J Sci Food Agric 1991,54,495-511

Review* Recent Advances in Lipid Oxidation

Edwin N Frankel

Department of Food Science & Technology, University of California, Davis, California 956 16, 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 pathway of the autoxidation of methyl linolenate, peroxyl radicals of the internal hydroperoxides undergo rapid 1,3-cyclisation to form hydroperoxyepidioxides. Because linolenate hydroperoxides are relatively unstable, free radical antioxidants are much less effective in linolenate oils than in linoleate oils, Tocopherols and carotenoids effectively inhibit photosensitised oxidation of vegetable oils. Direct gas chromatographic analyses of malonaldehyde do not correlate with the TBA test. Model fluorescence studies indicate that malonaldehyde may not be so important in crosslinking with DNA. In contrast to oxidised methyl linoleate, oxidised trilinolein does not form dimers. Although trilinolein oxidises with no preference between the l (3)- and 2-triglyceridepositions, the n-3 double bond of trilinolenin oxidises more in the l(3)- than in the 2-position. Synthetic triglycerides oxidise in the following decreasing relative rates: LnLnL, LnLLn, LLnL, LLLn (Ln = linolenic and L = linoleic). To estimate the flavour impact of volatile oxidation products their relative threshold values must be considered together with their relative concentration in a given fat .

Key words: Lipids, free radical autoxidation, hydroperoxides, photo- sensitised 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 the 1990 International Lecture addressed to the SCI’s Oils and Fats Group in London, 11 April 1990.

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J Sci Food Ayric 0022-5142/91/$03.50 0 1991 SCI. Printed in Great Britain

496

INTRODUCTION

E N Frankel

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

In the presence of initiators, unsaturated lipids (LH) form carbon-centred alkyl radicals (Lo) and peroxyl radicals (LOO-), which propagate in the presence of oxygen by a free radical chain mechanism to form hydroperoxides (LOOH) as the primary products of autoxidation (Frankel 1980).

LH -+ L*

L* +o, --* LOO*

LOO*+LH -+ LOOH+L* (3) In the presence of light, unsaturated fats can also form hydroperoxides by reacting with singlet oxygen produced by sensitised photooxidation, which is a non-free-radical process (Gollnick 1978).

Lipid hydroperoxides are readily decomposed into a wide range of carbonyl compounds, hydrocarbons, ketones and other materials that contribute to flavour deterioration of foods. Much work has been reported on the volatile oxidation products of unsaturated lipids (Frankel 1982, 1985; Grosch 1987) because they cause rancidity in foods and cellular damage in the body. Different volatile decomposition products are formed according to the relative thermal stabilities of the lipid oxidation precursors and resulting carbonyl products. To evaluate the oxidative and flavour stability of unsaturated edible oils, it is essential to know the structures of the oxidation products, how they decompose, the amounts of volatile compounds produced and the flavour significance of the volatiles.

A better understanding of the mechanisms of oxidation of linoleic and linolenic acids may lead to improved methods for control of flavour deterioration in vegetable oils. Several reviews of the literature have appeared (Frankel 1980, 1985, 1988; Chan 1987; Grosch 1987; Gardner 1989). The mechanism of autoxidation of linoleic acid and esters has received special attention (Porter 1986). This paper summarises recent progress made in understanding the mechanism by which polyunsaturated edible oils can undergo oxidative and flavour deterioration.

FREE RADICAL AUTOXIDATION Linolenate esters

Since 1961, when the isolation of pure hydroperoxides of methyl linolenate was first reported (Frankel et a1 1961), considerable advances have been made in

Lipid oxidation

X- XH

497

initiators . 00. /

14 11

Unoienate

a-Tocopherol

% B

9(16)-00H 12(13)-00H (25%) Monohydroperoxldes (5090

O 2 F L H

L a .702 Hydroperoxy C Epidloxidss *+& E

& OOH OOH OOH

Dlhydroperoxidss D (25%)

Fig 1. Free radical autoxidation of methyl linolenate.

