J Sci Food A.gric 1991. S4, 495-511

Review*Recent Advances in Lipid Oxidation

Edwin N Frankel

6588

Department of Food Science & Technology, University of California. Davis.California 95616. and Northern Regional Research Center. Agricultural Research Service.

US Department of Agriculture. Peoria. Illinois 61604. USA

(Received. 13 May 1990: revised version received 31 August 1990:accepted 1 October 1990)

ABSTRACT

In a major parhway ofthe auto.r:idarion ofmethyl lino/enare. peroxyl radicalsof the inrernal hydroperoxides undergo rapid 1,J.<.:yciisarion to formhydroperoxyepidioxides. Because linolenare h.vdroperoxides are relativelyUlfSraiJ/e. free radical amio:wianrs are much less effective in linolenate oilsthan in linoleare oils. Tocopherols and carotenoids effectively inlrihi'tphotosensitised oxidation of lJegetaiJ/e oils. Direct gas chromaiographicanalyses of malonaldeh.vde do nor con'elare wirh the iBA tesr. Modelfluorescence studies indicate that malona/dehyde ma.v not be so importanrin cross/inking wirh DNA. In contrasr to oxidised methyllinoleare. oxidisedtn/inolein does not form dimers. A/though tri/inoletn oxidises with nopreference between the 1(3)· and 2-triglyceride positions. the n·J double bondof trilinolenin oxidises more in the 1(3)· than in the 2-position. Synthetictrig/ycerides oxidise in the following decreasing relative rates: LnLnL.LnLLn. LLnL. LLLn (Ln =lino/enic and L = linoleic). io estimare theflavour impact of 1J0iarile oxidarion products thetr relative threshold lJa/uesmusr be considered together with their relative concentrarion in a givenfar.

Key words: Lipids. free radical autoxidation. hydroperoxides. photo­sensitised oxidation. aldehydes. volatiles.linolenate. cyclisation. epidioxides.tocopherol. carotene. antioxidants. malonaldehyde. dimerisation. tri·glycerides. trilinolein. trilinolenin. gas chromatography, stability, sensory,flavour significance. flavour reversion. sensory assessment. aldehydes.vegetable oils.

• This revIew IS based on [he 1990 InternanonaJ Lecture addressed [0 [he SeT's Oils and Fats GrouoIn London. t 1 Apni 1990.

495

J SCI Fooa .-1qnc 0022-5142/91/$03.501.' 1991 SCI. Printed in Great Bntam

Supplied by U.S. Dept. of Agric.,National Center for AgriculturalUtilization Research, Peoria, IL

496

INTRODUCTION

£ .V Frankel

Oxidation of polyunsaturated fatty acids is one of the most fundamental reactionsin lipid chemistry. Investigators working with polyunsaturated fatty acids andlipids have to be seriously concerned with their oxidation as the products havebeen implicated in so many vital biological reactions. The revival of the field oflipid oxidation in the last 10-15 years can be attributed in large pan to theaccumulating evidence that free radicals and reactive oxygen species panicipatein tissue injuries and in diseases. However, whether free radical species are thecause or the effect of these diseases is a question that has been very difficult toanswer.

In the presence of initiatorS, unsaturated lipids (LH) form carbon-<:entred. alkylradicals (L·) and peroxyl radicals (LOO-), which propagate in the presence ofoxygen by a free radical chain mechanism to form hydroperoxides (LOOH) asthe primary products of autoxidation (Franke! 1980).

LH - L·

L· +0% - LOa·

LOO-+LH - LOOH+L·

(1)

(2)

(3)

In the presence ofIig.h.t. unsaturated. fats can also form hydroperoxides by reactingwith singlet oxygen produced by sensitised photooxidation. which is anon-free-radical process (Gollnick 1978).

Lipid hydroperoxides are readily decomposed into a wide range of carbonylcompounds. hydrocarbons. ketones and other materials that contribute to flavourdeterioration of foods. Much work has been reported on the volatile oxidationproductS of unsaturated. lipids (Frankel 1982. 1985; Grosch (987) because theycause rancidity in foods and cellular damage in the body. Different volatiledecomposition products are formed according to the relative thermal stabilitiesof the lipid oxidation precursors and resulting carbonyl products. To evaluate theoxidative and flavour stability of unsaturated edible oils. it is t:ssential to knowthe structures of the oxidation products. how they decompose. the amounts ofvolatile compounds produced. and the flavour significance of the volatiles.

