can be seen, a similar pattern was obtained. 44 Table 7. Stabilization of peanut oil at 63°C. Oven Protective Syner- Antioxidant days* factor gism (days) 1. No antioxidant 13 1.0 2. Trolox C 0.01% 22 1.7 3. BHA 0.01% 17 1.3 4. BHT 0.01% 18 1.4 5. CA 0.005% 14 1.1 6. Trolox C 0.01% + BHA 0.01% 23 1.8 -3 7. Trolox C 0.01% + BHT 0.01% 23 1.8 -4 8. Trolox C 0.01% + BHA 0.01% + CA 0.005% 41 3.2 +14 9. Trolox C 0101% + BHT 0.01% + CA 0.005% 44 3.4 +16 *days to reach a weight gain of 0.2% Protective = Stability of the sample containing antioxidant factor Stability of the control sample 45 Rancid odor was noted in peanut oil when the weight gain reached 0.2%. This point was equivalent to a peroxide value of about 70 meq/kg and conjugated diene absorption of about 1.03. As indicated in Table 7, all antioxidants and their combinations had inhibitory effects to various degrees upon oxidative deterioration in peanut oil. The protective factor was calculated by the ratio of the stability of the sample containing antioxidant and the stability of the control sample, thus the higher the protective factor the greater the effectiveness of the antioxidant. Cort _t _l. (1975) revealed that 0.02% Trolox C in thin layer test at temperature of 45°C had two to four times the antioxidant activity of BHA and BHT in vegetable oils and animal fats. As can be seen from Table 7, the peanut oil treated with 0.01% Trolox C displayed good stability and required 22 days to reach a weight gain of 0.2%. The protective factor of Trolox C was 1.7. The peanut oil treated with BHA and BHT exhibited lower stability and required 17 and 18 days respectively to reach the same point. The protective factor of BHA was 1.3 while that of BHT was 1.4. Thus Trolox C, in the petticup test at 63°C in peanut oil, had 1.2 to 1.3 times the antioxidant activity of BHA and BHT. These were not as high as the values reported by Cort gt gt. (1975) and may be due to the higher temperature used as well as lower concentrations of antioxidants. It was postulated by Cort gt gt. (1975) that high temperature destroyed part of the efficacy of 46 Trolox C. Cort _t _l. (1975) showed synergism of Trolox C in lard and soybean oil with ascorbic acid and ascorbyl palmi- tate as measured by the active oxygen method. Ascorbic acid is not soluble in oil but very active. The lack of solubility actually enhances the stability of ascorbic acid in oil. Ascorbates convert Fe3+ to Fe2+ and Cu2+ to the lower oxidation states, thus the conversion of Trolox C to its quinone form which is catalyzed by these trace metals, can be inhibited. As shown in Table 7, 0.01% Trolox C showed a negative synergism in combination with either 0.01% BHA or 0.01% BHT in peanut oil. The addition of 0.005% citric acid provided a positive synergism. The citric acid probably had a role similar to that of ascorbic acid. As displayed in Figure 5, the control sample exhibited the highest rate of peroxide development with time, which in turn was followed by the samples containing citric acid, BHA, BHT and Trolox C respectively. Trolox C in combination with BHA had a slightly higher rate than