Pray. Lipid Res. VoL 19. pp. 1-22(l Pergamon Press Ltd 1980. Printed in Great Britain

LIPID OXIDATION

E. N. FRANKEL

0163~ iS17;SOn 00 1-000 1$05.00;0

Northern Regional Research Center, Agricultural Research,Science and Education Administration, U.S. Department of Agriculture,

Peoria, Illinois 61604, U.S.A.

CONTENTS

I. INTRODUCTIONA. AutoxidationB. AntioxidationC. Early studies in lipid oxidation

II. FATTY ACID FREE RADICAL AUTOXIDATIONA. OleateB. LinoleateC. Linolenate

III. FATTY ACID OXIDATION BY SINGLET OXYGENIV. OXIDATION OF MIXTURES OF FATTY ACIDS AND VEGETABLE OILSV. DECOMPOSITION OF HYDROPEROXIDES

VI. FLAVOR REVERSIONA. Causes of flavor reversion

I. Linolenic acid2. Phosphatides3. Nonglyceride components

B. Control measures1. Metal inactivators2. Processing with minimum exposure to oxidation3. Hydrogenation

VII. CONCLUSIONS

VIII. REFERENCES

1. INTRODUCTION

1I233444588

15IS151717171717181919

The problem of oxidative deterioration is of greatest economic importance in theproduction of lipid-containing foods. Oxidation of unsaturated lipids not only producesoffensive odors and flavors but can also decrease the nutritional quality and safety by theformation of secondary reaction products in foods after cooking and processing. Con­siderable attention has been given to the evaluation and assessment of oxidative andflavor deterioration, but not enough effort has been made to look into the basic sourcesof offensive odors and flavors in oxidized fat. This review summarizes mechanistic con­cepts of oxidation with the belief that new knowledge in these areas would lead to moreuseful methods of controlling lipid deterioriation. Soybean oil, which provides now themost important world source of vegetable food fat, develops a very complex type of lipiddeterioration known incorrectly as "flavor reversion". The last section of this review dealswith this flavor problem, which still troubles the soybean oil industry and the consumerdespite the many advances made in processing and packaging.

A. Autoxidationi8.82.155

Oxygen reacts with many organic substrates (RH) to yield hydroperoxides and otheroxygenated compounds. This oxidation is in most instances a free radical chain reactionthat can be described in terms of initiation, propagation, and termination processes.

Initiation: RH __i_oit_ia_'o_r_) R·

This production of free radicals may take place by direct thermal dissociation (thermo-

2 E. N. Frankel

lysis), by hydroperoxide decomposition, by metal catalysis and by exposure to light(photolysis) with or without the intervention of photosensitizers.

Propagation: R· + 02'=;: ROO· (2)ROO, + RH ........ ROOH +R· (3)

The susceptibility of organic substrates to autoxidation depends on their relative ease todonate a hydrogen by reaction (3). With unsaturated fats, susceptibility to autoxidation isdependent on the availability of aIIyIic hydrogens for reactions with peroxy radicals.

2 3

ROO· + R-eH 2-CH=CH-R' ........ R-CH-CH=CH-R' + ROOH. (A)

Valence bond structure (A) may be represented as a hybrid (B) with a partial free radicalat each end of the allylic system. Reaction of oxygen occurs at end carbon positions ofthe aIIyIic system to produce a mixture of isomeric hydroperoxides.

2 3

(A) == R-CH-CH-CH-R'-----------6' 6'

(B)! 3

~ R-CH(OO' )-CH=CH-R' + R-CH=CH-eH(OO' )-R'

RH 1Allylic 1- and 3-hydroperoxides

Termination: ROO, + ROO, ........ nonradical products (4)

The most important termination process for secondary peroxy radicals at room tempera­ture is that proposed by Russell! 2

7 and involving a tetroxide intermediate to produce aketone, an alcohol, and oxygen.

I __2 C-OO,

I --

/0,

,,9 9 I \ IC O-C-H -- C:O + HO-C-H + O2

~ ~H I / I

There is evidence that the oxygen produced above is activated in the singlet state. 79 Therole of singlet oxygen in oxidation will be discussed in Section III.

B. Antioxidation 2

Autoxidation can be inhibited or retarded by adding low concentrations of chain­breaking antioxidants (AH) that interfere with either chain propagation or initiation.

ROO· + AH <=t ROOH + A·

A· +A ROOA } nonradical products. + ........

(5)

(6)

Chain-breaking antioxidants include phenol and aromatic amino compounds hinderedwith bulky alkyl substituents. These compounds are designed to form either A· radicalso(Joose molecular complexes that are too unreactive to propagate the chain.

ROO, + AH<=t[AH-ROO·J

[AH-ROO' J + ROO ........ nonradical products

Lipid oxidation 3

Common chain-breaking antioxidants used in food lipids include butylated hydroxyani­sole (BHA), butylated hydroxytoluene (BHT), butylated hydroquinone (TBHQ), and pro­pyl gallate. These compounds generally lose their efficiency at elevated temperaturesbecause of homolytic decomposition of hydroperoxides formed by reaction (5) andbecause of a reaction of the antioxidants with oxygen (7).

AH + O2-> free radicals (7)

Preventive antioxidants overcome these disadvantages by acting to reduce the rate ofchain initiation. The most important initiation suppressors are metal inactivators thatcoordinate with metal ions catalyzing chain initiation. Metal inactivators used for stabi­lizing edible fat and lipid-containing foods include citric, phosphoric and ascorbic acid.Other types of preventive antioxidants are the peroxide destroyers, which react withhydroperoxides to give stable products by nonradical processes. These inhibitors includesulfur compounds, phosphites and phosphines, which reduce hydroperoxides into themore stable alcohols. Whether or not these compounds can be used in foods has notbeen established.

One more type of preventive antioxidant includes ultraviolet light deactivators thatabsorb irradiation without formation of radicals. Examples are pigments like carbonblack, phenyl salicylate, and a-hydroxy-benzophenone.