understanding linolenate autoxidation by the application of new powerful separation and analytical tools. The n-3 unsaturation of linolenate provides a key mechanistic feature affecting the nature of its primary and secondary oxidation products. In the presence of free radical initiators, such as heat, metals, irradiation or light, hydrogen transfer occurs with a suitable radical acceptor X- from the two activated doubly allylic methylene groups on carbon-11 and carbon-14 to form two pentadienyl radicals (Fig 1). Reaction with oxygen at the end carbon positions produces a mixture of four peroxyl radicals leading to the corresponding conjugated dienoic 9-, 12-, 13- and 16-hydroperoxides containing an isolated double bond. The fact that the external 9- and 16-hydroperoxides are formed in amounts significantly higher than the internal 12- and 13-hydroperoxides has been known for a long time (Frankel et a1 1961, 1977; Chan and Levett 1977). Only recently has it been possible to explain this uneven distribution of isomeric hydroperoxides of methyl linolenate. The peroxyl radicals of internal 12- and 13-hydroperoxides undergo rapid 1,3-cyclisation (A B) to form five-membered hydroperoxyepidioxides (C, Fig 1) (Coxon et a1 1981; Neff et a1 1981). This rapid cyclisation is a major pathway which accounts for the lower concentrations of the internal 12- and 13-hydroperoxides (25%) relative to the external 9- and 16- hydroperoxides (50%) (Frankel et a1 1961, 1977). By adding 5% a-tocopherol as a hydrogen donor, Peers et a1 (1981) showed that this cyclisation was completely inhibited, methyl linolenate producing an even distribution of the 9-, 12-, 13- and 16-hydroperoxide isomers. A mixture of dihydroperoxides (D and E) is formed in smaller concentrations than the hydroperoxyepidioxides, by a reaction competing

498 E N Frankel

0-0

B

I

0-0 OOH d - C

Hydroperoxy Epidioxides

oon F / Bicycloendoperoxides 0 0 w

0 Malonaldehyde

Fig 2. Formation of bicycloendoperoxides and malonaldehyde from oxidised methyl linolenate.

with cyclisation (Neff et a1 1981). Coxon et a1 (1984) showed that in the presence of 10% a-tocopherol the 9J6-dihydroperoxide (E) was formed selectively during oxidation of methyl linolenate.

The intermediate free radical (B) formed after cyclisation can either cyclise again to form bicycloendoperoxides (F), structurally related to the prostaglandins, or undergo cleavage to produce malonaldehyde (G) and to give a positive thiobarbituric acid (TBA) test (Fig 2) (Dahl et a1 1962; Pryor et a1 1976). In contrast to methyl linoleate (Porter 1986; Chan 1987), the cis, trans-hydroperoxides of methyl linolenate are not readily isomerised to the trans,trans configuration, apparently because cyclisation is favoured much more than geometric isomerisation (Porter et a1 198 1). The bicycloendoperoxides from oxidised linolenate were shown by O’Connor et al(1984) to have mainly cis substituents in contrast to the natural trans stereochemistry of the enzymically derived prostaglandins. The physiological importance of this structural difference has not been established.

Inhibition

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

LOO. +AH + LOOH + A* (4)

L * + Q * -+ LQ* (5 )

The first class of antioxidants (AH) includes hindered phenols such as butylated hydroxyanisole, butylated hydroxytoluene and a-tocopherol. To be effective, these compounds must compete with the unsaturated lipid substrate (reaction 3) which is normally present in the highest concentration. The second class of antioxidants (Q- ) includes quinones such as ubiquinone and a-tocopheroquinone which must compete with 0, in the fast reaction (2). These compounds may therefore only be active in biological systems where the oxygen pressure is relatively low.

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

Lipid oxidation 499

decomposed to form alkoxyl radical intermediates (LO.) and (LOO-), which can effectively propagate the free radical chain:

LOOH+M" + LO*+OH-+Mn"+l (6) LOOH+M"+' -+ LOO*+H++M" (7)

The catalytic effect of metals will be greatly enhanced in methyl linolenate because linolenate hydroperoxides are much more readily decomposed than linoleate hydroperoxides (Frankel 1962). In the presence of metals, the activity of free radical-acceptor antioxidants is also significantly diminished because their reactivity toward LO* is only one order of magnitude higher than that of the unsaturated lipids (Erben-Russ et al 1987). For these reasons, phenolic and other antioxidants are much less effective in inhibiting the oxidation of linolenate- containing oils, such as soya bean and rapeseed oils, than that of linoleate-containing oils, such as sunflower and safflower oils.