A better understanding of the mechanisms of oxidation of linoleic and linolenicacids may lead to improved. methods for control of /lavour deterioration invegetable oils. Several reviews of the literature have appeared (Frankel 1980. 1985.1988; Chan 1987; Grosch 1987; Gardner 1989). The mechanism of autoxidationof linoleic acid and esters has rea:ived special attention (Porter 1986). This papersummarises recent progress made in understanding the mechanism by whichpolyunsaturated. edible oils can undergo oxidative and /lavour deterioration.

FREE RAOlCAL AUTOXIDAnONLinolenate' esters

Since 1961. when the isolation of pure hydroperoxides of methyl linoienate wasfirst reported (Frankel ec ai 1961l. considerable advances have been made in

Lipid oxidation

0-0 OOH

rvrV-<C

1"**......., " 1 1d.~

F"II I. Free raciica1 autoxidation of methyl linolenate.

497

understanding linolenate autoxidation by the application of new powerfulseparation and analytical tools. The fie] unsaturation of linolenate provides a keymechanistic feature affecting the nature of its primary and secondary oxidationproducts. In the presence of free radical initiators. such as heat. metals. irradiationor light. hydrogen transfer occurs with a suitable radical acceptor X· from thetwo activated doubly allytic methylene groups on carbon- (1 and carbon-14 toform two pentadienyl radicals (Fig i). Reaction with oxygen at the end carbonpositions produces a mixture of four peroxyl radicals leading to the correspondingconjugated dienoic 9-, 12-, 13- and 16-hydroperoxides containing an isolateddouble bond. The fact that the external 9- and 16-hydroperoxides are formed inamounts significantly higher than the internal 12- and 13-hydroperoxides has beenknown for a long time (Frankel er at 1961, 1977: Chan and Levett (977). Onlyrecently has it been possible to explain this uneven distribution of isomerichydropeToxides of methyl linolenate. The peroxyl radicals of internal 12- and13-hydropeToxides undergo rapid 1.3-eyclisation (A - B) to form five-memberedhydroperoxyepidioxides (C. Fig 1) (Coxon er ai 1981; Neff er ai 1981). This rapidcyciisation is a major pathway which accounts for the lower concentrations of theinternal 12- and 13-hydroperoxides (25%) relative to the external 9- and 16­hydroperoxides 150%) (Frankel er ai 1961. 1977). By adding 51% :C-l:Qcopherol asa hydrogen donor. Peers er at (1981) showed that this cyciisation was completelyinhibited. methyl Iinolenate producing an even distribution of the 9-. 12-. 13- <lnd16-hydroperoxide isomers. A mixture of dihydroperoxides (0 and E) is formed insmaller concentrations than the hydroperoxyepidioxides. by a reaction competing

498

0-0_ 0~·

~a

E .V Frankel

0-0 OOH- '--J '--'-./- -~

o 0

VQ

~

Fig 2. Formation of bicycJoendopetOltides and malonaldehyd.e from oltidised. methyl linolena!e.

with cyclisation (Neff et al 1981). Coxon et al (1984) showed that in the presenceof 10% ~-tocopherol the 9.16-dihydroperoxide (E) was formed seiectiveiy duringoxidation of methyl linolenate.

The intenned.iate free radical (B) formed after cyclisation can either cyciise againto form bicycioendoperoxides (F), structurally reiated to the prostagiandins. orundergo cieavage to produce maionaJdehyde (G) and to give a positivethiobarbituric acid (TBA) test (Fig 2) (Dahl et al 1962; Pryor et al 1976). Incontrast to methyl Iinoleate (Porter 1986; Chan 1987), the cis, trans-hydroperoxidesof methyl linolenate are not readily isomerised to the crans.trans configuration.apparently because cyclisation is favoured much more than geometric isomerisation(Porter et al 198 I). The bicycioendoperoxides from oxidised 1inolenate were shownby O'Connor et al (1984) to have mainly cis substituents in contrast to the naturaltrans stereochemistry of the enzymically derived prostaglandins. The physiologicalimportance of this structural difference has not been established.

Inhibirion

Free radical autoxidation may be interrupted by several kinds of antioxidantswhich can react with either chain-carrying peroxyl radicals or the alkyl radicalintermediates (Scott 1985).

