V O L A T I L E L I P I D O X I D A T I O N P R O D U C T S
E. N. FRANKEL
Northern Re,~tional Research Center, A~lricultural Research Service, U.S. Department of A.qriculture, Peoria, Illinois 61604
II.
C O N T E N T S
I. l NTROI)U f'TION
DECOMPOSITION OF HYDROPEROXIDES A. Alkyl hydroperoxides B. Allyl hydroperoxides C. Fatty ester hydroperoxides
IlI. VOLATILE PRODUCTS FROM OXIDIZED LIPIDS A. Oleate B. Linoleate C. Linolenate D. Soybean oil E. Hydrogenated oils
IV. DECOMPOSITION OF SECONDARY PRODUCTS A. Aldehydes B. High-molecular-weight materials C. Hydroperoxy epoxy, cyclic peroxides and other oxygenated compounds
V. FLAVOR SIGNIFICANCE OF VOLATILES
VI.
VII.
VOLATILES FROM l,~, ' I'IVO OXIDATION
CON('LUSIONS
REFERENCES
1
2 2 3 5
7 8
11 15 17 18
19 20 22 23
26
27
28
29
I. I N T R O D U C T I O N
The field of lipid oxidation has recently undergone a renaissance due to increased aware- ness of the importance of polyunsaturated lipids in foods and biological systems. The development of objectionable flavors and odors by oxidation has obvious detrimental consequences on food quality and consumer acceptability. This classical economic prob- lem of lipid oxidation has now been overridden by the biological problems.X71 Unsatur- ated lipids are important for the structural and functional integrity of biological mem- branes. For this reason, lipid oxidation can cause biological damage that may be derived from changes in the diet and our environment. Changes in the diet such as increases in polyunsaturated fats without adequate protection, or increased intakes of tocopherols and other antioxidants, may cause or accelerate in vivo oxidation with potential damage to membranes and vital cell functions. 2°3 Similarly, atmospheres contaminated with traces of ozone and nitrous oxide accelerate lipid oxidation with potential damage to stored foods and biological systems. 14a'~75 Prostaglandin-related cyclic peroxides and other biological oxidation products such as leucotrienes and epoxy unsaturated fatty acids direct many important physiological activities including a role in the inflammatory pro- cess. 162,163,173,174- There is also the universally important problem of aging, which has been related to various forms of biological lipid oxidation. 155 Finally, the toxicity of thermally oxidized oils has attracted considerable attention. 2,16°,166 Heat-abused oils can be toxic to experimental animals, but the mechanism of toxicity of thermally and oxidatively derived cyclic and secondary oxygenated products has not been clarified.
Mechanistic concepts of lipid oxidation were previously reviewed. 55'56 Since these reviews were published, two important papers have appeared that throw further light on the mechanism and stereochemistry of linoleic and arachidonic acids oxidation. ~64,165
1 J.p.t.k. 22,1 A
2 E .N. Frankel
A monograph was published on volatile odor and flavor compounds derived from lipids, and analytical problems were surveyed. 52 Studies on the source of volatile lipid oxidation products are still controversial and often difficult to interpret. In this review, the sources of volatile oxidation products will be considered in detail to provide the basis for a better understanding of the mechanism of flavor deterioration. It is hoped that new knowledge in these areas would lead to improved methods of controlling flavor deterioration of lipid-containing foods. Much attention has been given recently to the analyses of hydro- carbons in the breath of experimental animals as a sensitive index of in I:ivo lipid oxi- dation. ~;2'2°3 A better understanding of the origin of these volatile oxidation products may also elucidate the mechanism of biological lipid oxidation.
I1. D E C O M P O S I f l O N O F H Y D R O P E R O X I D E S
The hydroperoxidation of unsaturated fatty esters has been reviewed in detail pre- viously, 53'55 Although fat hydroperoxides are generally tasteless and odorless, their products of decomposition have a great impact on flavor. Some volatile aldehyde clea- vage products are extremely potent and can affect the flavor of vegetable oils at concen- trations lower than I ppm (Section V).
Hydroperoxide decomposition involves a very complicated set of reaction pathways. A complex multitude of volatile and nonvolatile products are formed. Much attention has been given to the volatile decomposition products, but the impact of the nonvolatile secondary oxidation products on flavor deterioration is not well understood.
