Samples ojpalm olein were irradiated with y-rays up to 12kGy. The extent ojperoxidation in irradiated samples was

determined by conjugable oxidation products (COP) assay and the result were compared with the UV absorbance at 232

nm. The two parameters were poorly correlated (r = 0.6321) within the range ofdoses used. The ifJect ofy-irradiation

is mainly to oxidise linoleic acid (C18:2) as this component is the major diunsaturatedJatty acid in palm oil

Endinkeau, Kuang

Woodward, T. W.

Universiti Putra Malaysia Institutional Repository

Pertanika 13(1), 101-106 (1990)Application of Conjugable Oxidation Products Assay inAssessment of Gamma-Irradiated Palm OleinK. E DI KEAU 1 and T.W. WOODWARD21Department of ChemistryFaculty of Science and Environmental StudiesUniversiti Pertanian Malaysia43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.2Department oj Chemistry and Applied ChemistryUniversity of SalfordSalford M5 4WT, England.Key words: Palm olein, gamma-irradiation, conjugable oxidation products.ABSTRAK.Olein sawit didedahkan kepada sinar-y sehingga dos 12 kGy. Pembentukan peroksida di dalam sampelyang disinardiukur dengan kaedah hasil pengoksidaan terkonjugat (COP assay) dan nilainya dibandingkan dengan nilai-nilaiyangdiperolehi dari kaedah serapan ultralembayung pada 232 nm. Kedua-dua parameter tidak menunjukkan korelasi yangbaik (r = 0.6321) dalam julat dos yang digunakan. Kesan utama sinar-y adalah mengoksidakan asid linoleik yangmerupakan asid lemak di tak tepu utama dalam minyak sawit.ABSTRACTSamples ojpalm olein were irradiated with y-rays up to 12kGy. The extent ojperoxidation in irradiated samples wasdetermined by conjugable oxidation products (COP) assay and the result were compared with the UV absorbance at 232nm. The two parameters were poorly correlated (r = 0.6321) within the range ofdoses used. The ifJect ofy-irradiationis mainly to oxidise linoleic acid (C18:2) as this component is the major diunsaturatedJatty acid in palm oil.INTRODUCTIONRecently, there has been much concern about thequality of oil palm fruits sent to the mills. Poorquality fruits will produce low quality oil which isnot only costly to refine but also fetches lowmarket prices. Factors such as the degree ofripeness, severity of bruising of harvested fruitsand delays between harvesting and sterilizationdetermine the quality ofoil palm fruits. Bruised orover-riped fruits will contain a high percentage offree fatty acid (ffa.) partly due to the action ofalipase enzyme from microorganisms or fungi. Inconsidering these problems, it was thought thatradiation within a preservation dosage ofless than10 kGy (Webb 1985; Diehl 1977 might beeffective in reducing the microorganisms or fungiand also deactivating the enzyme.However, lately there has been increasingconcern about the quality and safety of irradiatedfood. Gamma-irradiation of food, besidesimproving its quality, might also lead to changesin its physical and chemical properties. It hasbeen known that irradiation of fat-containingfood forms lipid peroxides, particularly in thepresence of unsaturated fats (Hammer et al. 1979;Wolters et al. 1987) and lowers the quality of thefood. In view of these problems, knowledge aboutthe extent of deterioration is very important ifradiation is to be used to improve the quality ofpalm oil. Therefore, the objective of thisinvestigation was to study the effect of gamma-irradiation on the quality of palm oil using aConjugable Oxidation Products (COP) assay(Parr et al. 1976). The COP as ay gives moreK. E'DI KEAU