LOO-+AH - LOOH+A·

L'+Q- - LQ·

(4)

(S)

The first class of antioxidants (AH) includes hindered phenols such as butylatedhydroxyanisole. butylated hydroxytoluene and ~-cocopheroLTo be effective. thesecompounds must compete with the unsaturated lipid substrate (reaction 31 whichis normally present in the highest concentration. The second ciass of antioxidants(Q.) includes quinones such as ubiquinone and 'Z-cocopheroquinone which mustcompete with 0: in the fast reaction (2). These compounds may therefore onlybe active in biological systems where the oxygen pressure is relatively low.

In the presence of trace amounts of transition metals. hydroperoxides are readily

L.ipid oxidation 499

decomposed to form alkoxyi radicai intermediates (La·) and (LOa·), which caneffectively propagate the free radical chain:

LOOH + M" - La· +OH- + Mnn.,.l (6)

LOOH+M,,+l - LOa' +H'" +M" (7)

The catalytic effect of metals will be greatly enhanced in methyl linoienate becauselinoienate hydroperoxides are much more readily decomposed than linoleatehydroperoxides (Frankei 1962). In the presence of metais, the activity of freeradical-acceptor antioxidants is also significantly diminished because theirreactivity toward LO' is oniy one order of magnitude higher than that of theunsaturated lipids (Erben-Russ er ai 1987). For these reasons, phenolic and otherantioxidants are much less effective in inhibiting the oxidation of linolenate­containing oils, such as soya bean and rapeseed oils, than that oflinoJeate-containing oils, such as sunflower and saffiower oils.

Metal chelators act as preventive antioxidants by complexing metai ions andthus retarding free radical formation and hydroperoxide decomposition. Becauselinolenate hydroperoxides are so readily decomposed in the presence of metaicatalysts. metal chelators are particularly effective in preventing linolenateoxidation. Metal chelators are thus more effective than phenolic antioxidants incontrolling oxidative deterioration of soya bean oil that contains linoienate(Frankel er ai 1959). Antioxidant synergism is a process by which the antioxidanteffect of multi-component systems is reinforced. Significant synergism is generallyobserved between free radical acceptor antioxidants .and metal chelators.Antioxidant synergism is particularly important between natural tocopherols foundin soya bean oil and metal chelators. such as citric acid. which are essential toensure oxidative stability (Frankel er ai 1959). Another type of antioxidantsynergism is produced by reducing agents such as ascorbic acid (Frankel 1989).

PHOTOSENSITISED OXIDAnON

Linolemue esters

Oxygen becomes excited. into the singiet state by an energy transfer mechanismfrom a sensitiser (such as chlorophyU) that has been exposed to light energy (Foote1968). The resulting singlet oxygen reacts with methyllinoleate at least 1500 timesfaster than normal oxygen (Rawls and Van Santen 1970) to form hydroperoxides.The breakdown of hydroperoxides produced by singlet oxygen may go on toinitiate normal free radical autoxidation (Rawls and Van Santen 1970). Eachcarbon-Qrbon double bond of the fatty acids reacts directly with singiet oxygenby a concerted. 'ene' addition to produce hydroperoxides with a double bondshifted to an aliylic position and isomerised to the crans contiguration (Gollnick1978: Frankel 1980, 1982). Methyllinolenate thus forms six Isomers. 9-, lO-, [2-,13-, 15- and 16-hydroperoxides. by singiet oxygen addition at each unsaturatedcarbon. According to the ene addition mechanism an even distribution of theseisomeric hydroperoxides would be expected. However, an uneven distribution wasobserved (Frankel er al 1979). The internal 10-, 12-, 13- and 15-hydroperoxide

500

Fie 3. Formation of bis-etndioxicie:s from oxidisedmethyl Iinoleftate.

EN Frankel

0-0

~(CH2)eCOOCH3H 00'