Metal chelators act as preventive antioxidants by complexing metal ions and thus retarding free radical formation and hydroperoxide decomposition. Because linolenate hydroperoxides are so readily decomposed in the presence of metal catalysts, metal chelators are particularly effective in preventing linolenate oxidation. Metal chelators are thus more effective than phenolic antioxidants in controlling oxidative deterioration of soya bean oil that contains linolenate (Frankel et a1 1959). Antioxidant synergism is a process by which the antioxidant effect of multi-component systems is reinforced. Significant synergism is generally observed between free radical acceptor antioxidants and metal chelators. Antioxidant synergism is particularly important between natural tocopherols found in soya bean oil and metal chelators, such as citric acid, which are essential to ensure oxidative stability (Frankel et a1 1959). Another type of antioxidant synergism is produced by reducing agents such as ascorbic acid (Frankel 1989).

PHOTOSENSITISED OXIDATION Linolenate esters

Oxygen becomes excited into the singlet state by an energy transfer mechanism from a sensitiser (such as chlorophyll) that has been exposed to light energy (Foote 1968). The resulting singlet oxygen reacts with methyl linoleate at least 1500 times faster than normal oxygen (Rawls and Van Santen 1970) to form hydroperoxides. The breakdown of hydroperoxides produced by singlet oxygen may go on to initiate normal free radical autoxidation (Rawls and Van Santen 1970). Each carbon-carbon double bond of the fatty acids reacts directly with singlet oxygen by a concerted 'ene' addition to produce hydroperoxides with a double bond shifted to an allylic position and isomerised to the trans configuration (Gollnick 1978; Frankel 1980, 1982). Methyl linolenate thus forms six isomers, 9-, lo-, 12-, 13-, 15- and 16-hydroperoxides, by singlet oxygen addition at each unsaturated carbon. According to the ene addition mechanism an even distribution of these isomeric hydroperoxides would be expected. However, an uneven distribution was observed (Frankel et a1 1979). The internal lo-, 12-, 13- and 15-hydroperoxide

500 E N Frankel

(CH&COOCH, H 00.

Bicycloendoperoxides F

I 0-0

l o 2 + J 0-0 - 0-0 Fig 3. Formation of bis-epidioxides from oxidised Hydroperov bis-epidioxldes

methyl linolenate. K

isomers of methyl linolenate were found in lower concentrations than the external 9- and 16-hydroperoxide isomers. The peroxyl radicals of these internal isomeric hydroperoxides are readily cyclised in methyl linoleate and methyl linolenate (Mihelich 1980; Frankel et a1 1982; Neff et al 1982) into hydroperoxyepidioxides because they have a unique homoallylic unsaturation similar to the peroxyl radicals of the internal hydroperoxides in autoxidised methyl linolenate (Coxon et a1 1981 ; Neff et al 1981). Although singlet oxygen participates in the formation of the hydroperoxides, the cyclisation is a facile free radical process occurring as a side reaction that is not photosensitised (Frankel et a1 1982). In methyl linolenate, serial cyclisation (H + I) produced hydroperoxy-bis-epidioxides (I -+ J + K) and hydroperoxybicycloendoperoxides (F) (Neff et a1 1982) (Fig 3).

Inhibition

a-Tocopherol is highly reactive toward singlet oxygen and inhibits photosensitised oxidation by both physically quenching singlet oxygen (ie by preventing activation of oxygen into singlet oxygen) and by reacting with it to form stable products. Other natural quenchers such as carotenoids protect lipids against photosensitised oxidation by an energy transfer mechanism (Foote et a1 1970). Carotenoids can also react with the triplet state of the excited sensitisers by a similar energy transfer mechanism (Fujimori and Livingston 1957; Krinsky 1979).