when in combination with BHT. Trolox C in combination with either BHT or BHA and citric acid had the lowest rate. A similar trend was noted, when it was evaluated by conjugated diene absorption (Figure 6). The order of antioxidant efficacy in decreasing order was Trolox C, BHT, BHA and citric acid. Table 8 exhibits stabilization of corn oil at 63°C based on peroxide measurement. The stability was calculated 47 .uomo “a mmmeopm mcwcsv P_o pzcmma Lo m:_m> mumxogma an; a m2; 0 n N w b P .P b $000.0 (0 + 890.0 5.26 + $3.0 03—20. R0000 (0 .7 R30 (10 .o. R 3.0 O 3203—. R 3.0 #10 + I «0.0 0 £30.... 890.0 (In .7 $90.0 One-eh 8000.0 (0 R 3.0 #30 $5.0 (In $90.0 O “0.0.... “000.3393 02 440. 0.0.0 .IIIII|I|I|III.II.IIII. \HM . 000 (Bx/bow) anm aauxouaa .m weaned 48 .uomo um mangoum m=_L:0 F00 “3:000 00 cowuagomam m=0_0 umummancou no acacia w}: 13 a 3005+ 50.0 50+ a .00 0.3.0.: «0090 <0 + a .00 $3... a 6.0 o 6.0: «.00 50+ a 5.0 o 3.0.» R50 (:0 +flp00 0 :29; x m000 (0 u 5.0 #10 x '00 (In x 5.0 o 3.0:. .§0_x0_.:< 0: 0 0 OIOOOC .06 “N 883 1' NOLLdHOSBV 3N3“! OBLVON‘NOO .o mg:m_u 49 Table 8. Stabilization of corn oil at 63°C. Oven Protective Syner- . . days* factor ggsm ) Ant1ox1dant ays 1. No antioxidant 7 1.0 2. Trolox c 0.01% 15 2.1 3. BHA 0.01% 12 1.7 4. BHT 0.01% 10 1.4 5. CA 0.005% 8 1.1 6. Trolox C 0.01% + BHA 0.01% 16 2.3 -4 7. Trolox c 0.01% + BHT 0.01% 16 2.3 -2 8. Trolox C 0.01% + BHA 0.01% + CA 0.005% 18 2.6 -3 9. Trolox C 0.01% + BHT 0.01% + CA 0.005% 18 2.6 -1 *days to reach a peroxide value of 70 meq/kg Protective = Stability of the sample containing antioxidant factor Stability of the control sample 50 as the number of days required to reach the peroxide value of 70 meq/kg. This end point corresponded with a weight gain of 1.2% and conjugated diene absorption of 1.431. Corn oil treated with 0.01% Trolox C had a higher stability than the samples with BHA, BHT and citric acid. The order of effectiveness in decreasing order was Trolox C, BHA, BHT and citric acid. Trolox C had about 1.2 to 1.5 times the antioxidant activity of BHA and BHT. This value was lower than that reported by Cort _t _l. (1975) probably by the greater temperature employed. It was interesting to note that BHT had greater effectiveness than that of BHA in peanut oil. On the other hand BHA showed higher activity in corn oil than did BHT. In corn oil, 0.01% Trolox C in combination with either 0.01% BHA or BHT displayed a nega- tive synergism. The addition of 0.05% citric acid to these samples reduced the negative value of the synergism. Figure 7 shows the development of oxygen uptake as measured by weight gain in corn oil. Here it is seen that the amount of oxygen absorbed varied with antioxidants throughout the storage period. Figure 8 displays the absorbance at 233 nm due to diene conjugation. It seemed that a similar trend existed. Results of tests performed with coconut oil appear in Table 9, Figure 9 and Figure 10. Since Trolox C in combina- tion with another antioxidant never reached an end point over a period greater than two months, synergism in coconut oil could not be calculated. Table 9 shows mean conjugated .uomc um mmmgoum mcwcau P00 :000 *0 0000 050003 .N 002000 Ana-003. mi; 51 $500 <0 + $ 5.0 .50 + $50 0 3.0:. $000.0 (0 + $ 5.0 (20 + $5.0 0 £0.00». $5.0 #20 +$5.0 0%... $50 (10 + $50 0 0.0:. $ 000.0 (0 a 5.0 .23 $ p00 <20 $ 50 0 .33: 05312.0( 02 10.? (X) NIVS 1119131111 90000040. 