~~

~J 0-0

~~~

0-0, til a't: _K

isomers of methyl linolenate were foimd in lower concentrations than the external9- and 16-hydroperoxide isomers. The peroxyl radicals of these internal isomerichydroperoxides are readily cyclised in methyl linoleate and methyl linolenate(Mihelich 1980; Frankel et ai 1982; Neff et ai 1982) into hydroperoxyepidioxidesbecause they have a unique homoallylic unsaturation similar to the peroxyl radicalsof the internal hydroperoxides in autoxidised methyllinolenate (Coxon et ai 1981:Neff er ai 1981). Although singlet oxygen participates in the formation of thehydroperoxides. the cyclisation is a facile free radical process occurring as a sidereaction that is not photosensitised lFrankel ItC ai (982). In methyl linolenate.serial cyciisation (H - I) produced hydroperoxy-bis-epidioxides (I - J - K) andhydroperoxybicycioendoperoxides (F) (Neff er ai (982) (Fig 3).

IDhiDition

~-Tocopherol is highly reactive toward singlet oxygen and inhibits photosensitisedoxidation by both physica.Uy quenching singlet oxygen (ie by preventing activationof oxygen into singlet oxygen) and by reacting with it to form stable products.Other natural quenchers sucb as carotenoids protect lipids against photosensitisedoxidation by an energy transfer mechanism (Foote er ai 1970). Carotenoids canalso react with the triplet state of tbe excited sensitisers by a similar energy transfermechanism (Fujimori and Livingston 1957: Krinsky 1979).

In many foods carotenoids are bleached during processing. In distilled soyabean oil esters cHocopherol was found to be more efficient than ,lJ-earotene ininhibiting oxidation photosensitised by chlorophyll (Frankel er ai 1979). Thisgreater activity was attributed to the- dual effect of tocopherol in quenching andreacting with singlet oxygen. With distilled soya bean oil esters a protective effectfor ,lJ-earotene was shown at a concentration of I g kg - l _ Later studies showed

Lipid oxidation 501

soya bean oil that contains natural tocopherols and citric acid to be adequatelyprotected against light oxidation by ,B-carotene at concentrations < 20 mg kg - l

(Warner and Frankel 1987). However. when soya bean oil was stored in the dark.,B-carotene promoted peroxide development. At concentrations > 20 mg kg - l

carotenoids can produce objectionable colour and flavour. and can form secondaryproducts that initiate and promote free radical autoxidation.

DECOMPOSmON OF MONOHYDROPEROXlDES

MechanismFragmentation of hydroperoxides occurs by homolytic and heterolytic cleavagemechanisms (Fraiucel 1982). Homolytic ,B-scission produces alkoxyl radicalintermediates (L and M. Fig 4) that undergo further carbon-earbon splitting.Homolytic cleavage a on one side of the aikoxy carbon forms pentane plus methyl13-oxo-9,11-tridecadienoate from the 13-hydroperoxide of methyl linoleate. andmethyl oetanoate pius 2.4-decadiena! from the 9-hydropcroxide of methyl linoleate(Fig 4). Homolytic cleavage b forms hexana! and methyl 9-oxononanoate fromthe respective 13- and 9-hydropcroxides of methyllinoleate. Under acid conditions,heterolysis produces ether carbocation intermediates (N and O. Fig 4) which cleaveselectively to form the same products as those of the homolytic pathway b. namelyhcxana1 and methyl 9-oxononanoate (Frankel er aJ 1984) (Fig 4).

The literature is not clear on the effect of antioxidants on the decomposition ofhydroperoxides. In one study. ::-tocopherol and butylated hydroxyanisole changed.the carbonyl products formed from the 9-hydroperoxide of linoleic aciddecomposed with copper but not from the corresponding 13-hydroperoxide isomer(Grosch er at 1981). In another study, ,;-tocopherol promoted the formation of

Fig "'- Homolytic and heterolytic scissionmechanisms for the decomposition of hydro.

perOXides.

502 E .V Frankel

~ ..... 9-OiOIU...1I

, ODH

v==v=v;+1RZ.4.7~ '" Me OctllllMa

HOG

Fie S. Main volatile decomposition productS of linolenate ~Rhyciroperoxides. Pr..-I

dienals that produce fishy flavours in the copper-atalysed oxidation of butterfat(Swoboda and Peers 1971). Recently a;-tocopherol and l.~ciohexadiene wereinvestigated to determine how they ati'ect the relative amounts of thermaldecomposition products formed from linoleate hydroperoxides (Frankel andGardner 1989). These hydrogen-donor compounds diminished the relativepercentages of pentane and methyl octanoate and increased the relative percentagesof hexanal and methyl 9-oxononanoate. This effect of 2-tocopherol and1.4-cyclohexadiene was explained by their inhibition of homolytic .B-scission of analkoxyl radical intermediate (cleavage a. Fig 4). and promotion of heterolyticcleavage (Fig 4).