Synergism is known for the reinforcing effect of multi-component stabilizer systemsexhibiting a greater combined effect than the sum of their individual effects. Significantsynergism is generally observed when chain-breaking and preventive antioxidants areused together because they suppress both initiation and propagation. The synergisticeffect of common antioxidants in combination with metal inactivators in foods has beenknown for a long time. 18

C. Ear!.v Studies in Lipid Oxidation

Previous reviews3.74.102.1 03.1 09.116.11 7.128.138.140.141.144.145.147 covered the studies of

early workers in the field. Farmer and his group at the British Rubber Producers'Research Association6-8.33-36 developed the generally accepted free radical mechanismof autoxidation involving attack of oxygen at the allylic position with the formation ofunsaturated hydroperoxides. Other pioneers in this field and their major contributionsinclude: Lundberg, Holman, Privett and Chipault at the Hormel Insti­tute: 18.74-76.88.101-103.121-123 autoxidation mechanism and kinetics of antioxidation ofunsaturated fatty acids; Lea and Swoboda at the Low-Temperature Research Station inCambridge95-98.148 (now at the Food Research Institute in Norwich): methodology forperoxides and volatiles, tocopherols and antioxidants; Dutton, Kawahara, Cowan andEvans at the Northern Regional Research Laboratory:9.22.24.28.56.57.85.157.158 separ-ation of oxidation products by countercurrent distribution, volatile carbonyl compounds,flavor reversion and analysis of volatiles; Swern, Knight and Riemenschneider at theEastern Regional Research Laboratory :89.90.137.143-147 trans isomerization in autoxi­dized oleate, decomposition products of fatty acid oxidation, and antioxidants; Tappeland Olcott at the University of California at Davis:113-115.149.150 vitamin E, in vivooxidation, antioxidants and marine lipid oxidation. The early contributions of these andother workers in lipid oxidation were summarized in the Symposium on Food: Lipidsand Their Oxidation held at Oregon State University in 1961,131

II. FATTY ACID FREE RADICAL AUTOXIDATION

The susceptibility of unsaturated fatty acids to autoxidation varies according to thelability of their allylic hydrogens. The hydroperoxidation of unsaturated fatty acids hasbeen reviewed in detail previously.44.45 This subject will only be summarized here as abasis for further discussion of autoxidation of unsaturated fats (Section IV) and de­composition of hydroperoxides (Section V).

9·00H

11·00H

4 E. N. Frankel

I-H, OOH Hydroperoxides

.0\2+ ~ ~___ -.-I ~ 10-00H

~ tH' 98

Or --....~ 8-00H

OOH

10 OOH02 'J!--(

I ..--1/ "'-.-F\.- tH· 9

"02 --.... --<LOOH

FIG. I. Mechanism of oleate autoxidation_

A. Oleate

The most accepted mechanism of autoxidation involves hydrogen abstraction on car­bon-8 and carbon-II with formation of two allylic radicals (Fig. 1). These radical inter­mediates react with oxygen at the end carbons to produce a mixture of 8-, 9-, 1O~, andII-allylic hydroperoxides.

12

~9I Ii H I

+-H·

01 .........ry.~/01

I I~I ItH/ "..tHo./ ~ 12 10

HOO~9 ~OOH

13-Hydroperoxide 9-H ydro peroxide

FIG. 2. Mechanism of linoleate autoxidation.

B. Linoleate

Hydrogen abstraction on the reactive doubly allylic carbon-II produces a pentadienylradical (Fig. 2). This intermediate radical reacts at both ends with oxygen to produce amixture of conjugated 9- and 13-diene hydroperoxides.

C. Linolenate

Hydrogen abstraction on the two active methylenes on carbon-ll and carbon-I4produces two pentadienyl radicals (Fig. 3). These intermediates react with oxygen at theend carbons to produce a mixture of conjugated diene-triene 9-, 12-, 13-, and I6-hydro­peroxides.

Quantitative studies of isomeric hydroperoxides showed that only for linoleate did theproduct distribution correspond to that predicted by the free radical mechanism(Table I). For oleate, the 8- and II-hydroperoxides were slightly but consistently higherthan the 9- and IO-hydroperoxides. The difference between these pairs of isomers wasgreatest (II %) when oleate was autoxidized at 40cC and smallest (6%) at 80Ge. Whetherthis difference is due to greater oxygen attack at carbons-8 and -II than at carbons-9 and-10 or to allylic rearrangement of hydroperoxides has not been established.45

Lipid oxidation 5

12· + 16· Hydroperoxides 9· + 13·

FIG. 3. Mechanism of linolenate autoxidation.

For linolenate, the 9- and 16-hydroperoxides were formed in significantly higher pro­portions than the 12- and 13-hydroperoxides. Several explanations can be advanced forthe reduced yields of 12- and 13-hydroperoxides: (a) the pentadiene radicals of linolenateprefer to react with oxygen at the end carbon-9 and carbon-16 either because autoxida­tion is regioselective1 2 ,49 or because of steric effects;45 (b) the 12- and 13-hydroperoxidesare more easily decomposed;49 and (c) the 12- and 13-hydroperoxides undergo eitherl,4-cyclization into six-membered peroxide hydroperoxides or 1,3-cyclization into pros­taglandin-like endoperoxides. 55 A more detailed discussion of the stereochemistry of fatautoxidation is presented in a previous review.45

III. FATTY ACID OXIDATION BY SINGLET OXYGEN

In the most stable triplet state, oxygen is not so reactive with unsaturated compounds.The activation of oxygen by electronic excitation forms singlet oxygen, which reacts

TABLE 1. Isomeric Hydroperoxides of Oleate, Linoleate andLinolenate53- 55

Temperatureof oxidation Peroxide

(0C) value

25 46140 20060 28280 355

40 26140 68660 50580 1249

25 49540 33760 21280 466

Isomeric distributionrelative per cent

Methyl oleate8-0H 9-0H lO-OH ll-OH

26.6 24.3 22.3 26.827.5 22.9 22.1 27.527.1 23.0 22.8 27.126.4 23.8 23.4 26.4

Methyl linoleate9-0H 13-0H

51.7 48.349.7 50.351.5 48.552.5 47.5

Methyllinolenate9-0H 12-0H 13-0H 16-0H

31.7 10.7 10.9 46.730.4 11.9 13.0 44.826.6 10.3 ILl 52.034.8 13.2 10.5 41.4

6

'0 •Oleate~

E. N. Frankel

9

~1~o=iJ'-

tOOH

~10 • 11 9 11 9

llnoleate~ ~j=\ ~\~-r';O=iJv '- volo~r-

OOH t ~ OOH H

~+~O~ O~

~+~

FIG. 4. Reaction of singlet oxygen with oleate and linoleate.

readily with unsaturated fatty acids. This reaction of singlet oxygen proceeds by a differ­ent mechanism than free radical autoxidation.40 Singlet oxygen reacts directly withdouble bonds by concerted addition, the so-called "ene" reaction. Oxygen is thus insertedat either end carbon of a double bond, which is shifted to yield an allylic hydroperoxidein the trans configuration (Fig. 4).