Free radical mechanisms established for the decomposition of hydroperoxides are based largely on extensive studies of tert-butyl hydroperoxide, which is relatively stable compared to the hydroperoxides of unsaturated fatty esters. Little information is avail- able on the less stable allylic hydroperoxides that are related to fat hydroperoxides. Generally, accepted schemes for tert-butyl hydroperoxide and other alkyl and allyl hydro- peroxides will be summarized here as a basis for subsequent discussions of unsaturated fatty esters.
A. Alkyl Hydroperoxide~s
Alkoxy radicals are the precursors of most stable oxidation products. The early schemes advanced by Bell et al. 7 for the oxidation of paraffin hydrocarbons are still generally accepted today. Accordingly, alkoxy radicals may be formed by three routes: homolytic cleavage of a hydroperoxide (1 I, interaction of peroxy radicals (2), or homoly- tic cleavage of a peroxide (3).
ROOH a , R O . + .OH
ROO. + ROO. * 2RO. + 02
ROOR A,2RO.
Secondary alkoxy radicals may cleave to form aldehydes (4).
R '2CH--O' , R 'CHO + R'.
(1)
(2)
(3)
(4)
This unimolecular decomposition of a radical is also known as/¢-scission or elimination because it can be regarded as the reverse of radical addition to a multiple bond (5).109, 111,171
R- + C - - C ~ R - - C - - C ' (5)
Alkoxy radicals may also react with the substrate (6), or with the corresponding alkyl radical (7), or interact with another alkoxy radical (8).
Volatile lipid oxidation products 3
Rr~ , RICHOH + R" (6)
R I C H _ O ' R , RiCO + RH (7)
, R o RiCO + ROH (8)
Since reactions (4) and (6) form alkyl radicals, they propagate the chain. Reactions (7) and (8) are terminating by formation of stable products. Hydroperoxides are readily decomposed thermally or in the presence of metal ions. 97
Thermal decomposition is regarded as one of the most complex of free radical reactions. 94'95 Thermal homolysis of the O--O bond (1) is complicated by induced decomposition in which radicals attack hydroperoxides (9) and (10).
RO. + ROOH ~ ROH + ROO. (9)
2ROO.---, 2RO. + 02 (I0)
Other routes for radical production include bimolecular decomposition of hydroperoxides (l 1-13), 211
2ROOH ~ RO" + ROO. + H20 (1l)
ROO. + RH ~ ROOH + R. (12)
R. + O 2 ---* ROO. (13)
or
ROOH + RH + 0 2 ~ RO. + ROO. + H20 (11 + 12 + 13)
reaction of hydroperoxy radicals with alkenes (14), 214 with surfaces and trace metals.
ROO. + CHz~-----CH--R --o ROO--CH2--~H--R (14)
Metal-catalyzed decomposition 96"9~ involves metal initiators acting by one-electron transfers with hydroperoxides (15) and (16).
M" + ROOH---~ M "+1 + RO" + OH- (15)
M "+1 + ROOH---~ M" + ROO" + H + (16)
or
2ROOH--~ RO. + ROO. + H20 (15 + 16)
Metal catalysts may be coordinated with ligands as complexes or may exist as dimers or higher molecular weight species.178 Metals may also complex with hydroperoxides 117'178 which catalyze autoxidation and decomposition. Other materials form strong complexes with the metals and inactivate their effect in promoting hydroperoxide decomposition. Homolytic decomposition of hydroperoxides is often considered metal-catalyzed because it is very difficult or nearly impossible to eliminate trace of metals that can act as potent catalysts for hydroperoxide decomposition. 94
B. Allyl Hydroperoxides
Decomposition of allyl hydroperoxides is accompanied by the formation of alcohols, carbonyl compounds, scission and secondary reaction products between hydroperoxides and olefins especially in the presence of metal catalysts, 125 between hydroperoxides and unsaturated ketones, condensation of hydroperoxides and aldehydes, oxidation of alde- hydes to acids (Section IV. A), and production of esters from alcohols, acids, and alde- hydes. Hawkins and Quin 92 oxidized various primary and secondary olefins and ident- ified among the oxidation decomposition products carbonyl and hydroxy compounds containing both the same number of carbon atoms as the original olefins and less carbon
4 E .N . Frankcl
atoms than the original olefins. The mechanism postulated for scission of allylic hydro- peroxides involved the intermediate formation of alcohol ethers (171 and 1181,
R - - C H - - C H ~ C H a
OH \ ~
- R - - O - - C H - - C H = C H , 1 ~ ROH + C H 2 ~ - C H - - C H O tl7~
6. _I - R - - C H - - O - - C H = C H 2 ] --* RCHO + CH 3 CH O {181
By this mechanism, chain scission may occur on either side of the carbon atom initially carrying the hydroperoxide group. Products were also identified that result from fission across the double bond including aldehydes and ketones, as well as esters, undistilled residues, CO2 and water.