Significant differences were found between the composition of products fromIinolenate hydroperoxides decomposed thermaHy at 150°C and catalytically withferric chloride and ascorbic acid (Frankel er ai 198Th). Figure 5 shows the mainvolatile compounds expected from the 9-. 12-. 13- and 16-hydroperoxide isomersof methyl 1inolenate. Thermal decomposition produced more methyl octanoateand 2.4,7-decatrienal. and less 2.4-heptadienai, methyl 9-oxononanoate andpropanal.. than catalytic decomposition. Aithough these products represent a: smallportion of the total decomposition materials (7'4% by thermal decomposition and2'1 % by catalytic decomposition). they have an important impact on the ITavoura.nd biological effects of lipid oxidation (Frankel [982. (988).

Malonajdebyde fonnation

Malonaldehyde (G. Fig 2) has been assumed to be an important lipid oxidationproduct in foods and biological systems but many studies in the literature havebeen based on the non-specific TBA test. To determine maionaldehyde moredetinitively, a GC procedure was developed based on the stable acetal derivativesformed under mild acid conditions (Frankel and Neff (983). Tn dilute

Lipid oxidation 503

HCl/methanol. hydroperoxides are readily cleaved to the diacetal derivatives andmaionaidehyde is converted to the tetramethyl acetals. This acid decomposition­acetaiation procedure was used to study how much malonaldehyde is formed fromvarious primary and secondary lipid oxidation products.

As expected. the five-membered hydroperoxyepidioxides of methyl linolenate(compound C. Fig 2) provided rich sources of maionaidehyde (Frankel and Neff1983). The bicycloendoperoxides of methyl linolenate (compound F. Fig 2) werealso good sources of maionaidehyde. as predicted in the literature (Dahl ec ai1962; Pryor et ai (916). The bis-epidioxides of methyl linolenate (compound K.Fig 3) and the mono-epidioxides of methyl linoleate. oxidised with singlet oxygen.were better sources of maionaidehyde than the bicycloendoperoxides of methyllinolenate. There was. however. no correlation between the TBA values and theamounts of malonaidehyde found by the GC procedure. The 10.11- and13.15-dihydroperoxides and 9.12- and 13.16-<iihydroperoxides. from methyllinolenate oxidised with singlet oxygen. were important precursors ofmaionaidehyde. As expected. the 9.16- and lO.16-dihydroperoxides did not formany maionaidehyde as measured by the GC method. On the other hand. highvalues were obtained by the TBA test for ail the dihydroperoxides. From the lackof correlation between the direct GC anaiyses for maionaldehyde and the TBAtest. Frankel and Neff (1983) concluded that the importance of malonaldehydemay have been exaggerated in the literature.

The interactions between lipid oxidation products. DNA. metals and reducingagents were investigated by determining the fluorescence formed in a model system(Fujimoto et ai 1984). Hydroperoxyepidioxides (e. Fig 1l. hydroperoxy­bicycloendoperoxides (F. Fig 2). dihydroperoxides (D and E. Fig [) andhydroperoxy-bis-epidioxides (K. Fig 3) from oxidised methyl linolenate were allrich sources of DNA fluorescence in the presence of iron and ascorbic acid.Unsaturated aldehydes were much less active than their corresponding precursorsmethyl linolenate hydroperoxides in forming DNA fluorescence in the presenceof iron and ascorbic acid (Frankel ItC ai 1981al. In the presence of DNA. metalsand reducing agents. maionaldehyde produced very little or no fluorescence andthe TBA test did not correlate with fluorescence formation. Therefore.malonaldehyde may not be so important in its crosslinking properties with DNA.

A rapid headspace capillary GC method was recently developed to determinehexanal as an important volatile product of n-6 polyunsaturated lipid oxidationin rat liver samples (Frankel Itt ai 1989). Total volatiles were also determined bythis method as a measure of total lipid oxidation. This rapid and convenientmethod is a more direct measure of lipid oxidation than the TBA test. which isnon-specific and subject to interference by many substances (Slater 1984l.