52 .uomo 00 mmmgopm 000030 Fwo 0000 00 cowpagomnm wcmwu umpmmancou b A0800): m2: $000.0 (0 +$ 5.0 #10 + $5.0 O 0.0.9.... $000.0 (0 + $ p00 (10 + $5.0 0 x200. $5.0 P10 +$5.0 00.0.... $5.0 (10 +$5.0 0 3.0:. $ 000.0 (0 $ 5.0 5.10 $ p00 (10 $ 5.0 0 5.00. 00035.0( 01 9.0.0460. "N 882 1' NOIldUOSBV 394310 OBLVDHI‘NOO .m 00=m_0 53 -Pupzz 3mz m.:muc:a any ucmgm00wu appcmquwcmwm Ho: «gm: mgmuump :oEEou mcwgmzm mm=~o>u .Nmoo.o A>\zv oooFup yo cowp=_wu a pm umpup V 7V \’ Vii! VIKrrV I? V V .D'VVV" V 7 u <0 pqmuxm N_o.o u :u—mu new 5: mmm mo npmcmp m>oz use pm cmgzmoms* .moo.o n mammz mo soggy usmucmum cowumgucmucou acmuwxowucm** Fo.o v a Aummh magma mpg on L m$N¢P.o hwmmp.o wsmm—.o wmpm~.o wwvunmmp.o Nm¢.o owrm.o xhpmp.o $muunemp.o acmnwxowucm 02 m o , -- -»,¢ , , N o . pcmuwxowp=< Amxmmzv mswp *« mxmmz m so» come as mpmgoum mcwgsu Fwo uncouou mo co_pagomnm m:m_u umummzncou cum: .1 .m mpnmk 54 .A>m An umgsmmmsv mxmmz xwm Lo» uomo um vmgoum Fwo uncouou 0o cowuw~_pwamum .m wgsmwu - , , 0 m3? mvmxogma 3.5.2: .IEI—Lilllllllll.lu I... _ g .1: d m 4H: m X . . . . . cc n. amoo 0 <9 + N_o 0 him + RPO o u xopogh 0 IL a “moo.o <0 + &_o.o «:m + fipo.o u xopogp .; ;IL _2 m Npo.o Him + N_o.o u xopogp .m u m. fl_o.o <=m + N_o.o u xopogh .0 u . cm a xmoo.o «u .m IL w . . b N_o o hzm c m I, x. xpo.o u 00000 0~0.0 u 0000000000000 0000.53.30...0 .0... 00.0. 00.0. 00.000.0 00.0. .wm 0mmmm.m 00... Pm... 000000.m 0m... om 00000o.m gm..o_ sumo.o. 000000.m 0m.o_ @— 0000.m mwwuum.m 00000.m 00000.m m00000~.m N. 00000.0 000000.0 00000.0 000.0.0 00000.0 0 00m.w 00000.m 0000m.m 00m.m 0000.m 0 0000.0 00000.0 00000.0 0000.0 00.0 0 0:00 0:0 <20 0 x0.00h .000000 Amxmmzv 05.0 .0000.x0.0e< . .0000: <~ 000 Auommuomv 00000000500 5000 00 000000 .00 0000000 00 00.0> 000x0000 000: .0. 0.000 61 .00.0000000 000.0 0000000000 00 00000005 000 00003 00 000 00000000500 5000 00 000000 ..0 0000000 00 00.000...000m 0No.o xmo.o 0~o.o 0No.c 0:0. .10 <20 0 x0.00. .000000 0000000000 .0.0.0. III ... ‘ 1| o..o m..o o~.o ... 0000.0 mu £53 19 aauquosqV 62 .moo.o u meow: $0 goggm nsmucmpm _o.o v a Apmmh manna m_awppaz 3mz m.:muc=o may pcmgwmmwv xpucmowwwcwwm uoc mgmz mgwupmp cossou mcwgmgm mmzpm>nm xmo.o u cowpogucmucou ucmnwxo_ucm«« A>\3V coopnp we cowpzpwu m an umumFaupmu new 5: mmm pa umgammms* hwemm.o somm.o opow.o nwmpm.o ommw.o «N xwom~.o xpmm.o pmnmé chom.o Femm.o om h2:135 _o_m.o xmmm.o $mcom~.o :m_¢.o m_ $muumem.o mmu~m~.o P;_om.o wmuunmoema xfiomm.o N_ muunqmmm.o mmcuneem.o mwmmmm.o muunoom~.o smmmm.o m muunmm_~.o $muunmmmw.o mmuunammw.o uunmm.m.o ¥muunomm~.o e mmo~.o ammom.o unmmp~.o mmo~.o amopm.o o oxmp Hzm <=m o xo_ogp Foggcou Amxmmzv «EFF *iucmuwxowuc< «Auom~-omv mgzpmgmasmu Eco; “a umgopm on usccma mo cowpqgomnm mam?u umummzncou cam: ._P m_nmh 63 .Am=~m> mnvxogma an nmgammws mmv mxmmz em Lo$ mgaumgmqsmg Eco; um umgoum Fwo uzcmma *o comum~w_mnmum .N_ wgamwu o