According to this mechanism, oleate produces a mixture of 9- and lO-hydroperoxides,linoleate a mixture of 9-, 10-, 12- and 13-hydroperoxides, and linolenate a mixture of 9-,10-, 12-, 13-, 15- and 16_hydroperoxides.lO.2o.152 Although the relative amounts of theseisomers are well established for oleate (50~~ each of 9- and 10-isomers), no reliablequantitative study has been reported for linoleate and linolenate until recently. Quanti­tative analysis obtained by gas chromatography-mass spectrometry52 showed for lino­leate: 32% 9-, 17% each 10- and 12-, and 35% 13-hydroperoxides. For linolenate, theisomeric distribution showed: 23% 9-, 12 to 14% each 10-, 12-, 13- and 15-, and 25%16-hydroperoxides.

Singlet oxygen can be generated in a great variety of ways. Two recent reviews sum­marize twelve different potential sources of singlet oxygen, many of which are of biologi­cal importance.93 .94 Another report presents a partial list of fifty-three known and postu­lated interconversion reactions of singlet oxygen and related species. 139 With the evi­dence and indication that singlet oxygen is involved in many biochemical processesS.41.94and produced by oxygen sensitizers in environmental pollutants, it is not surprising thatthis aspect of oxidation chemistry has become an intensively active area of research andthat much controversy has resulted.

The most important way of generating singlet oxygen is by exposure to light in thepresence of a photosensitizer. Rawls and van Santen 124 presented evidence that singletoxygen (generated either directly in a radio frequency gas discharge or by photosensitiza­tion with natural pigments) catalyzes the oxidation of methyl linoleate. Singlet oxygenreacted with methyl linoleate at a rate at least 1500 times faster than normal tripletoxygen. 125 It was, therefore, concluded that singlet oxygen may play an important role ininitiating the normal free radical autoxidation of unsaturated fats.

Vegetable oils frequently contain natural photosensitizers such as chlorophylls orheme compounds that yield singlet oxygen in the presence of visible light. In general,olefins undergo photosensitized oxidation by a mechanism in which the sensitizer in thetriplet state is excited by light energy to the singlet state followed by intersystem crossingto an activated triplet state (Fig. 5, mechanism I). Energy is then transferred from thetriplet sensitizer to oxygen to give excited singlet oxygen. In another postulated mechan-

Lipid oxidation 7

Mechanism 1

Sens~ ISens '~ 3S ens '

303S ens'-4 101' + lSens

IA = acceptor]

Mechanism 2

3S ens + 301 ---.. 1ISenS·Ol]

1ISenS·Ol] + A _ A01 + lSens

FIG. 5. Mechanisms of photosensitized oxidation.

ism, the triplet sensitizer forms a sensitizer-oxygen complex that reacts with a substrateacceptor to give a peroxide and regenerates the sensitizer4o.41 (Fig. 5, mechanism II).

Deterioration of unsaturated vegetable oils by singlet oxygen can be effectively reducedby removal of natural photosensitizers during refining and bleaching. However, Clementset a/. 19 found that refined soybean oil can act as a sensitizer in the photooxidation of amodel diene, 4,7-undecadiene. As the soybean oil was well refined, they believed thatchlorophyll-like sensitizers may be unimportant and that other minor unidentified sensi­tizer(s) may be involved. However, even commercially refined soybean oil contains resi­dual porphyrins. Refining and bleaching also remove singlet oxygen quenchers such asthe carotenoids. The restoration of carotenoids may effectively protect lipids againstsinglet oxygen deterioration if the resulting yellow coloration of the oil is not objection­able to the consumer. Another obvious approach is the use of suitable packaging or acontainer that is absorbent to the light energy necessary for photosensitization or thatprevents such light from reaching the oil.

There is evidence for other mechanisms of activating oxygen to the singlet state that donot require light. Russel's mechanism of free radical termination via a tetroxide inter­mediate was discussed earlier (Section I. A). The oxygen thus produced is activated in thesinglet state. 79

Metals can initiate fatty acid oxidation by reaction with oxygen. 154 The anion thusproduced can either lose an electron to give singlet oxygen or react with a proton to forma peroxy radical, a good chain initiator.

Many oxygenated complexes of transition metals have now been isolated and used ascatalysts for oxidation of 0Iefins.60.104.105 Recent evidence supports the initiation ofautoxidation through formation of a metal-hydroperoxide catalyst complex.4 There isalso evidence that singlet oxygen participates in the propagation of lipid hydroperoxida­tion. Thus, Hawco et a/69 have reported that the breakdown of linoleic acid hydroperox­ides by hemeproteins produces significant amounts of singlet oxygen.

Cyclic peroxides and endoperoxides have already been mentioned as secondaryproducts of linolenate autoxidation (Section II. C). These cyclic products are related tothe biosynthetic precursors of prostaglandin from the enzymatic oxidation of arachidonicacid.68 Endoperoxides can also be important intermediates producing singlet oxygen ifthis activated oxygen is originally involved in the catalysis.94 Further research is neededto ascertain more directly whether or not singlet oxygen is involved in various catalyticprocesses including those by metals, unstable oxygen adducts and endoperoxides.