In many foods carotenoids are bleached during processing. In distilled soya bean oil esters a-tocopherol was found to be more efficient than 0-carotene in inhibiting oxidation photosensitised by chlorophyll (Frankel et a1 1979). This greater activity was attributed to the dual effect of tocopherol in quenching and reacting with singlet oxygen. With distilled soya bean oil esters a protective effect for p-carotene was shown at a concentration of 1 g kg-l. Later studies showed

Lipid oxidation 501

soya bean oil that contains natural tocopherols and citric acid to be adequately protected against light oxidation by /?-carotene at concentrations < 20 mg kg- (Warner and Frankel 1987). However, when soya bean oil was stored in the dark, /?-carotene promoted peroxide development. At concentrations > 20 mg kg- carotenoids can produce objectionable colour and flavour, and can form secondary products that initiate and promote free radical autoxidation.

DECOMPOSITION OF MONOHYDROPEROXIDES

Mechanism Fragmentation of hydroperoxides occurs by homolytic and heterolytic cleavage mechanisms (Frankel 1982). Homolytic /?-scission produces alkoxyl radical intermediates (L and M, Fig 4) that undergo further carbon-carbon splitting. Homolytic cleavage a on one side of the alkoxy carbon forms pentane plus methyl 13-0x0-9,ll-tridecadienoate from the 13-hydroperoxide of methyl linoleate, and methyl octanoate plus 2,4-decadienal from the 9-hydroperoxide of methyl linoleate (Fig 4). Homolytic cleavage b forms hexanal and methyl 9-oxononanoate from the respective 13- and 9-hydroperoxides of methyl linoleate. Under acid conditions, heterolysis produces ether carbocation intermediates (N and 0, Fig 4) which cleave selectively to form the same products as those of the homolytic pathway b, namely hexanal and methyl 9-oxononanoate (Frankel et al 1984) (Fig 4).

The literature is not clear on the effect of antioxidants on the decomposition of hydroperoxides. In one study, a-tocopherol and butylated hydroxyanisole changed the carbonyl products formed from the 9-hydroperoxide of linoleic acid decomposed with copper but not from the corresponding 13-hydroperoxide isomer (Grosch et al 1981). In another study, a-tocopherol promoted the formation of

k.0. ksoH Homolytic

L R' L R R&M

Y k Y k Pentane Hexanal Methyl octanoate Methyl S-oxo-

M&h iSOx0- t 2,4-Decadienal nonanoate 9,i i-mdL3dienoate _______________-_______________

H,O Heterolytic "$ H,O

N

H20 J- "t Hexanal 9.0xmonanoate Fig 4. Homolytic and heterolytic scission

mechanisms for the decomposition of hydro- R L. (CH&COOCH, R' (CH,),CH, peroxides.

502 E N Frankel

Me 9.0xononanoate

2,4,7.0ecatrienal* Me octanoate

OOH

2,CHeptadienal r /

HOO

*R 2.I3-Hexenal 2

HOO

Fig 5. Main volatile decomposition products of linolenate hydroperoxides. Propanal

dienals that produce fishy flavours in the copper-catalysed oxidation of butterfat (Swoboda and Peers 1977). Recently a-tocopherol and 1,4-~yclohexadiene were investigated to determine how they affect the relative amounts of thermal decomposition products formed from linoleate hydroperoxides (Frankel and Gardner 1989). These hydrogen-donor compounds diminished the relative percentages of pentane and methyl octanoate and increased the relative percentages of hexanal and methyl 9-oxononanoate. This effect of a-tocopherol and 1 ,Ccyclohexadiene was explained by their inhibition of homolytic p-scission of an alkoxyl radical intermediate (cleavage a, Fig 4), and promotion of heterolytic cleavage (Fig 4).