w m3: 320ng ECLEIIIIIIIIIIIIIIIII w x _ , 1 ON” D. U n a 1 SM fimo.o ozmp .m .b _L w 6 fimo.o Hzm .o u /\ xmo.o no cowumcucmocou ucmn_xowucaa« A>\3v ooopnp we cowuapwu m we umumpaopmu vcm E: mmm um cmsamwwsa xwmo.o =Pmm.o =mmm.o comm.o aam_._ em “comm.o Pem~.o ENNw.o meo~¢.o omem.o om meaem¢.o upmm.o _m_~.o cao~¢.o Empm.o up auao_¢.o _;oom.o xpme.o anua~o¢.o xmmm.o N_ acunaemm.o aaao_¢.o canowe.o auanammm.o gmmme.o m naomm.o canapsm.o acunaamm.o unammm.o cmoFme.o e naomm.o aeem.o anammm.o nacmm.o unamem.o o I. oxmh elm «rm 9 xo_ocp _oepeou Amxaazv ..acauwxo_p=< asap .Auomm-omv mcauagmaEmu Eco; an umcopm Fwo cgou mo cowpqcomam mcmwn umammawcou com: .N— man» 66 Amapm> mu_xocma ma umgammme mmv mxmm3 em cow mgaumcmgamp Eco; um nmcoum P_o ccoo mo cowum~w_wnmum m:_m> mcwxocoa meumcw xwo.o 01m» xmc.o Him &No.o <=m $No.o u xerox» pocucou om cc om om oo— .mp aeamwe (Bx/ham) BHLPA aptxouad 67 order was Trolox C, TBHQ, BHT and BHA with respect to inhi- bition of peroxide development. As mentioned, all the oils stored at room temperature ranging from 20 to 28°C underwent oxidative changes during storage periods. These oxidative changes varied with the oils. Neither coconut nor peanut oils reached a peroxide value of 70 meq/kg during a storage period of 24 weeks, while only the control sample achieved this end point in corn oil. This phenomenon, of course, could be accounted for by the higher polyunsaturated fatty acid content of corn oil compared to that of peanut and coconut oil. The stability of control samples might be due to the residual tocopherols present in these oils. The antioxidants added to vegetable oils inhibited oxidative deterioration during storage at room temperature. Both BHA and BHT seemed to be least effective as antioxi- dants in these vegetable oils. This finding was in agree- ment with the data reported by Berger (l975). Trolox C as well as TBHQ exhibited better activity than either BHA or BHT. The superiority of Trolox C over BHA and BHT was con- sistent with the finding reported by Cort gt a1. (l975). The greater efficacy of TBHQ compared to either BHA or BHT confirmed the data reported by Cort gt 11. (1975), Sherwin and Luckadoo (l970) and also Chahine gt El- (1974). It is well known that rancidity is a major problem in fats and oils. This problem is more significant in tropical countries such as Indonesia where the daily high temperatures 68 accelerate oxidative deterioration. As a consequence of this factor, rancidity occurs in tropical climates in a relatively shorter time than in a temperate climate. Besides, lack of advanced technology of storage, inappro- priate handling and distribution system of food through marketing channel in most developing countries has empha- sized the need of antioxidant treatment. The selection of an appropriate antioxidant for particular foods such as vegetable oils is highly important in order to obtain the best results. Trolox C seemed to be the most suitable antioxidant for vegetable oils. SUMMARY Investigations were made on the use of Trolox C, butylated hydroxyanisole, butylated hydroxytoluene, citric acid and tertiary butylhydroquinone