8 E. N. Frankel

IV. OXIDATION OF MIXTURES OF FATTY ACIDS AND VEGETABLE OILS

Although natural fats contain mixtures of different unsaturated fatty acids, the ques­tion of how these fatty acids interact during oxidation has been largely overlooked. Earlyreports indicated that small amounts of linoleate in a mixture with oleate greatly acceler­ated the oxidation. 65 . 143 A more recent kinetic study showed that, initially, oleic acidacted as a diluent for the oxidation of linoleic acid. 126 At more advanced stages ofoxidation, the rate followed a nonlinear function of the oleate concentration and wasaffected by both propagation and termination rates. However, kinetic data alone cannotbe used directly to predict the thermodynamic stability of hydroperoxides. It is necessaryto analyze for different hydroperoxides to determine how easily hydrogen abstrac.tionoccurs from unsaturated fatty acids compared to their relative rate of disappearance dueto hydroperoxide decomposition and secondary oxidation. These factors are importantin mixtures containing linolenate, which is particularly sensitive to oxidative decompo­sition.49

New methodology has recently been developed to attack this problem. Hydroperox­ides have been determined by gas-chromatography (GC) of the allylic hydroxy estersfrom oxidized mixtures of oleate and linoleate,156 and by spectrophotometric analysis of"conjugatable oxidation products" from different polyunsaturated fats.39.118 In ourlaboratory we developed, with the collaboration of scientists at Queen Mary College inLondon, a quantitative gas chromatography-mass spectrometry (GC-MS) method todetermine the isomeric hydroperoxide composition from oxidized mixtures of oleate,linoleate and linolenate.53- 55 In autoxidized mixtures of oleate and linoleate, linoleatehydroperoxides dominated the products at low levels of oxidation. In an equal mixture ofoleate, linoleate and linolenate, more linolenate hydroperoxides were produced at aperoxide value (PV) of 114 than linoleate hydroperoxides, and their ratio was reversed athigher PVs.

With soybean oil esters containing a mixture of oleate (23%), linoleate (52%) andlinolenate (7%), an unusual isomeric distribution of hydroperoxides was found at lowPVS.51 An unexpectedly high proportion of the 12-hydroperoxide isomer was found atPVs below 50. This unusual isomeric composition seems unique to soybean oil esters,because pure methyl linolenate oxidized at a PV of 20 gave the same isomeric compo­sition as observed at much higher PVs (Table 1). Photosensitized oxidation of linoleatewas invoked as a possible source of the 12-hydroperoxide in weakly oxidized soybean oilesters. The decrease in concentration of the 12-hydroperoxide at higher PVs wasexplained by the quenching effect of oxygen toward triplet sensitizers.

We now have evidence for the presence of varying amounts of 12-hydroperoxide incottonseed, safflower and corn oil esters when oxidized to low levels. 52 Since these oilsdo not contain linolenate, the likely source of the 12-hydroperoxide is photosensitizedoxidation of linoleate. The importance of photosensitized oxidation in soybean oil esterswas further demonstrated by the effect of singlet oxygen quenchers (f3-carotene andiX-tocopherol) causing a decrease in 12-hydroperoxide, and the effect of photosensitizers(methylene blue and chlorophyll) producing an increase in 12-hydroperoxide. TheGC-MS approach to determine isomeric hydroperoxides has thus proved to be a usefuldiagnostic tool to study the role of singlet oxygen in the oxidation of unsaturated fats.

V. DECOMPOSITION OF HYDROPEROXIDES

A variety of volatile and nonvolatile secondary products are formed from hydroperox­ides when lipid oxidation is carried out to high conversion or at elevated temperatures. Avery complicated set of reaction paths has been recognized for hydroperoxide de­composition. Although there is extensive literature on the nature of secondary lipidoxidation reactions, the decomposition products are so complex that much of thesestudies can be regarded as only qualitative accounts.

Lipid oxidation 9

In the following reaction sequence, the first step of the decomposition of an unsatur­ated hydroperoxide is the homolytic cleavage of the oxygen-oxygen bond (i) to yield analkoxy and hydroxy radical.

---+. R--eHOaldehyde

isomerizedROOH

R-CH-R'-+

Io--r-(i)oIH

R' = fatty ester end.

+ 'OH

/liii)

R'-CHOaldehyde ester

Carbon-earbon cleavages (ii) and (iii) lead to aldehyde esters and aldehydes, respect­ively.44 Carbon-oxygen cleavage (iv) may provide a path for positional isomerization ofunsaturated hydroperoxides (e.g. 9-linoleate-OOH +::± 13-linoleate-OOH).11 Additionalaldehydes or aldehyde esters are formed from allylic hydroperoxides by cleavage (ii) or(iii), producing 1-0lefin radicals that react with hydroxy radicals to form 1-enols tauto­merizing to the corresponding aldehydes.

iI 'OH

R-CH=CH+CH-R' ----+ R--eH=CH· --> [R-CH=CH-OH]

: I 1ii o· R-CH2--eHO

The cleavage products obtained by thermal decomposition of oleate hydroperoxidescorrespond to those expected by carbon-carbon scissions (ii) and (iii) (Table 2).132 Withlinoleate hydroperoxides, the same volatile cleavage products were obtained from eitherthe 9- or 13-isomers. 13 These results were explained by suggesting a degree of mobility ofthe peroxy group resulting from the decomposition by carbon-oxygen scission (iv).Among the nine scission products expected from the 9- and 13-hydroperoxides (Table 3),hexanal, methyl octanoate, 2,4-decadienal, methyl 9-oxononanoate (methyl azelaaldehy­date),13.119.153 3-nonenal1.27 and pentane30.77 have been reported in oxidized linoleate.2-Heptenal is another commonly reportedJ.6J.1l9 decomposition product that isexpected from photosensitized oxidation of linoleate (Table 3).

Linolenate hydroperoxides are easily decomposed into a very complex mixture ofsecondary products. The primary decomposition products expected are given in Table 4.Evidence for many of the aldehydes has been reported in the literature,I.61.85 but most ofthe other volatile compounds have not yet been identified. The instability of linolenatehydroperoxides can be explained by the presence of active methylene groups (Fig. 3).These structural features would promote the formation of dihydroperoxides, whichwould in turn decompose into dialdehydes.44 Alternatively, dialdehydes may be derivedfrom hydroperoxides of unsaturated aldehydes.