Significant differences were found between the composition of products from linolenate hydroperoxides decomposed thermally at 150°C and catalytically with ferric chloride and ascorbic acid (Frankel et a1 1987b). Figure 5 shows the main volatile compounds expected from the 9-, 12-, 13- and 16-hydroperoxide isomers of methyl linolenate. Thermal decomposition produced more methyl octanoate and 2,4,7-decatrienal, and less 2,4-heptadienal, methyl 9-oxononanoate and propanal, than catalytic decomposition. Although these products represent a small portion of the total decomposition materials (7.4% by thermal decomposition and 2.1 YO by catalytic decomposition), they have an important impact on the flavour and biological effects of lipid oxidation (Frankel 1982, 1988).

Malonaldehyde formation

Malonaldehyde (G, Fig 2) has been assumed to be an important lipid oxidation product in foods and biological systems but many studies in the literature have been based on the non-specific TBA test. To determine malonaldehyde more definitively, a GC procedure was developed based on the stable acetal derivatives formed under mild acid conditions (Frankel and Neff 1983). In dilute

Lipid oxidation 503

HCl/methanol, hydroperoxides are readily cleaved to the diacetal derivatives and malonaldehyde is converted to the tetramethyl acetals. This acid decomposition- acetalation procedure was used to study how much malonaldehyde is formed from various primary and secondary lipid oxidation products.

As expected, the five-membered hydroperox yepidioxides of methyl linolenate (compound C, Fig 2) provided rich sources of malonaldehyde (Frankel and Neff 1983). The bicycloendoperoxides of methyl linolenate (compound F, Fig 2) were also good sources of malonaldehyde, as predicted in the literature (Dahl et a1 1962; Pryor et al 1976). 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 malonaldehyde than the bicycloendoperoxides of methyl linolenate. There was, however, no correlation between the TBA values and the amounts of malonaldehyde found by the GC procedure. The 10,12- and 13,15-dihydroperoxides and 9,12- and 13,16-dihydroperoxides, from methyl linolenate oxidised with singlet oxygen, were important precursors of malonaldehyde. As expected, the 9,16- and 10,16-dihydroperoxides did not form any malonaldehyde as measured by the GC method. On the other hand, high values were obtained by the TBA test for all the dihydroperoxides. From the lack of correlation between the direct GC analyses for malonaldehyde and the TBA test, Frankel and Neff (1983) concluded that the importance of malonaldehyde may have been exaggerated in the literature.

The interactions between lipid oxidation products, DNA, metals and reducing agents were investigated by determining the fluorescence formed in a model system (Fujimoto et al 1984). Hydroperoxyepidioxides (C, Fig l), hydroperoxy- bicycloendoperoxides (F, Fig 2), dihydroperoxides (D and E, Fig 1) and hydroperoxy-bis-epidioxides (K, Fig 3) from oxidised methyl linolenate were all rich sources of DNA fluorescence in the presence of iron and ascorbic acid. Unsaturated aldehydes were much less active than their corresponding precursors methyl linolenate hydroperoxides in forming DNA fluorescence in the presence of iron and ascorbic acid (Frankel et al 1987a). In the presence of DNA, metals and reducing agents, malonaldehyde produced very little or no fluorescence and the 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 determine hexanal as an important volatile product of n-6 polyunsaturated lipid oxidation in rat liver samples (Frankel et aE 1989). Total volatiles were also determined by this method as a measure of total lipid oxidation. This rapid and convenient method is a more direct measure of lipid oxidation than the TBA test, which is non-specific and subject to interference by many substances (Slater 1984).

DIMERISATION OF HYDROPEROXIDES

Peroxide-linked dimers were identified during the initial autoxidation of methyl linoleate at room temperature (Miyashita et a1 1982a,b). Peroxide or ether dimers isolated from methyl linoleate hydroperoxides were composed of unsaturated fatty

504 E N Frankel

,---- Propanal

P I ' :L Me 9-Oxononanoate

10 Q >t---, Me 9-Oxononanoote

I , Propanal ---' '.- Me ktonoate

Fig 6. Thermal decomposition of methyl linolenate dimers. R = (CH&COOCH,

ester units containing hydroperoxy, hydroxy and 0x0 groups (Miyashita et a1 1985). By gel permeation chromatography analyses before and after sodium borohydride reduction, peroxide dimers were identified as main products from methyl linoleate and methyl linolenate autoxidised at 40°C (Neff et a1 1988). The dimers formed at 150°C were entirely ether or carbon-carbon linked. Dimers formed in the presence of ferric chloride and ascorbic acid consisted of both types of linkage. Other dimers from hydroperox yepidioxides and dihydroperoxides were mainly peroxidic in nature.