in coconut, peanut and corn oils. The peroxide value, conjugated diene absorption and weight gain methods were used to follow the oxidative changes of the samples. Excellent correlation was obtained between peroxide value and conjugated diene absorption. A good correlation was found between peroxide value and weight gain as well as between conjugated diene absorption and weight gain, but these were weaker than that between peroxide value and con- jugated diene absorption. . The oxidative changes in the vegetable oils followed similar trends as evaluated by the three methods. The study suggested that shifts of double bonds occured along with hydroperoxides formation. The amount of oxygen uptake was proportional to the hydroperoxide produced. Trolox C has been found to be the most effective among the antioxidants used in both natural aging and accelerated tests. The order of effectiveness in decreasing order was Trolox C, TBHQ, BHA or BHT and citric acid. As the concen- tration increased, the effectiveness was generally increased. 69 70 BHA, BHT and citric acid when used alone appeared to provide little additional stability to vegetable oils. Trolox C, when used in combination with BHA or BHT, exhibited a negative synergism in both peanut and corn oils. However, Trolox C in combination with BHA or BHT and citric acid exhibited a small negative synergism in corn oil and displayed highly positive synergism in peanut oil. The use of the highly effective antioxidant such as Trolox C for vegetable oils seemed to be applicable in tropical countries such as Indonesia. BIBLIOGRAPHY BIBLIOGRAPHY Angelo, A.J. ST., R.L. Dry and L.E. Brown. 1975. Compari- son of methods for determining peroxidation in processed whole peanut products. J. Am. Oil Chem. Soc. 52:34. Berger, K.G. 1975. Catalysis and inhibition of oxidative process. Chem. and Industry. p. 194. Boland, J.L., and P. Ten Have. 1947. Kinetic studies in the chemistry of rubber and related materials. IV. The inhibitory effect of hydroquinone on the thermal oxidation of athyl linoleate. Trans. Faraday Soc. 43: 201. Boozer, C.E., G.S. Hammond, C.E. Hamilton and J.N. Sen. 1955. Air oxidation of hydrocarbons. II. The stoi- chiometry and fate of inhibitors in benzene and chloro- benzene. J. Am. Chem. Soc. 77:3233. Brown, C.F. and C.M. Gooding. 1955. Stabilization of glyceridic oils. U.S. Pat. 2,699,395. Jan. 11. Cannon, J.A., K.J. Zilch, S.C. Burket and H.J. Dutton. 1952. Analysis of fatty acid oxidation products by counter current distribution methods. IV. Methyl linoleate. J. Am. Oil Chem. Soc. 29:447. Chahine, M.H., and R.F. MacNeill. 1974. Effect of stabili- zation of crude whale oil with tertiary butyl hydro- quinone and other antioxidants upon keeping quality of resultant deodorized oil. A feasibility study. A. Am. Oil Chem. Soc. 51:37. Cooney, P.M., C.D. Evans, A.N. Schwab and J.C. Cowan. 1958. Influence of heat on oxidative stability and on effectiveness of metal inactivating agents in vegetable oils. J. Am. Oil Chem. Soc. 35:152. Cort, H.M. 1974. Antioxidant activity of toc0pherols, ascorbyl palmitate and ascorbic acid and their mode of action. J. Am. Oil Chem. Soc. 51:321. Cort, W.M., J.H. Scott and J.H. Harley. 