The volatile compounds described in many studies include only those most amenableto the separation techniques used. We still know little on the nature of the intermediateprecursors of decomposition products. The origin of these products is further obscuredby secondary oxidation of unsaturated aldehydes,42,44.99.1oo.107 by dimerization of hyd­roperoxides48,56,84 and by reactions between hydrop~roxides and the unsaturated sub- .strates. 142

Only recently significant progress has been made in elucidating the nature of nonvola­tile secondary products from lipid oxidation. From 'autoxidized 0leate53 .113 we identifiedallylic ketoenes, saturated epoxy esters, dihydroxyenes (l,2- and 1,4-) and saturated

Alkoxy radical

TABLE 2. Decomposition of Oleate Hydroperoxides132

Cleavage Decomposition products

o

(ii) O' (iii)

I I ICH3(CH2h-CH=CH-I-C1-I-l-(CH2)6COOMe

I 8 II I

(ii) O' (iii)

I I II ICH3(CH2),,-CH=CH-l-CH-I-(CH2hCOOMe

I 9 II I

(ii) O' (iii)I I II I

CH3(CH2h-l--CH-I-CH=CH-(CH2)6COOMeI 10 II I

(ii) O. (iii)

I I II I

CH 3(CH 2),,-I-CH-l-cH=cH-(CH2hCOOMeI II 11 II I

(ii)(iii)

(ii)(iii)

(ii)(iii)

(ii)(iii)

Decanal + Me 8-oxooctanoate2-Undecenal + Me heptanoate

Nonanal + Me 9-oxonanoate2-Decenal + Me octanoate

Octane + I-octanol + Me lO-oxo-8-decenoateNonanal + Me 9-oxononanoate

Heptane + I-heptanol + Me ll-oxo-9-undecenoateOctanal + Me IO-oxodecanoate

!Tl:z::;J"'::>7<"!:!:.

Alkoxy radical

TABLE 3. Decomposition of Methyl Linoleate Hydroperoxides

Cleavage Expected decomposition products

(ii) O' (iii)

I I II I

CH3(CH2)4-CH=CH-CH=CH-j-CH-I---(CH2hCOOMeI 9 II I

(ii) O' (iii)

I I II I

CH3(CH2)4-I-CH-I-CH=CH-CH=CH--(CH2hCOOMeI II 13 II I

(ii) O' (iii)

I I II . ICH3(CH2)4-CH=CH-CH2-I-CH-I-CH=CH--(CH2)6COOMe*

: 10 II I

(ii) O' (iii)

I I II ICH 3(CH 2!J-CI-I=CH-I-CH-I-CH2-CH=CI-I--(CI-I 2hCOOMe*

I 12 I .I I

* Produced by photosensitized oxidation.

(ii)(iii)

(ii)(iii)

(ii)(iii)

(ii)(iii)

3-Nonenal + Me 9-oxononanoate2,4-Decadienal + Me octanoate

Pentane + l-pentanol + Me l3-oxo-9, ll-tridecadienoateHexanal + Me l2-oxo-9-dodecenoate

2-0ctene + 2-octen-l-ol + Me IO-oxo-8-decenoate3-Noncnal + Me 9-oxononanoatc

I-1exanal + Me 12-oxo-9-dodecenoate2-Heptenal + Me 9-undecenoate

r­oO'0.:o><0:~o'::>

Alkoxy radical

TABLE 4. Dccomposition of Mcthyl Linolcnatc Hydropcroxides

Cleavage Expected decomposition products

IJ

(ii) O' (iii)I I II I

CH3CH 2-CH=CH-CH2-CH=CH-CH=CH-I-cH-I-(CHzhCOOMeI II 9 II I

(ii) O' (iii)I I II I

CH3CH 2-CH=CH-cH-cH-I-eH-I--cH2-cH=CH-(CH 2 hCOOMeI II 1Z II I

(ii) O' (iii)

'I .I II

CH 3CH 2-CH=CH-CH 2--+-CH-j--CH=CH-CH-CH-(CHzhCOOMe: 13 :

(ii) O' (III)

I I II I

CH 3CH 2-I-CH-I-CH=CH-CH=CH-CHz-CH=CH-(CHzhCOOMeI II 16 II I

(ii) O' (iii)I I II I

CH3CHz-CH=CH-CHzCH=CH-CHz-I-CI-I-I-CH-CH--(CHzl"COOMe'I II 10 II I

(ii) O' (iii)I I II I

CH 3CH=CH-I-CH-I-CHz-CH=CH-CH 2-CH=CH-(CH zhCOOMe'I 15 II I

• Produced by photosensitized oxidation.

(ii)(iii)

(ii)(iii)

(ii)(iii)

(ii)(iii)

(ii)(iii)

(ii)(iii)

3,6-Nonadienal + Me 9-oxononanoate2,4,7-Decatrienal + Me octanoate

3-Hexenal + Me 12-oxo-9-dodecenoate2,4-Heptadienal + Me 9-undecenoate

2-Pentene + 2-penten-l-ol + Me l3-oxo-9, Il-tridecadienoate3-Hexenal + Me l2-oxo-9-dodecenoate

Ethane + ethanol + Me 16-oxo-9, 12, 14-hexadecatrienoatePropional + Me 15-oxo-9, 12-pentadecadienoate

2,5-0ctadiene + 2,5-octadien-l-ol + Me lO-oxo-8-decanoate3,6-Nonadienal + Me 9-oxononanoate

Propional + Me 15-oxo-9, 12-pentadecadienoate2-Butenal + Me 9, 12-butadecadienoate

rn:z:'"T1-.Il>::>:><"~

Lipid oxidation

Oleate + OI·OOH~ 0

~/" I H20

-HO· 'O~ V

Olea/t...." • 1

1~HO~

H. OH

HO~d·OOH HO~/

I 0 I ~' OH

OH.OOH~ '"'"HO~

FIG. 6. Origin of methyl oleate secondary oxidation products.

13

dihydroxy esters (1,2-). The allylic ketoenes may be formed by a two-step dehydration ofthe hydroperoxides.

-·OH -H'-CH=CH-eH- ---+l -eH=CH-eH----+, -eH=CH-e-

I I IIOOH O' 0

Epoxy esters may come either from the reaction between oleate and hydroperoxides 142or from the hydroperoxides themselves (Fig. 6). The 1,2-dihydroxy esters may comeeither from the epoxy esters by hydration or from the alkoxy intermediate. The unsatur­ated 1,2- and l,4-dihydroxy esters may originate either from an allylic alkoxy radicalintermediate or from dihydroperoxides or hydroxy-hydroperoxides (Fig. 6).