AND T.W. WOODWARDinformation on oxidation products and alsodifferentiates between the oxidation of dienoicsand those of more highly unsaturated polyenoicfatty acids.MATERIALS AND METHODSMaterialsPalin olein was prepared from crude palm oil(CPO) as previously described (Endinkeau et al.1989). Isooctane (HPLCgrade), propan-2-01 andabsolute ethanol (AR) were obtained from BDHLtd. Sodium borohydride (NaBH4 ) waspurchased from Sigma Chern. Co. All othersolvents w'ere of reagent grade and were usedwithout further purification.Preparation of ReagentsA stock solution of reducing reagent was preparedby dissolving NaBH4 (1 g) in propan-2-ol (100cm3). The solution was filtered before being usedto prepare an approximately 0.4% (w/v) freshsolution of NaBH4 In propan-2-01. Adehydrating reagent was prepared by mixingconcentrated sulphuric acid (5.43 cm3) Inabsolute ethanol to give a 20 0 0 (wi") solution in50 cm3.IrradiationSample of palm olein (2 x 20 g) were irradiatedwith y-rays in glass tubes (5.5 x 3.0 cm) at a doserate 65 GYlmin using a 60Co-gamma source at theniversity ofSalford, England. The dose rate wasdetermined by Fricke Dosimetry Oayson et al.1975). Samples of oxygenated and aeratedsolutions were used during irradiation. Inoxygenated solutions the oil was bubbled withoxygen 10 min prior to and during theirradiation. For air-saturated solutions, the oilwas irradiated as it was, i.e. in its natural form.Samples were irradiated to a full dose rangingfrom 2 to 12 kGy. At intervals of time, portions ofirradiated samples were taken and the COP assaywas performed immediately.MethodThe procedure of COP assay was based on that ofParr et al. (1976) and Swoboda (1981).Stock solutions of irradiated and unirradia-ted oil samples were prepared by dissolving aknown weight of each sample (about 0.1 g) in 5.0cm3 isooctane-ethanol (1:1 by volume). Aliquots(1.0 cm3) of the stock solution were thentransferred to each of three 10.0 cm3 stopperedvol u metric flasks which were rna rked '0'(original), 'R' (reduced) and 'D' (dehydration).To flask code '0' was added 10.0 cm3 isooctane-ethanol (1:1); to flask code 'R' was added 0.4 cm3reducing reagent (0.4 0 0 w/v) and held at 60°C for30 Olin, then cooled and made up to the markwith isooctane-ethanol; flask code 'D' was firsttreated as 'R' and subsequently 2.0 cm3 ofdehydrating reagent (20% w/v) H 2S04 inethanol) was added with the flask being heldagain at 60°C for 30 min. After cooling, thesolu tion was made up to 10.0 cm3 with isooctane-ethanol. The absorbance ofeach solution, '0', 'R'and 'D' at the appropriate wavelengths wasmeasured using a SP1800 Spectrophotometer.Solution '0' was measured at 275 nm (A275),solu tion 'R' was measured at 268,275 and 301°nm(A~68, A~75 and' A~I and solution 'D' wasmeasured at 268 and 301 nm (A268 and A30I ). A 1.0D ncm silica cell containing isooctane-ethanol waused as a blank. The UV absorbance at 232 nmconjugated diene) was also performed using aP1800 Spectrophotometer and 0.2 0 0 oil01 u tions in isooctane.RESULTS AND DISCUSSIONGamma-irradiation of unsaturated fats (RH)which contain a 1,4-diene structure produces freeradical CR) which reacts with oxygen to form apemxy radical (ROO'). Subsequent abstractionof a H atom from neighbouring molecule forms ahydroperoxide (ROOH). The co·mpound isunstable and decomposes into various secondaryoxidation products namely hydroxydienes,ketodienes and dienals. The ketodienes anddienals are described as oxodienes, (Frankel 1980)in Scheme 1.In conjugable oxidation product (COP)assay, the