1975. Proposed antioxidant exhibits useful properties. Food Tech- nology 29:46. 71 72 Cort, N.M., M. Araujo, N.J. Mergens, H.A., Cannalonga, M. Osadea, J.H. Harley, D.R. Parrish and H.R. Pool. 1975. Antioxidant activity and stability of 6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid. J. Am. Oil Chem. Soc. 52:174. Cowan, H.C., P.M. Cooney and C.D. Evans. 1962. Citric acid: Inactivating agent for metals or acid syner- gist in edible fat? J. Am. Oil Chem. Soc. 39:1. Dugan, L.R., Jr., H.R. Kraybill, L. Ireland and R.C. Vi- brans. 1950. Butylated hydroxyanisole as an anti- oxidant for fats and food made with fat. Food Tech- nology 4:457. Dugan, L.F., Jr., L. Marx, C.E. Weir and H.R. Kraybill. 1954. Butylated hydroxytoluene. A new antioxidant for food use. Am. Meat Inst. Foundation Bull. No. 18. Dugan, L.R., Jr. 1955. Stability and rancidity. J. Am. 011 Chem. Soc. 32:605. Dugan, L.R., Jr. 1961. Development and inhibition of oxi- dative rancidity in foods. Food Technology 15:10. Dugan, L.R., Jr. 1963. Antioxidants. In: Encyclopedia of chemical technology. Ed. R.E. Kirk and 0.F. Othmer. John Wiley and Sons Inc., New York, Vol. II, p. 588. Duncan, 0.3. 1955. Multiple range and multiple F tests. Biometrics 11:1. Farmer, E.H. and D.A. Sutton. 1943. The course of autoxi- dation reactions in polyisoprenes and allied com- pounds. Part IV. The isolation and constitution of photochemically-formed methyl oleate peroxide. J. Chem. Soc. 48:392. Fritsch, C.M., V.E. Weiss and R.H. Anderson. 1971.‘ Sta- bility of coconut oil in food product. J. Am. Oil Chem. Soc. 46:64. Fukuzumi, K. and N. Ikeda. 1969. Study on the effect of antioxidants in the autoxidation of methyl non-conju- gated octadecadienoates. J. Am. Oil Chem. Soc. 46:64. Fukuzumi, K., N. Ikeda and M. Egawa. 1976. Phenothiazine derivatives as new antioxidants for the autoxidation of methyl linoleate and their reaction mechanisms. J. Am. Oi1 Chem. Soc 53:623. Gooding, C.M., H.N. Vahlteich and R.H. Neal. 1950. Citric acid esters. U.S. Pat. 2,518,678. August 15. 73 Gray, J.I. 1978. Measurement of lipid oxidation: A review. J. Am. Oil Chem. Soc. 55:539. Holman, R.T. and O.C. Elmer. 1947. The rates of oxidation on unsaturated fatty acids and esters. J. Am. Oil Chem. Soc. 24:137. Ikeda, N. and K. Fukuzumi. 1977. Synergistic antioxidant effect of nucleic acids and tocopherols. J. Am. Oil Chem. Soc 54:360. Ke, P.J., D.M. Nash and R.G. Ackman. 1977. Mackerel skin dipids as an unsaturated fat model system for the deter- mination of antioxidative potency of TBHQ and other antioxidant compounds. J. Am. Oil Chem. Soc 54:417. Kraybill, H.F., L.R. Dugan, Jr., B.W. Beadle, F.C. Vibrans. V. Nona Schwartz, and H. Rezabek. 1949. Butylated hydroxyanisole as an antioxidant for animal fats. Lange, W. 1950. Cholesterol, phytosterol and tocopherol content of food product and animal tissue. J. Am. Oil Chem. Soc. 27:414. Lea, C.H. 1971. The effect of light on the oxidation of fats. Proc. Roy. Soc. B. 108:176. Lockwood, L.B. and W.E. Irwin. 