Hydroxyepoxy monoenes are major secondary oxidation products from linoleate hyd­roperoxides.62.63.66.67 Experiments with 180 2 labeled hydroperoxides supported a mech­anism involving elimination of 'OH from the hydroperoxy group and cis addition to theallylic double bond to form the epoxide.67

H

""?iIo

/R-CH 'OH 0 OH

""~ i / "" ICH=CH-eH=CH-R' ---> R-eH-CH-CH-eH=CH-R'

Gardner62 reviewed enzymatic and nonenzymatic decomposition mechanisms for linoleicacid hydroperoxides. The major pathways postulated for decomposition in the presenceof Fe(III)-cystein catalyst involve: (a) formation of an alkoxy radical by loss of .OH: (b)cyclization of the alkoxy radical to the et-unsaturation; (c) reaction of the epoxy allylicradical either with O2 to form an epoxyhydroperoxy monoene or with . OH to form theepoxyhydroxymonoene; (d) formation of ketodienes or hydroxydienes by either loss orgain of .H from the allylic alkoxy radical; and (e) formation of di- and trihydroxymonoenes by further reaction of hydroxy dienes with, OH by l,4-addition.

14 E. N. Frankel

In an alternate mechanism67 the ketodiene is formed by expulsion of 'OH from theperoxy radical in the presence of hemoglobin.

-eH-eH=CH-CH=CH- ........ -e-CH=CH-CH=CH-

I I00· OOH

........ -e-eH=CH-eH=CH- + 'OH

IIo

Another well-recognized route to keto and hydroxy products involves decomposition ofa tetroxide intermediate derived from the reaction of two peroxy radicals (Section 1. A).This route can also provide a path for the formation of singlet oxygen (Section III).

Thermal decomposition of linoleate hydroperoxides produced a multitude of com­pounds including di- and trihydroxy esters derived from corresponding keto, hydroper­oxy and epoxide. 15l From thermally oxidized methyl linoleate, we also identified amultitude of minor products including ketodienes (with CO on C-9 and C-13), epoxyhydroxymonoenes (9,10-/12,13-epoxy-ll-hydroxy and 9-/13-hydroxy-12,13/9,10-epoxy),dihydroxy (9,13-) and trihydroxy (9,10,13- and 9,12,13-) esters.51.54 These products can beexplained' by pathways similar to those suggested by Maier and Tappel lo6 andGardner62 (Fig. 7). The hydroxy epoxy monoenes are derived from an allylic epoxyradical undergoing 1- or 3-addition with .OH. Similarly, the trihydroxy monenes comeeither from allylic dihydroxy radicals undergoing 1- or 3-addition with .OH or from thecorresponding hydroxy epoxy monoenes by hydration.

Dimeric and higher molecular-weight materials are also formed by thermal decompo­sition of autoxidized lipids. Many studies have been published on these ma­terials. 31.38.48.l20.l34 The products we characterized from the decomposition of linoleatehydroperoxides at 210cC consisted of 82% dimer, monomers with decreased conjugateddiene, and 8-10~~ volatiles, half of which was water.48 Our evidence indicated that inmixture of hydroperoxides (22-90%) and unsaturated fatty esters only the hydroperox­ides polymerized. However, Mounts et a/. lll found that the chromatographic "dimer"fraction was derived half from hydroperoxide and half from l4C-labeled linoleate inheated mixtures containing 5-6% hydroperoxide. Many of these dimers and polymers areknown to be rich sources of volatile carbonyl compounds l5 .83 and to decrease flavor andoxidative stability of soybean oil. 29 However, these materials include such complex mix­tures that they have not been satisfactorily resolved and fully identified. Data are gener­ally scanty on the significance of these polymeric materials in lipid deterioration.

FIG. 7. Origin of methyl linoleate secondary oxidation products.

Lipid oxidation 15

VI. FLAVOR REVERSION

The development of offensive odors and flavors known as flavor reversion is character­istic of soybean oil and other linolenate-containing oils and occurs at low levels ofoxidation. This flavor deterioration is described as beany and grassy at the early stagesand as fishy or painty at the more advanced stages. Since none of these flavors character­ize the crude oil, reversion is a misnomer which, however, still persists.

A. Causes of Flavor Reversion

1. Linolenic Acid

Linolenic acid is widely accepted as the most important precursor of flavor reversion.In a classical experiment, Dutton et aes interesterified linolenic acid into cottonseed oil;when the oxidized product was examined by a taste panel, it could not be distinguishedfrom oxidized soybean oil.

The terminal pentene radical in linolenate, CH 3-eH 2-CH=CH-eH 2-, is gener­ally thought to be associated with the flavor reversion compounds of soybean oil. Thedecomposition of linolenate hydroperoxides is observed at relatively early stages of oxi­dation.44 A great number of odor and flavor compounds have been identified by differentworkers in oxidized soybean oil (Table 5). This number is continuously increasing as aconsequence of improved analytical methodology.

Hoffman7t isolated 3-cis- and trans-hexenal from reverted soybean oil and described

the odor of the cis isomer as green-beany. This aldehyde was considered to be the firstperceptible reversion flavor compound of soybean oil. This compound is derived fromthe decomposition of both 12- and 13-linolenate hydroperoxides (Table 4). In later work,Chang et al. J7 attributed the flavor of reverted soybean oil to a-pentyl furan, which theyassumed to be derived from linoleate oxidation. They suggested that linolenic acid maycatalyze the oxidation of linoleate. In a more recent report from the same laboratory,70ex-pentenyl furan was synthesized as a product expected from linolenate oxidation. These

TABLE 5. Partial List of Volatile Components Identified in Oxidized Soy­bean Oil*

Class

Aldehydes

Ketones

Alcohols

Ethyl estersHydrocarbons

Other compounds

Compounds

Alkanals: C b C2• C3• C4 • Cs• C6• C7 • Ca. Cg

AIkenals ,0.2 : C4 • Cs• C6 • C7 • Ca. Cg • C I O. C11

Alkenals ,0.3: C6

Dienals. ,0.2.4: C6• C7• Ca. Cg • C,O. C ,2

Dienals. ,0.2.5 : CaDienals. ,0.2.6: Cg

Trienals. ,0.2.4.7: C I 0

Dialdehydes: malonaldehyde. maleic dialdehyde.hexene- L6-dial

2-Alkanones: C4 • Cs. C6 • C7 • Ca3-Alkanones: CaUnsaturated: l-pentene-3-one. 4-octene-3-one.