hydroperoxide and oxodienes areconverted, by reduction and then dehydration,in to nonpolar triene or tetraene which arestrongly chromophoric and conveniently mea-sured in the ul tra violet region. Change inabsorbance resulting from their formationcorrespond to the amount present. The decreasein absorbance at 275 nm due to reduction ofcarbonyl compounds is defined as the oxodienevalue. The sum changes in absorbance at 268 and301 nm which measure the amount of triene and102 PERTA;\IKA \'01.. I~ NO. I, 1990CONJUCAHLE OXIDATION PRODUCTS ASSAY I ASSESSMENT OF GAMMA-IRRADIATED PALM OLEI- CH CH - CH 2 - CH = CH - CH -1- HCH - CH - CH - CH - CH - CH 2 -- CH 1()2- CH CH - CH = CH - CH - CH2 -IoIoIH- CHdecomposi tionJ!CH - CH = CH - C - HIIo reductionor- CH CH - CH = CH - C -~ IIooxodiene(275 nm)- CHdehydration~CH - CH = CH - CH CH-triene (268 nm)or- CH CH - CH = CH - CH = CH - CH CH-tetraene (301 nm)Scheme 1PERTA IKA YOLo 13 NO. 1,1990 103K. E D1NKEAU AND T.W. WOODWARDFig. 1. Absorb/ion spec/TO a/ OTl:ltinal (0), reduced (R) and dehydTOted(D) solutions of unirradiated palm olein.320300280o260240WAVELENGTH (om)2201.60.41.20.8Fig. 2. Absorb/ion spectra oforiginal (0) reduced (R) and dehydTOted(D) solutions of irradiated palm olein.by the higher absorption at 232 nm. Uponadd ition of the dehydrating reagent, thehydroxydienes were converted to conjugatedtrienes and thus, reduced the number of dienes(Scheme 1) as indicated by the decrease inabsorbance at 232 nm, in solution D. The mostsignificant changes in the COP assay are that thereduced 'R' spectrum change to the dehydrated'D' spectrum due to the formation of moreconjugated triene with absorption maximum at268 nm, whilst that of232 nm decreases. A typicalabsorption of nonpolar triene which has a tripletfine structure is shown in spectra of irradiated andunirradiated palm olein.A small broad absorption of tetraene at 301nm, arose from oxidised linolenic acid (C18:3)indicating the presence ofa small amoun t oftriene(linolenic acid) in palm olein (less than 0.2%).The larger change in absorbance at 268 nm,(conjugated triene) in irradiated compared tounirradiated samples means. that the efIect of y-irradiation is mainly induced autoxidation oflinoleic acid (C18:2) which comprises about 10%of the fatty acids in palm olein.The COP assay calculated from the changein absorbance at the three wavelengths (268, 275and 301 nm) was as follows (Parr el al. 1976):-Oxodiene value = (A275 - A275 ) -:-- 2w" R320300280260WAVELENGTH (run)2402202000.20.60.40.8The increase in absorbance at 232 nm (Figure2) in solution 'R' compared to '0' is due to theconversion of carbonyl compounds to conjugatedhydroxydienes, and these compounds togetherwith the initially formed hydroxydienes increasedthe concentration of conjugated dienes as shownFigures 1 and 2show the UV spectra of irradiatedand unirradiated palm olein recorded between210 and 320 nm for solutions '0', 'R' and 'D'. Thedecrease in absorbance at 275 nm from theoriginal '0' to the reduced 'R' spectra resultingfrom reduction with l aBH4 gives the oxodienevalue. The value corresponds to the amount ofunsaturated carbonyl compounds such asaldehydes and ketones produced from theoxidised palm oil. These products arose fromdecomposition of the initially formed hydropero-xides. A strong absorption of solu tion '0' at 268nm (Figures 1 and 2) is due to the presence ofsecondary oxidation products such as diketonesand conjugated trienes (Rossell 1983). However,the decrease in absorbance at this wavelength insol ution 'R', was due