1963. Citric acid. In. Encyclopedia of Chemical Technology. Ed. by R.E. Kirk and 0.F. Othmer. John Wiley and Sons Inc., New York, Vol. V p. 524. Lundberg, W.O. 1962. Mechanism. In: Lipid and their oxi- dation. Ed. by H.W. Schultz, E.A. Day and R.O. Sinhuber. The AVI Publishing Company Inc. Westport, Conn. p. 31. Mahon, J.H. and R.A. Chapman. 1953. The relative rate of destruction of propyl gallate and butylated hydroxy- anisole in oxidizing lard. J. Am. Oil Chem. Soc. 30:34. Mehlenbacher, V.C. 1960. The analysis of fats and oils. The Garrard Press Publisher. Champaign, 111. p. 219. Mertens, W.G., C.E. Swindells and B.F. Teasdale. 1971. Trace metals and the flavor stability of margarine. J. Am. Oil Chem. Soc. 48:544. Metcalfe, L.D., A.A. Schmitz and H.R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatography analysis. Analytical Chem. 38:514. Morawetz, H. 1949. Phenolic antioxidants for paraffinic materials. Ind. Eng. Chem. 41:1442. 74 Morris, S.G., J.S. Myers, Hr., M.L. Kip and R.W. Riemen- shneider. 1950. Metal deactivation in lard. J. Am. Oil Chem. Soc. 27:105. Neter, J. and W. Wasserman. 1974. Applied linear statis- tical model. Richard D. Irwin Inc. Homewood Ill. p. 21. Olcott, H.S. and H.A. Mattill. 1936. Antioxidants and autoxidation of fats. VII. Preliminary classification of inhibitors. J. Am. Chem. Soc. 58:2208. Olcott, H.S. and E. Einset. 1958. A weighing method for measuring the induction period of marine and other oils. J. Am. Oil Chem. Soc. 35:161. Privett, O.S., W.O. Lundberg, N.A. Khan, W.E. Tolberg and D.H. Wheeler. 1953. Structore of hydroperoxides obtained from autoxidized methyl linoleate. J. Am. Oil Chem. Soc. 30:61. Privett, 0.5. and F.W. Quackenbush. 1954. The relation of synergist to antioxidant in fats. J. Am. Oil Chem. Soc. 31:321. Rawls, H.R. and P.J. van Santen. 1970. A possible role for singlet oxygen in initiation of fatty acid autoxida- tion. J. Am. Oil Chem. Soc. 48:121. Reiners, R.A. and C.M. Gooding. 1970. Corn oil. In: Corn: culture, processing products. Ed. by C.E. Inglett. The AVI Publishing Company Inc. Westport, Conn. p. 241. Sattar, A., J.M deMan and J.C. Alexander. 1976. Light induced oxidation of edible oils and fats. Food Science & Technology 9:149. Schonberg, V.A. 1935. Notiz uber die photochemische bildung von biradikalen. Justus Liebigs Annalen der chemie, p. 299. Scott, J.W., J.H. Harley, D.R. Parrish and G. Saucy. 1974. 6-hydroxychroman-2-carboxy1ic acid: Novel antioxidants. J. Am. Oil Chem. Soc. 51:200. Sherwin, E.R. and J.W. Thompson. 1967. Tertiary butyl- hydroquinone- An antioxidant for fats and oils and fat containing foods. Food Technology 21:912. Sherwin, E.R. 1968. Methods for stability and antioxidant measurement. J. Am. Oil Chem. Soc. 45:632A. 75 Sherwin, E.R. and B.M. Luckadoo. 1970. Studies on anti- oxidant treatment of crude vegetable oils. J. Am. Oil Chem. Soc. 47:19. Sherwin, E.R. 1972. Antioxidants for food fats and oils. J. Am. Oil Chem. Soc. 49:468. Sherwin, E.R. 1976. Antioxidants for food fats and oils. J. Am. Oil Chem. Soc. 53:430. Skinner, W.A. and P. Alaupovic. 1963. Vitamin E oxidation with alkaline ferricyanide. Science 140:803. Skinner, W.A. and R.H. Parkhurst. 