Aldehydes are important products of the autoxidation of butcncs. When 1-butenc-2- hydroperoxide was thermally decomposed, no formation of acetaldehyde or propionalde- hyde was observed. '1 Because these aldehydes are principal products of autoxidation, Brill '° concluded that they arise by tin addition rather than abstraction mechanism. It was not clear whether aldehydes were formed by cleavage of a polyperoxidc {19t.
O \ \
R O O - - C H - - C H - - O O - - C H - - ( ~ H - - - , R O . + 2CHO + C H - - C H (19)
or via an alkoxy radical intermediate (20):
RO--CH2--CH--R ' - - -* R'CHO + ROCH,2 (20)
In a detailed study of the oxidation of acyclic alkenes, Van Sickle et al. 21221-~ observed an addition abstraction competition between 1"2 and 2:1 depending on structural and steric factors. They, suggested that much of the discrepancies in the literature regarding the ratio of addition and abstraction products of oxidation may be due to thermal decomposition obscuring product analyses and the presence of secondary products.
Hydroperoxides as v, d l as carbonyl compounds, acids, and epoxides have also been identified among the oxidation products of n-butenes. 2'~'25'14-''1~ The relative contri- bution of addition and abstraction processes may again be obscured according to whether the products can be regarded as primary or secondary, the latter product becoming significant at high temperatures and high conversions. Also, acetaldehyde and metal catalysts contribute to the formation of epoxide from alkenes. 2~3
Brill ~ demonstrated that the isomeric hydroperoxides from 4-methyl-2-pentene arc readily interconverted in dilute organic solvents or olefin at 40 C. He proposed a mech- anism for the propagation step involving rearrangement through a cyclic peroxide inter-
mediate 121} and (22t:
C C ('
C - - C - - C = C - - C ~ C - - C - - C - - C - - C ~ C - - C ~ C - - C - - C ( 21 }
I 1 I .o l o OO- O O
C C C C
[ { I ] C - - C = C - - C - - C + C - - C - - C - - C - - C ~ C--C~-~-C--C--C + C - - C - - C ~ C - - C (22)
-o o I I I O O H H O O O O . (A} (B)
Volatile lipid oxidation products 5
At 100 °, hydroperoxides (A) and (B) were equally decomposed in olefin solvent and the decomposition preceded isomerization. However, when rearrangement was retarded by using 70~o aqueous acetone, hydroperoxide (A) decomposed less readily than hydroper- oxide (B).
Basic free radical studies with olefins have generally been too limited to define the relationship between the allylic hydroperoxides and their secondary oxidation products on one hand, and between their rearrangement and ease of decomposition on the other hand. A better understanding of free radical reactions of allylic hydroperoxides is now emerging from work recently published on the rearrangement of linoleic acid hydroper- oxides, and on the stereochemistry of autoxidation of linoleic and arachidonic acids 18'55'a6'*'165 discussed below.
C. Fatty Ester Hydroperoxides
Early studies showed that methyl linoleate hydroperoxides oxidize more rapidly than either methyl linoleate or its conjugated cis,trans-9,11-diene isomer. 169 The conjugated dienol obtained by reduction was relatively stable and even more resistant to oxidation than methyl linoleate. Evidently free radical fragmentation and propagation is dimin- ished by reduction of the hydroperoxide. Oxidation of linoleate hydroperoxides increased with temperature (activation energy 16.2 Kcal/mole) and oxygen pressure. The decrease in cis,trans-diene, formation of trans,trans-diene and isolated trans, and increase in mol- ecular weight were explained by formation of dimers, polymers, and scission products.116,169
By using iSO-enriched hydroperoxides, it was shown recently that the rearrangement of 9- and 13-hydroperoxide cis,trans-diene isomers of linoleate proceeds by complete exchange of peroxy oxygen with atmospheric oxygen.18 This result rules out the 1,3-cycli- zation scheme (21) suggested previously 9 and supports a mechanism involving cleavage of the carbon-oxygen bond and the formation of the same pentadienyl intermediate as that recognized for linoleate autoxidation (23).18'53"55
9 --CH--CH~-----CH--CH~---CH-- ~ - - C H - - C H - - C H - - C H - - C H - -
(~O. +
.OO. 13
--CH~---CH--CH~---CH--CH-- (23)
However, the stereochemistry of the pentadienyl radical was not defined above and the formation of trans,trans-diene isomeric hydroperoxides was not explained. 18 Recent kin- etic studies showed that the isomerization from cis,trans- to trans,trans-diene hydroper- oxides of linoleic acid is inversely related to the concentration of the substrate and effectively inhibited by a hydrogen donor such as p-methoxyphenol.~65 How these factors may affect the decomposition and types of products expected from linoleic acid hydro- peroxides remains to be investigated. The volatile decomposition products from each hydroperoxide isomer of oxidized linoleate are discussed below (Section III. B).