OIMERISATION OF HYDROPEROXIDES

Peroxide-linked dimers were identified during the initial autoxidation of methyllinoleate at room temperature (Miyashita ec ai [98a.b I. Peroxide or ether dimersisolated from methyllinoleate hydroperoxides were composed of unsaturated fatty

504

F"II 6. Thermal decomposition of methyllinolenate dialers.

£ .V Frankel

ester units containing hydroperoxy, hydroxy and oxo groups (Miyashita et ai1985). By gel permeation chromatography analyses before and after sodiumborohydride reduction. peroxide dimers were identified as main produet5 frommethyllinoleate and methyllinolenate autoxidised at 40°C (Neff et ai 1988). Thedimers formed at 1S0°C were entirely ether 0' carbon-carbon linked. Diptersformed in the presence of ferric chloride and ascorbic acid consisted of both typesoflin.lcage. Other dimers from hydroperoxyepidioxides and dihydroperoxides weremainly peroxidic in nature.

Significant differences were found between the volatile produet5 from thermaland catalytic decomposition of monomers and corresponding dimers from oxidisedlinolenate (Frankel et ai 1988). Major volatile decomposition products expectedfrom dimer structures P and Q are shown in Fig 6. Cleavage between the peroxidelink and the olefinic side of the 9- and 16-hydroperoxide groups produces methyl9-oxononanoate, which is the most substantial thermal volatile decompositionproduct. C1eavages on the opposite side of the peroxide links form methyl octanoateon one side and propanal on the other side of the first monomer unit of dimer P(Fig 6). Dimer Q undergoes cleavage on the right to produce methyl9-oxononanoate and methyl octanoate and cleavage on the left to producepropanal.

TRIGLYCERIDE AUTOXIDATION

Trilinoiein and trilinoienin were used as models for oxidation studies of vegetableoil trig.lycerides (Frankel et ai 1990: Neff et ai (990). The main autoxidationproducts from trilinolein were identified as mono-, bis- and tris-hydroperoxideswhich are formed by sequential oxygen addition. The mono-hydroperoxides werefurther oxidised to prodUce a mixture of I.J- and 1.2-bis-hydroperoxides. whichwere also oxidised to tris-hydroperoxides (Fig 7). The hydroperoxides werecomposed of a mixture of cis,trans- and crans,rrans-9- and -13-isomers. The

Lipid o:cidalion 505

2-Monoo

-

l.{l.OOH . l.{l.l. 1.00101

l-l«lnO- 3-MI:lrIo-~CWClelI

102,~{~.{:

1.2-811- 1.3-811-I"",,*OIWCIiiidIe

F"11 1. Mcchani.sm of trilinolein autoxidation.

~{~--{:1~ ~

~.QIJWCilCIIII

I.,......,.·· ..

1.2... 1.3-811-IIjOQIJWCilCIIII

102,--{::m..

1olydI0iM' _

F"11 8. Mechanism of trilinolenin autoxidation.

triglyceride position of monohydroperoxides was determined by HPLC and bypancreatic lipolysis. The ratios of the 9- and I3-1inoleate hydroperoxides in the1(3)- relative to the 2-triglyceride position averaged a value of 2. Therefore. theoxidation of trilinolein had no positional preference between the 1(3). and2-triglyceride positions (Neff er a1 1990).

Trilinoienin produced. on autoxidation. 1(3)- and 2-monohydroperoxides.1.3- and 1.2-bis-hydroperoxides and tris-hydroperoxides by sequential oxidation(Fig 8) (Frankel er at 1990). However. in addition to hydroperoxidcs. trilinolcninproduced significant amounts ofhydroperoxyepidioxidcs formed by 1.3-cyclisation(Fig 1). The isomeric composition was the same as that of methyl linolenate(Frankel 1980), 9-. 12-. 13- and 16-hydroperoxidcs. The cyclic peroxides weremixtures of 9- and 16-hydroperoxyepidioxidcs. By HPLC the ratio of thecis,trans 16-linoienate hydroperoxide in the 1(3)- relative to the 2-triglycerideposition was found to be higher (2'3) than that for the corresponding cis.lrans9-linoienate hydroperoxides (l·8). This evidence supports the small preferentialoxygen attack of the n-3 double bond oflinolenate in the 1(3 )-triglyceride positions.

rn contraSt co methyl linoleate and its hydroperoxides. which form SIgnificantamounts of dimers (Miyashita e! ai 1982a.b. 1984. 1985). no evidence was foundfor dimer formation in highly oxidised trilinoiein (NetT e! at 1990). Also. no dimerformation was found when che purified monohydroperoxides of crilinolein were

506 £ tv Frankel