Significant differences were found between the volatile products from thermal and catalytic decomposition of monomers and corresponding dimers from oxidised linolenate (Frankel et al 1988). Major volatile decomposition products expected from dimer structures P and Q are shown in Fig 6. Cleavage between the peroxide link and the olefinic side of the 9- and 16-hydroperoxide groups produces methyl 9-oxononanoate, which is the most substantial thermal volatile decomposition product. Cleavages on the opposite side of the peroxide links form methyl octanoate on 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 methyl 9-oxononanoate and methyl octanoate and cleavage on the left to produce propanal.

TRIGLYCERIDE AUTOXIDATION

Trilinolein and trilinolenin were used as models for oxidation studies of vegetable oil triglycerides (Frankel et a1 1990; Neff et a1 1990). The main autoxidation products from trilinolein were identified as mono-, bis- and tris-hydroperoxides which are formed by sequential oxygen addition. The mono-hydroperoxides were further oxidised to produce a mixture of 1,3- and 1,2-bis-hydroperoxides, which were also oxidised to tris-hydroperoxides (Fig 7). The hydroperoxides were composed of a mixture of &,trans- and trans,trans-9- and -13-isomers. The

Lipid oxidation 505

L i : L f T + L 1 l o H + HOoL{' L

Trilinolein 1-Mono- s-~ono- 2-MWO- Hydroperoxides

U M H

H o o ~ ~ ~ + LfLooH - HooL{

1,2-BS- 1,3-Bis- Tris- LOOH WOH

Hydroperoxides Hydroperoxides

Fig 7. Mechanism of trilinolein autoxidation.

Ltl{T: HOOLn{h % Ln

1 (3)-MOnO- 2-Mono- Hydroperoxides

i

1: + LnWHEpi +TEPi 2-MOno-

Trilindenin

1 (3)-Mono- Hydroperoxy epidioxides

HOOLn c :TH 1,Z-BlS- 1.3-Bis- hOOH

Hydroperoxides

i" HOOLn 4

LnOOH Tris-

Hydroperoxides

Fig 8. Mechanism of trilinolenin autoxidation.

triglyceride position of monohydroperoxides was determined by HPLC and by pancreatic lipolysis. The ratios of the 9- and 13-linoleate hydroperoxides in the 1(3)- relative to the 2-triglyceride position averaged a value of 2. Therefore, the oxidation of trilinolein had no positional preference between the l(3)- and 2-triglyceride positions (Neff et al 1990).

Trilinolenin produced, on autoxidation, l(3)- and 2-monohydroperoxides, 1,3- and 1,2-bis-hydroperoxides and tris-hydroperoxides by sequential oxidation (Fig 8) (Frankel et al 1990). However, in addition to hydroperoxides, trilinolenin produced significant amounts of hydroperoxyepidioxides 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-hydroperoxides. The cyclic peroxides were mixtures of 9- and 16-hydroperoxyepidioxides. By HPLC the ratio of the cis,trans 16-linolenate hydroperoxide in the l(3)- relative to the 2-triglyceride position was found to be higher (2.3) than that for the corresponding cis,trans 9-linolenate hydroperoxides (1.8). This evidence supports the small preferential oxygen attack of the n-3 double bond of linolenate in the 1(3)-triglyceride positions.