2-methyl-5-octene-4-one. 7-methyl-2-octene-4-one.3.6-nonadiene-5-one

Alkadiones. 2,3-: C4 • CsAlkadiones. 4.5-: CaSaturated: C2• C3• C4 • Cs. iso Cs. C6 • C7

Unsaturated: l-pentene-3-ol. l-octene-3-o1C b C2

Saturated: C2• C3• Cs• C6, Ca. Cg• C,O. CllUnsaturated: 2-pentene. I-hexene, 2-octene. I-decene.

I-decyne2-pentyl furan. lactones. benzene. benzaldehyde, aceto­

phenone. water

*Key references. 43. 72. 133. 142

16 E. N. Frankel

17CH3-CH-CH=CH- +

IOOH

15CH3-CH==CH-CH--­

IOOH

16CH3-CH2-CH-CH=CH- +

16 14 ICH3-CH2-f~=-ClI=~H-~ OOH 14I' CH3-CH2-CH==CH-CH-

02 02 60H

FIG. 8. Autoxidation of linolenate dimers.

workers now claim that the cis and trans forms of cx-pentenyl furan produce flavor andodor reminiscent of reverted soybean oil.

Other linolenic acid derivatives implicated as causes of flavor reversion include isolino­leic acid and oxidative polymers. Isolinoleic acid is a mixture of isomeric dienes formedby hydrogenation of linolenate in soybean oil and will be discussed later in this section.Oxidative polymers refer to a complex mixture of oxygen-containing compounds formedby polymeric decomposition of the labile linolenate hydroperoxides. These high­molecular-weight and polar compounds are easily decomposed and generate volatilealdehydes 16

.83 and other compounds contributing to the flavor deterioration occurring

even in the absence of oxygen and at low temperatures.Dimeric compounds with carbon-earbon linkage between fatty acid molecules were

produced by thermal decomposition of linolenate hydroperoxides48 (Fig. 8). If there is asingle double bond in the fatty acid and it is on carbon-IS, free radical oxidation wouldproceed, as in oleate (Fig. 1), to produce a mixture of allylic 14-, 15-, 16- and 17-hydro­peroxides as shown on Fig. 8. Decomposition of these four hydroperoxides would pro­duce by (iii) cleavage a mixture of acetaldehyde, propional, 2-butenal and 2-pentenal andby (ii) cleavage a mixture of methane (+ methanol), ethane (+ ethanol). propional andbutanal (Fig. 9). Many of these compounds have been identified in oxidized soybean oil(Table 5).

The spontaneous decomposition of oxidative polymers has been referred to as "hiddenoxidation" because it cannot be measured by peroxide value. For this reason we devel­oped chromatographic methods for their estimation.28

•3

1,50 A good correlation wasshown between flavor or oxidative stability and the content of oxidative dimers andother polar materials determined chromatographically. Although these "dimers" havebeen suggested as the immediate precursors of flavor reversion, the problem is compli­cated by other factors.

'17 \CH3~CH~CH==CH---, I \

t OOH \~

Methane Acetaldehyde(methanolj

'16 \CH3-CH27LCH~\CH=CH--

, I \/ OOH \

~

Ethane PropionalIethanolj

'15 \CH3-CH=CH~CH~CH2--, I \

I OOH \Propional 2·Butenal

'14 \CH3-CH2-CH=CH~CH~CH2-­

'I \/ OOH \r ~

Butanal 2-Pentenal

FIG. 9. Decomposition of linolenate dimer hydroperoxides.

Lipid oxidation [7

2. Phosphatides

Phosphatides were implicated as precursors of flavor reversion in the early daysbecause improperly refined soybean oil produced fishy flavors on heating. This type ofdeterioration would be expected from the high content of polyunsaturates and linole­nates of phosphatides. However, oxidized amino compounds may also playa role, andChang et al. 14 have reported as much as O.7~/~ nitrogen compounds among the flavorreversion material isolated from soybean oil. They suggested the presence of carbonyland noncarbonyl compounds with dimethyl amino groups. More recently, from bitter­tasting soybean phosphatides, Sessa et al.135.136 identified oxygenated products similarto the decomposition products from linoleate hydroperoxides (Section V) and5-(pentenyl)-2-furaldehyde. Therefore, any small amounts of these phosphatides in soy­bean oil would be expected on oxidation and decomposition to contribute to the offen­sive flavor.

3. Nonglyceride Components

Nonglyceride components were also implicated in the early days. lOB They playa rolewhich apparently has not been fully determined. In some of our early studies, the stab­ility of soybean oil was improved by lowering the tocopherol content to about one-halfof its original value of 1500 pg/g.46 Later work in our laboratory showed that addition ofunsaponifiables lowered flavor stability.73 Although sterols had little effect, squalenelowered stability. The effect of pigments such as chlorophyll and carotenoids has alreadybeen discussed in Section III. Growing evidence indicates that a sufficient amount ofphotosensitizers is left in bleached-deodorized soybean oil to contribute to its lightinstability.19.52.124

B. Control A1easures

Although much progress has been made in the control of reversion, there are stillproblems caused by light and thermal instability in processed soybean oil. The greatestimprovements have been achieved through the use of metal inactivators, processing andselective hydrogenation. Other approaches investigated without too much successinclude selective removal of linolenate triglycerides, prevention of oxidation by antioxi­dants, and breeding soybeans to produce oil low in linolenic acid.