further oxidised. Dimerisation is evidently significant only in the methyl esters ofunsaturated fatty acids because intermolecular condensations of peroxyl radicalsare favoured. On the other hand. further oxidation of the monohydroperoxidesof trilinolein to bis- and tris-hydroperoxides is apparently the preferred reaction.fntramolecular hydrogen abstraction from the linoleoyl residues can evidentlyoccur more favourably than intermolecular condensation of the peroxyl radicals toform dimers. No evidence was found for dimerisation of tris-hydroperoxides. Thiswork therefore demonstrates that simple esters of unsaturated fatty acids do notaecessarily provide valid models for the oxidative dimerisation of unsaturatedtriglycerides.

Autoxidation of synthetic triglycerides containing linoleate and linolenate indifferent· known positions formed monohydroperoxides and hydroperoxy­epidioxides as the main products (Miyashita er ai 1990). By reversed phase HPLCthe linolenate triglyceride components were found to be oxidised twice as much asthe linoleate components. However. the relative triglyceride positions of thelinolenate components had no influence on the rates of cyclisation of their internal12- and 13-monohydroperoxides. LaLaL oxidised faster than LaLLa (L =linoleate. La = linolenate) and LLaL oxidised faster than the corresponding LLLa.The easier interactions between the two linolenoyl residues in LaLnL may explainits lower oxidative stability than LaLLa. On the other hand. the easier interactionsbetween linolenoyl and linoleoyl residues in LLaL may explain its lower oxidativestability than LLLn.

FLAVOUR SIGNIFICANCE OF VOLATILES

The genesis of volatile lipid oxidation products. their flavour and their biologicalsignificance were reviewed previously (Frankel 1980. 1982). The types of flavourimparted by lipid oxidation in foods is extremely difficult to assess because thereis wide variation in the sensory impact of different volatile products. in the methodsused for their determination and in the vocabulary used by taste or odour panelsto describe their defects.

Gas clII'omatographic: methods

Three commonly used capillary GC methods were compared. to determine volatileoxidation compounds in vegetable oils (Snyder er ai 1988). Each method produceddifferent volatile profiles with oxidised soya bean oil. The weighted percentagesof each volatile were calculated in Table 1 on the basis of l-octen-3-ol which hasthe lowest threshold value (Forss 1972) (defined as the lowest concentration of acompound that a pane! can detect). By the direct injection method.crans.cis-2.4-decadienal was the most flavour significant followed by crans.crans­2.4-<iecadienal. l-octen-3-o1. crans.crans-?.4-heptadienal. hexanal and crans.cis-2A­heptadienal. 2-Pentylfuran ranked tenth in importance. and pentane had the leastt1avour significance. By the dynamic headspace method. crans.cis-2.4-<iecadienalwas also the most t1avour significant. followed by crans.rrans-2.4-<iecadienai.crans.cis-2.4-<iecadienal. l-octen-3-o1. hexanal and crans.cis-2A-heptadienai. By the

Lipid o.:cUUuiol'l 507

TABLE 1Flavour significance of volatiles in oxidised soya bean oilG

Major volatiles TH Rei % Weighted %b Relative ordervalues

Df DHS SHS Df DHS SHS Df DHS SHS

t,l-2.4-Decadicna1 O'lO 46-9 4O'S 0'3 4·7 4'[ 0-03 2 2 7t.c-2.4-Decadicna1 0-02 23·8 21'S [-() [,1'9 to·8 (}S I I 3t,l-2.4-Hcptadicna1 (}04 6·5 13·3 2-{) . ['6 3'3 (}5 3 3 3t-2-Hcptcna1 0'20 3·1 6·7 8'3 () 16 (}33 0'4 7 7 St.c-2.4-Hcptadienal O'lO H 5'4 2·5 (}31 (}S4 0'25 6 6 6,,-Hexanal 0-{)8 6·9 5'4 24·7 0'86 (}68 3-[ 5 5 2,,-Pentane 340 4·8 3·7 38'6 (}[4C (}IIC I'[C [ 1 lO 10t-2-Pentenal 1·00 1·9 1'4 1·2 0-02 0-01 0-01 9 8 8I-O<:ten-3-o1 0-01 1·4 1'1 0'3 1·4 1·1 0'3 4 4 42-Pentylfuran 2-00 1·2 I-{) o-S 60QC 60QC 2·5c 10 9 9,,-Pt'opanal (}{)6 0·5 20'6 0-08 3-4 8 I

GTH::athre:shold values (Forss 1972). DI=direa injection. DHS=dynamic headspace.SHS ::& static headspac:. t,l-::a t7'altSPaltS-. t.c - t7'altS.cis-.b Calculated on the basis of I-octen-3-o1 which has the lowest threshold value.C x lO-.o.

static headspac: method.. propanal was the most important flavour volatilefollowed by hexanal. crallS.cis-2.4-decadienal and cl'aIIS.cis-2.4-heptadicnal.Therefore. the amounts of each volatile compound found varied according to themethod. used. To estimate the flavour impact of volatile oxidation products. notonly their relative concentration in a given fat must be known. but also theirrelative threshold values.