In contrast to methyl linoleate and its hydroperoxides, which form significant amounts of dimers (Miyashita et al 1982a,b, 1984, 1985), no evidence was found for dimer formation in highly oxidised trilinolein (Neff et al 1990). Also, no dimer formation was found when the purified monohydroperoxides of trilinolein were

506 E N Frankel

further oxidised. Dimerisation is evidently significant only in the methyl esters of unsaturated fatty acids because intermolecular condensations of peroxyl radicals are favoured. On the other hand, further oxidation of the monohydroperoxides of trilinolein to bis- and tris-hydroperoxides is apparently the preferred reaction. Intramolecular hydrogen abstraction from the linoleoyl residues can evidently occur more favourably than intermolecular condensation of the peroxyl radicals to form dimers. No evidence was found for dimerisation of tris-hydroperoxides. This work therefore demonstrates that simple esters of unsaturated fatty acids do not necessarily provide valid models for the oxidative dimerisation of unsaturated triglycerides.

Autoxidation of synthetic triglycerides containing linoleate and linolenate in different known positions formed monohydroperoxides and hydroperoxy- epidioxides as the main products (Miyashita et al 1990). By reversed phase HPLC the linolenate triglyceride components were found to be oxidised twice as much as the linoleate components. However, the relative triglyceride positions of the linolenate components had no influence on the rates of cyclisation of their internal 12- and 13-monohydroperoxides. LnLnL oxidised faster than LnLLn (L = linoleate, Ln = linolenate) and LLnL oxidised faster than the corresponding LLLn. The easier interactions between the two linolenoyl residues in LnLnL may explain its lower oxidative stability than LnLLn. On the other hand, the easier interactions between linolenoyl and linoleoyl residues in LLnL may explain its lower oxidative stability than LLLn.

FLAVOUR SIGNIFICANCE OF VOLATILES

The genesis of volatile lipid oxidation products, their flavour and their biological significance were reviewed previously (Frankel 1980, 1982). The types of flavour imparted by lipid oxidation in foods is extremely difficult to assess because there is wide variation in the sensory impact of different volatile products, in the methods used for their determination and in the vocabulary used by taste or odour panels to describe their defects.

Gas chromatographic methods

Three commonly used capillary GC methods were compared to determine volatile oxidation compounds in vegetable oils (Snyder et a1 1988). Each method produced different volatile profiles with oxidised soya bean oil. The weighted percentages of each volatile were calculated in Table 1 on the basis of l-octen-3-01 which has the lowest threshold value (Forss 1972) (defined as the lowest concentration of a compound that a panel can detect). By the direct injection method, trans,cis-2,4-decadienal was the most flavour significant followed by trans,trans- 2,4-decadienal, l-octen-3-01, trans,trans-2,4-heptadienal, hexanal and trans,cis-2,4- heptadienal. 2-Pentylfuran ranked tenth in importance, and pentane had the least flavour significance. By the dynamic headspace method, trans,cis-2,4-decadienal was also the most flavour significant, followed by trans,trans-2,4-decadienal, trans,cis-2,4-decadienal, I-octen-3-01, hexanal and trans,cis-2,4-heptadienal. By the

Lipid oxidation 507

TABLE 1 Flavour significance of volatiles in oxidised soya bean oil"

Major volatiles TH Re1 YO Weighted YO Relative order

DI D H S S H S DI D H S SHS DI D H S S H S values

t,t-2,4-Decadienal 0.10 46.9 40.5 0.3 4 7 4.1 0.03 2 2 7 t,c-2,4-Decadienal 0.02 23% 21.5 1.0 11.9 10.8 0.5 1 1 3 t,t-2,4-Heptadienal 0.04 6.5 13.3 2.0 1.6 3.3 0.5 3 3 3 t-2-Heptenal 0.20 3.1 6.7 8.3 0.16 0.33 0.4 7 7 5 t,c-2,4-Heptadienal 0.10 3.1 5.4 2.5 0.31 0.54 0.25 6 6 6 n-Hexanal 0-08 6-9 5.4 24-7 086 0.68 3.1 5 5 2 n-Pentane 340 4.8 3.7 38.6 0.14' 0.11' 1.1' 11 10 10 t-2-Pentenal 1.00 1.9 1.4 1.2 0.02 0.01 0.01 9 8 8 1-Octen-3-01 0.01 1-4 1.1 0.3 1.4 1.1 0.3 4 4 4 2-Pent ylfuran 2.00 1-2 1.0 0 3 6.0' 6.0' 2.5' 10 9 9 n-Propanal 0.06 0.5 - 20.6 0.08 - 3.4 8 - 1