1. Metal Inactivators

Many compounds have been tested26.130 and the most commonly used metal inactiva­tors include citric acid and phosphoric acids. These additives are very effective for im­proving storage stability at normal temperatures. However, they decompose and do notprovide protection at cooking temperatures (l5Q-200"C). Although metal inactivatorsmay be used in early stages of processing to protect the oil against oxidation, they aremore effective after the oil is heated. 21 They are, therefore, added on the cooling cycle ofthe deodorization step. Apparently, metal catalysts occur in the oil in the form of prooxi­dant complexes with oxygen or hydroperoxides4.154 and these complexes must be des­troyed by heat before the chelating agent becomes effective. 21

2. Processing with Minimum Exposure to Oxidation

Any peroxide development during processing is detrimental to flavor and oxidativestability of the finished oil. The last step in soybean oil processing is the deodorization,which involves heating at 2OQ-210°C in a good vacuum. If hydroperoxides or otheroxidation products are present before this step, they will polymerize or condense and theresulting oxidative polymers and polar products are, as indicated before, detrimental to

J.P.LR. 19,1-2-a

t~ E. N. Frankel

flavor stability. Therefore, good processing measures include careful control of refiningtemperature, vacuum bleaching, nitrogen blanketing and protection from light. Bleachingis known to lower oxidative stability of partially processed soybean oil.64 Oxidation canreadily occur in this step by exposure of large surface area to air at elevated tempera­tures. Furthermore, bleaching may upset the natural balance between de-stabilizing pho­tosensitizers and stabilizing quenchers.

3. Hydrogellatioll

This step is probably the most important for maintaining flavor stability and is thebasis for the shortening, margarine and salad oil industries. However, hydrogenationalone has not produced a liquid oil sufficiently stable to heat and light to be suitable asboth a salad and cooking oil. Partially hydrogenated soybean oil develops offensiveodors and flavors on heating32 and light oxidation. I 10 There remains the problem ofexplaining how flavor reversion develops even after selective reduction of linolenate.

Hydrogenation of linolenate in soybean oil produces isomeric dienes called isolino­feate. Because they have two isolated double bonds that cannot be conjugated, they areunreactive and accumulate in the hydrogenated products. They are also less reactivetoward oxygen and behave like two oleate functions in the same molecule. However, thepentene function of isolinoleate can oxidize to produce offensive odors and flavors.

Oxidation of isolinoleate would be expected to produce hydroperoxides by allylicattack of oxygen on each double bond. Using 9,15-diene as a model, the expectedhydroperoxides produced by attack on the Li9 double bond would be the same as oleate:8-, 9-, 10- and 11-00H. Similarly, attack on the Li15 double bond would produce amixture of 14-, 15-, 16- and 17-hydroperoxides, as shown previously for the oxidation oflinolenate dimers (Fig. 8). Decomposition of the external 14-, 15-, 16 and 17-hydroperox­ides would also produce the same volatile cleavage products (Fig. 9), all of which havebeen identified in nonhydrogenated soybean oil (Table 5). However, the flavor problem isfurther complicated by the decomposition products arising from the internal dienoic 8-,9-. 10-, II-hydroperoxides.

15 8I

CH3-CH2-CH=CH-(CH2)4-CH=CH-CH-\-R

I IOOH I

12,8-undecadienal

15 9I

CH3-CH2-CH=CH-(CH2h-CH=CH-CH-I-CH2-R

I IOOH I

12,7-decadienal

15 10 8I

CH3--eH2-CH=CH-(CH2)4--eH-l-CH=CH-R

I IOOH I

16-nonenal

15 II 9I

CH3--eH2-CH=CH-(CH2h-CH-I-CH=CH-CH2-R

I IOOH I

15-octenal

Lipid oxidation 19

Evidence for some of these aldehydes has appeared in the literature.37.86.87 These com­pounds supposedly impart a hardening or hydrogenated flavor to soybean oil.

One important development is the copper-bearing hydrogenation catalysts that have alinolenate to linoleate selectivity as high as 13.92 It is possible with these catalysts toreduce most or all of the linolenate and still produce a liquid oil without need ofwinterization. Soybean oil hydrogenated with copper catalysts had significantly higherroom odor and flavor scores than nickel hydrogenated soybean oil. 23 However, evenwhen the linolenate content was reduced to 0% with copper catalyst, the flavor stabilitywas diminished. This problem arises from the isolinoleic acid produced in relatively highproportion (5-{j%) by copper catalysts which reduce linolenate almost entirely by aconjugation mechanism.91

Hydrogenation has therefore provided only a partial solution to the flavor reversionproblem of soybean oil. By converting linolenate into isolinoleate and monoenes, thestability of the oil is increased by virtue of decreased susceptibility of the products tooxidation. However, these oils contain enough isolinoleate and monoenes with doublebonds between the C14 and C16 positions to produce compounds similar to those oflinolenate and to impart objectionable odor and flavor compounds on thermal oxidation.The ultimate solution is to find a catalyst that can selectively reduce the ~15 doublebond oflinolenate to convert it to linoleate. Much of our research on heterogeneous andhomogeneous hydrogenation catalysts47 has been directed toward this goal, but this hasnot yet been achieved.

VII. CONCLUSIONS

The problem of flavor deterioriation of soybean oil has motivated much of ourresearch on autoxidation aimed at determining the precursors of offensive odors andflavors and on hydrogenation aimed at removing linolenic acid as the main cause ofdeterioration. New work on the different isomeric hydroperoxide distribution observedin soybean oil esters at low levels of oxidation suggests that it may contribute to theunique flavor deterioration of this oil. 51.5 2 Further research is needed to ascertain moredirectly the role of singlet oxygen and the significance of secondary oxidation products assources of offensive odors and flavors in deteriorated lipids.

Although many chain-breaking and preventive antioxidants, peroxide destroyers, andV.V. deactivators are used effectively in rubber and petroleum products, they cannot beadded to foods because of established or potential health hazards. One approach toreduce this hazard is to use polymeric antioxidants that are claimed to be unabsorbed intest animals. Many reports have now been published on this approach,58,59,129 but it hasnot been approved for food as yet.

Despite the many catalytic metal complexes and photosensitizers found in plants andanimals, nature has provided them with effective inhibitors, quenchers of singlet oxygen,and reducing enzymes for effective protection against hazards and toxicity from lipidhydroperoxidation, For example, plant leaves are loaded with linolenic acid,8o,81 and yetplants have a mechanism for protection against oxidation, Research on natural antioxi­dants and singlet oxygen quenchers may unravel the mechanism plants have for protec­tion against linolenate oxidation, Processing of vegetable oils may upset the naturalbalance between photosensitizers and quenchers, resulting in decreased stability. Futureresearch should address the problem of how processing can be changed to improve thisbalance in favor of quenchers,

(Received 21 January 1980)

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