A GC sniffing procedure was recently employed by Ullrich and Grosch (1987.1988a.b) and Guth and Grosch (1989) to assess the flavour impact of volatiles inoxidised fatty acids. esters and soya bean oil by an aroma extract dilution analysis.The most potent flavour volatiles found in oxidised linoleic acid included hexanal.cis-2-octenal. cl'aIIS-2-nonenal. l-octen-3-o1 and l-octen-3-one (Ullrich and Grosch1987). The relative contribution of these volatiles depended on the level ofoxidation. with crallS-Z-nonenal being most potent after 24 h oxidation. andhexanal. Z.4-nonadienal and cis-2-octenal being produced in greater amounts after48 and 72 h oxidation. The most significant volatile compounds found in oxidisedmethyl linolenate included crallS.cis-2.6-nonadienal. l.cis-S-oetadien-3-one.crallS.cis-3.'s-oetadien-Z-one and cis-3-hexenal (Ullrich and Grosch 1988a).'Reverted' soya bean oil is defined as having a characteristic flavour defect occurringat low oxidation levels. usually below a peroxide value of 10 (Frankel 1980). Themost flavour potent volatiles found in a .reverted' soya bean oil indudedcis-3-hexena!. octana!. l-octen-J-one. l.cis-5-octadien-J-one. nonana!. crans-Z­nonena!. cis-2-nonena!. cis-J-nonena! and crans-2.cis-6-nonadienal (Ullrich andGrosch 1988b I. In this study the "reverted' soya bean oil was prepared by storageat room temperature under diffused daylight and the volatiles were concentratedby distillation at 50°C prior to capillary GC and sniffing at the GC exit port. fn

508 £ N Frankel

a later study by the same group, nonan-2.4-dione and 3-methyl-nonan-2.4-dionewere identified in a 'reverted' soya bean oil that had been stored at 21-23"C undera northern light exposure (Guth and Grosch 1989), Although these studies provideimportant qualitative data on the flavour impact of certain volatile compoundsin unsaturated fats. they are difficult to compare with other studies in the literaturebecause of the complexity of flavour formation in different unsaturated oils oxidisedunder different conditions and analysed by different methods. Under the conditionsof direct injeaion (Snyder et al 1988) and dynamic headspace (Selke and Frankel1987) capillary GC, the volatile profiles included only four of the potent compoundsreponed by Ullrich and Grosch (1988b) (2-/3-hexenal. octanal. nonanal and2-nonenal) in soya bean oil stored at room temperature in the dark. However,these results on major volatiles that can be readily determined quantitatively bycapillary GC cannot be related to the results of UHrich and Grosch (1987, 1988a.b)and Guth and Grosch (1989. 1990) until an estimate of the concentration of theflavour-intensive volatile compounds found in soya bean oil can be made.

Sensory assessment

Because of the subjeaive nature of panel testing there is much variation in thevocabulary used in the literature by different workers to describe a given volatilecompound. The conditions used for storage are also critical in the assessment ofthe impact of tlavour compounds formed in vegetable oils. In a recent study Warneret a1 (1989) compared the flavour stability of different vegetable oils. Soya beanoil after storage iIi the dark at 60°C was described by a taste panel as grassy andbeany. and low-erucic rapeseed oil as characteristic of cabbage and sulphurflavours; both oils after exposure to intense light were described as grassy, sour,metallic or buttery. In a similar study by Guth and Grosch (1990) soya bean oilafter storage for 30 days at room temperature in daylight was described as strawy,lard-like. beany, green, hay-like. buttery and fatty, and rapeseed oil as green.strawy and fatty.

The diversity of sensory vocabulary used by different investigators to describethe same flavour defect in an edible oil has led to controversy as to what individualproduct or mixture of volatile oxidation products causes the so-<;ajled 'reverted'flavour in soya bean oil. Clearly, a greater understanding of flavour developmentin oxidised lipids is needed. Future progress in this area will require for theanalytical chemist to work more closely with the sensory investigators to correlatequalitative and quantitative flavour analyses with improv~ taste panel techniquesusing commonly agreed terms to describe flavours and odours from oils that havebeen stored under the same conditions.

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510 £ .V Frankel

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