to the reduction ofdiketonesto hydroxy compounds by sodium boron hydride.tetraene is defined as the conjugable oxidationproduct value (COP value), whereas the ratio ofabsorbance change at 301 to 268 nm is termed asthe COP ratio which measures the relativeproportion of tetraene to triene products.104 PERTANIKA VOL. I:i I O. 1,1990CON] CABLE OXIDATIO PRODUCTS ASSAY IN ASSESSMENT OF CAMMA-IRRADIATED PALM OLEIwhere w is the weight of the oil sample taken forpreparation of the 5.0 cm3 stock solu tion whichgave a strength of 2 x w per 100 cm3 of a finalsolu,tion in the 10.0 cm3 volumetric flask. Tables 1and 2 show the results obtained after correctionfor background (unirradiated sample).The COP value of irradiated palm olein at aconstant dose rate (65 Gy/min) increased steadilywith increasing irradition dose from 2 to 12 kGy(Table 1). I t is suggested that as the dose increasesTABLE 1COP assay of irradiated palm olein in the presence ofoxygenCOP Assay*Dose Oxodiene COP COP(kGy) Value Value Ratio2 0.06 1.05 0.044 0.11 1.67 0.046 0.19 2.80 0.088 0.20 3.36 0.0610 0.25 3.64 0.0812 0.32 3.90 0.10Unirrd. 0.05 0.20 0.13*COP = (COP;rrd. - COPunirrd.)more linoleic acid as well as linolenic acid will beoxidised. However, there is no significant changein oxodiene value, though the values increasedslightly with increasing dose. This shows that onlysmall amounts of carbonyl compounds areproduced after irradiation. Some of thesecompounds are volatile, especially those with lowmolecular weights and may be evaporated duringanalysis. This factor may also account for thelower yield of carbonyl compounds. Thesecompounds, known as odorous substances, areresponsible for the development of an unpleasantsmell in deteriorated oils and fats.Similarly, the COP ratio is very small sincethe oxidation is mainly of dienoic rather thantrienoic in nature. Theoretically, this value variesfrom zero for a pure triene to 2.8 for a puretetraene (Fishwick et ai. 1977). It has beenobserved that in the aerated solution (Table 2),the COP and oxodiene values were much lowerthan that of the oxygenated solution. It might beexpected that in the presence of low con-centrations of oxygen, less hydroperoxides areTABLE 2COP assay of irradiated palm olein in aerated solutionCOP Assay*Dose Oxodiene COP COP(kGy) Value Value Ratio2 0.19 0.87 0.054 0.19 0.95 0.036 0.17 1.13 0.068 0.15 1.25 0.0510 0.10 1.21 0.0612 0.15 1.33 0.07nirrd. 0.09 0.66 0.13formed, accounting for the lower COP andoxodiene values. The relationship between theCOP values and the UV absorbance at 232 nm isgiven in Figure 3. The results show little"r----------------~•••'!"",-.<:'------'--------'2-------''-----.JCOPYAlUESFig. 3, Correlation between COP values and UV absorbance at 232nm obto.inedfrom irradiated palm olein (r = 0.6321, Y =0.2434x - 0.0578)correlation between the two parameters (r =0.6321). This indicated that the yield ofconjugated dienes or hydroperoxides by gamma-irradiation is not paralleled with the changes ofCOP values. Presumably, in the presence ofexcessoxygen and high irradiation doses, somehydroperoxides undergo other types of reactions,forming non-conjugated products (Gilman 1953;Swern 1961). These reactions, therefore, red ucethe number of initially formed hydeperoxides andthus, the conjugated dienes.PERTA IKA VOL. 13 NO, I, 1990 105K. ENDINKEAU AND T.W. WOODWARDPrevious work (Parr et aL. 1976) has shownthat some of the commercially available foodstuffshave COP values ranging from 0.5 to 77. \ poorquality pork mince and kipper fillets, for example,have