TH = threshold values (Forss 1972), DI = direct injection, DHS = dynamic headspace, SHS = static headspace, t,t- = transpans-, t,c = trans,cis-. ' Calculated on the basis of 1-octen-3-01 which has the lowest threshold value. c x 10-3.

static headspace method, propanal was the most important flavour volatile followed by hexanal, trans,cis-2,4-decadienal and trans,cis-2,4-heptadienal. Therefore, the amounts of each volatile compound found varied according to the method used. To estimate the flavour impact of volatile oxidation products, not only their relative concentration in a given fat must be known, but also their relative 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 in oxidised 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, trans-Znonenal, 1-octen-3-01 and 1-octen-3-one (Ullrich and Grosch 1987). The relative contribution of these volatiles depended on the level of oxidation, with trans-Znonenal being most potent after 24 h oxidation, and hexanal, 2,4-nonadienal and cis-2-octenal being produced in greater amounts after 48 and 72 h oxidation. The most significant volatile compounds found in oxidised methyl linolenate included trans,cis-2,6-nonadienal, l,cis-5-octadien-3-one, trans,cis-3,5-octadien-2-one and cis-3-hexenal (Ullrich and Grosch 1988a). 'Reverted' soya bean oil is defined as having a characteristic flavour defect occurring at low oxidation levels, usually below a peroxide value of 10 (Frankel 1980). The most flavour potent volatiles found in a 'reverted' soya bean oil included cis-3-hexenal, octanal, l-octen-3-one, l,cis-5-octadien-3-one, nonanal, trans-2- nonenal, cis-2-nonena1, cis-3-nonenal and trans-2,cis-6-nonadienal (Ullrich and Grosch 1988b). In this study the 'reverted' soya bean oil was prepared by storage at room temperature under diffused daylight and the volatiles were concentrated by distillation at 50°C prior to capillary GC and sniffing at the GC exit port. In

E N Frankel 508

a later study by the same group, nonan-2,4-dione and 3-methyl-nonan-2,4-dione were identified in a ‘reverted’ soya bean oil that had been stored at 21-23°C under a northern light exposure (Guth and Grosch 1989). Although these studies provide important qualitative data on the flavour impact of certain volatile compounds in unsaturated fats, they are difficult to compare with other studies in the literature because of the complexity of flavour formation in different unsaturated oils oxidised under different conditions and analysed by different methods. Under the conditions of direct injection (Snyder et al 1988) and dynamic headspace (Selke and Frankel 1987) capillary GC, the volatile profiles included only four of the potent compounds reported by Ullrich and Grosch (1988b) (2-/3-hexenal, octanal, nonanal and 2-nonenal) in soya bean oil stored at room temperature in the dark. However, these results on major volatiles that can be readily determined quantitatively by capillary GC cannot be related to the results of Ullrich and Grosch (1987, 1988a,b) and Guth and Grosch (1989, 1990) until an estimate of the concentration of the flavour-intensive volatile compounds found in soya bean oil can be made.

Sensory assessment

Because of the subjective nature of panel testing there is much variation in the vocabulary used in the literature by different workers to describe a given volatile compound. The conditions used for storage are also critical in the assessment of the impact of flavour compounds formed in vegetable oils. In a recent study Warner et al (1989) compared the flavour stability of different vegetable oils. Soya bean oil after storage in the dark at 60°C was described by a taste panel as grassy and beany, and low-erucic rapeseed oil as characteristic of cabbage and sulphur flavours; 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 oil after 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 describe the same flavour defect in an edible oil has led to controversy as to what individual product or mixture of volatile oxidation products causes the so-called ‘reverted’ flavour in soya bean oil. Clearly, a greater understanding of flavour development in oxidised lipids is needed. Future progress in this area will require for the analytical chemist to work more closely with the sensory investigators to correlate qualitative and quantitative flavour analyses with improved taste panel techniques using commonly agreed terms to describe flavours and odours from oils that have been stored under the same conditions.

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