1970. Antioxidant pro- perties of alpha-tocopherol derivatives and relation- ship of antioxidant activity to biological activity. Lipids 5:184. Smith, G.J. and W.L. Dunkley. 1962. Initiation of lipid peroxidation by a reduced metal ion. Arch. Biochem. Biophys. 98:46. Spencer, 0.F. and S.F. Herb. 1976. Fatty acid composition as a basis for identification of commercial fats and oils. J. Am. Oil Chem. Soc. 53:94. Sreenivasan. 1968. Component fatty acids and composition of some oils and fats. J. Am. Oil Chem. Soc. 45:259. Strouse, J., S.W. Layten and C.E. Strouse. 1977. Struc- tural studies of transition metal complexes of tri- ionized and tetraionized citrate. Models for coordi- nation of the citrate ion to transition metal ions in solution and at the active site of aconitase. J. Am. Chem. Soc. 99:562. Stuckey, B.N. 1962. Antioxidants. In: Lipids and their oxidation. Ed. by H.W. Schultz, E.A. Day and R.O. Sinhuber. The AVI Publishing Company, Westport, Conn. p. 139. Swanholm, V., K. Bechgaard and V.D. Parker. 1974. Electro- chemistry in media of intermediate acidity. VIII. Reversible oxidation products of alpha-toc0pherol model compounds. Cation radical, cation and dication. J. Am. Chem. Soc. 96:2409. Swern, D. 1964. Bailey's Industrial Oil and Fat Products. John Wiley & Sons Inc. New York. p. 55. Tappel, A.L. 1955. Catalysis of linoleate oxydation by Copper-Proteins. J. Am. Oil Chem. Soc. 32:252. 76 Thampan, P.K. 1975. The coconut palm and its products. Green Villa Publishing House, Kerala, India. Thewalt, K., A. Pastura and G. Renckhoff. 1969. Fette Seifen Anstrich 71:85. Thompson, J.W. and E.R. Sherwin. 1966. Investigation of antioxidants for polyunsaturated edible oils. J. Am. Oil Chem. Soc. 43:683. Thompson, R.B. and T. Symon. 1956. Some derivatives of hydroxyhydroquinone as antioxidants. J. Am. Oil Chem. Soc 33:414. Tollenar, F.D. and H.J. Vos. 1958. Problems arising in connection with the use of antioxidants in the food industry. J. Am. Oil Chem. Soc. 35:448. Vioque, A., M.A. Albi and Ma del Pilar Villagran. 1964. Trace elements in edible fats. VIII. Soybean oil demetalization with cation exchange resins. J. Am. Oil Chem. Soc. 41:785. Vioque, A., R. Guttierrez, M.A. Albi and N. Nosti. 1965. Trace elements in edible fats. IX. Influence of demetalization on the oxidative and flavor stability of soybean oil. J. Am. Oil Chem. Soc. 42:344. Waters, W.A. 1946. General discussion. In: Processes in the oxidation of hydrocarbon fuels 1 by A.D. Walsh. Trans. Faraday Soc. 42:281. Wheeler, D.H. 1932. Peroxide formation as a measure of autoxidative deterioration. Oil & Soap 9:89. Woodroof, J.C. 1973. Peanuts: Production, Processing, Products. The AVI Publishing Company Inc., Westport, Conn. p. 247. Worthington, R.E. and R.O. Hammons. 1977. Variability in fatty acid composition among Arachis genotypes: A potential source of product improvement. J. Am. Oil Chem. Soc. 54:105A. Zeldes, H. and R. Livingston. 1971. Paramagnetic resonance study of liquids during photolysis. IX. Citric acid and sodium citrate in aqueous solution. J. Am. Chem. Soc. 93:1082. "IT'IIIIIII'IIIIIIII