COP values of 1.5 and 2respectively. Based on these values and the valuesobtained from irradiated palm olein, gamma-irradiation within the range of preservation doses(1-10 kGy) (Webb 1985, Kader 1986) can,therefore, be applied to preserve oil palm fruits.Furthermore, at these doses only small amounts ofcarbonyl compounds, which impart anunpleasant smell to irradiated samples, areformed.CONCLUSIONThe COP value ofirradiated palm olein increasedwith increasing irradiation dose. Although theCOP values obtained (1.05 to 3.9 in oxygenatedsolution) are acceptable, gamma-irradiation toorne extent promotes autoxidation of unsatura-ted fatty acid, particularly the linoleic acid. Thepoor correlation between COP values andabsorbance at 232 nm indicates that otherreactions may be involved in the early stages ofautoxidation of palm olein.This work covered only a small part ofquality assessment. I t should therefore beconsidered as a preliminary study. More detailedinvestigations im'olving lipase activity andpossibly, mutagenic and teratogenic studiesshould be carried out. Nevertheless, from theCOP point of view, the results indicate thepossibility for the commercial application ofgamma irradiation for preservation of oil palmfruits.REFERENCESDIEHL,j.F. (1977): Preservation of Food. Radiat. Phys.Chem. 9 193-206.ENDIKEAU, K. and T.W. Woodward. (1989):Preservation of Oil Palm Fruit: NonoxidativeElleets of Ionizing Radiation on Palm Olein andCrude Palm Oil. Pertanika (In press).FISHWICK, Lj. and P.A.T. SII'OBODA. (1977:Measurement of Oxidation of PolyunsaturatedFatty Acids by Spectrophotomnrie Assay ofConjugated Derivatives. J. Sci. Food Agric. 28: 387-393.FRANKEL, E.N. (1980): Lipid Oxidation. Prog. LipidRes. 19: 1-22.GILMAN. H. (1953): O/ganic ChflnlJtlY, Vol. :~ p. 199New York: john Wiley and Sons.HAMl'v1 ER, CT. and E.D. WILLS. (1979): The Effect ofIonizing Radition on the Fally Acid Compositionof Natural Fats and on Lipid Peroxide Formation:Int. J. Radiat. Biol. 935: 323-332.JAYSON, G.G., B.J. PARSONS and A.J. SWALLOW.(1975): Mechanism of Fricke Dosimeter. Int. J.Radiat. Phys. Chem. 7: 363-370.K.\\)ER, A.A. (1986 : Potential Application oflonizingRadiation in Postharvest HandlingofFresh Fruitsand Vegetables. Food Tee/mol. 40: 117-121.P.-\RR, L.J. and P.A.T. S\I'OB,\O.\. 1976: The Assay ofConjugable Oxidation Products Applied to LipidDeterioration in Stored Foods. J. Food Technol. 11:1-12.ROSSEI.L, j.B. 1983: l\lea~urement of Rancidity inOils and Fals. Scielllilic ilnd Technical Surveys, No.140. p. B. The British Food Manul~ll·turin~I ndustries Research Association, London.. IVER:'<. D. (1961): Fat., a.'ld OilJ. Pan 2, cd. MARKEI.EY.K.S. p. 14(n. New York: Intcrscicnce.SWOIlADA. P.A.T. (1981): U\'-VIS Spenrophotomet-I-ic Assays for Palm Oil Quality. Paper presented atthe Conference on Palm Oil Product Technology in the.t.'ighties, Kuala Lumpur, Malaysia.WEBB, T. 1985): Food Irradiation in Britain, p.ll.London: London Food Commission PromotionsLtd.WOLTERS, H., C.A.M. TILBURG and A.W.T.KO:\I:\G. (1987): Radiation-induced LipidPeroxidation: Influence of Oxygen Concentrationandl\Iembrane Lipid Composition. Int. J. Radiat.Biol. 51: 619-628.(Received 10 March, 1989).106 PERTA rKA VOL. 13 NO. I, 1990

Application of Conjugable Oxidation Products Assay in

Assessment of Gamma-Irradiated Palm Olein

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Application of Conjugable Oxidation Products Assay in Assessment of Gamma-Irradiated Palm Olein

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