The breakdown of secondary alkoxy radicals leading to/~-scission recognized for alkyl hydroperoxides (4)(Section lI. A) has also been generally accepted 53'12t,~5~ and authen- ticated for the allylic system of fatty ester hydroperoxides (24) and (25).
R2--CH~---CH--CHO + .CH2--R1 (24) 7
Rz--CH~----CH--CH--CH2--R 1
~)" "~Rz--CH~--------CH' + OHC--CHz--Ra (25)
6 E.N. Frankel
Our original suggestion 73 that the alkyl radical from reaction (241 yields a hydrocarbon by hydrogen abstraction or an alcohol by reaction with a hydroxyl radical has been amply confirmed. '*2'4s'v°T°°'~8s The formation of saturated aldehydes was also suggested by a mechanism involving production of 1-enols from the reaction of hydroxy radicals with l-olefins resulting from reaction (25), followed by tautomerism (26): 7~'~ 2~
R2--CH~--~-CH" + 'OH ---, R 2 - - C H ~ - C H - - O H ~ R 2 - - C H 2 - - C H O (26)
Another mechanism v,32 explaining the formation of lower aldehydes proceeds by reaction of the alkyl radical from reaction (241 with oxygen to produce primary hydro- peroxides (27):
R - - ( C H 2 ) . - - C H i o, , R__(CH2)n__CH2OO ' RH , R - - ( C H 2 ) , - - C H 2 O O H (27)
The primary alkoxy radical formed by loss of hydroxyl radical may form either an aldehyde by dehydration or lose a carbon by formaldehyde formation 3s (28) and (29):
R-- (CH2)n--CHO (28)
R__(CH2) __CH2OO H .oH , R__(CH2) _ _ C H 2 0 .
" ~ R--(CH2), ~CHfi + H CH O
(291
Lower aldehydes are formed from the alkyl radical of reaction (29) by repeating reaction sequences (27) and (28). The primary hydroperoxide from reaction sequence (27) can also disproportionate into a short chain fatty acid and hydrogen by the interaction with an aldehyde through a peroxyhydroxy intermediate 3~ (30):
OH !
R--CH2OOH + RCHO--~ R - -CH2OO- -~H- -R
H . . . . H
/ R - - C H C--R--- , RCHO + RCOOH + Hz (301
/ \XO____ O -
Reaction of vinyl radical from reaction (25) with oxygen producing a vinyl hydroperoxide is another pathway suggested T57 for the formation of saturated aldehydes (31):
R - - C H = C H . o, R, • " , R - - C H = C H O O ' ' R--CH~---CH--OOH
R , R - - C H 2 C H O + .OH (31)
Direct disproportionation of the vinylic radical would produce an alkyne 15 = ~97 (32):
R--CH~---CH. --~ R - - C ~ C H + H, (32)
In free radical chemistry, 110.128,17 o,183 the fragmentation reaction (4) is recognized to favor those products that are most stable. Therefore, reaction (24) may be favored because it produces a more stable saturated alkyl radical than the vinyl radical produced by reaction (25). However, the alkenal produced by reaction (24) (and the corresponding dienal from conjugated diene hydroperoxides) is less stable than the alkanal produced by reaction (25). Whether these two effects balance each other is an open question.
Other thermolysis schemes recognized in free radical chemistry and photochemistry have the advantage of not invoking unstable vinylic radicals. The fragmentation of ullylic
Volatile lipid oxidation products 7
hydroperoxides into two carbonyl products by reaction (33) has been referred 35'74'84 to as Hock-cleavage, after Hock 1°° who first observed that cyclohexene hydroperoxide is acid cleaved into butane and cyclopentene carboxaldehyde.
O--OH
\ / C ~ C - - C
/ \
\ / o \ ,7 C----C C
/ \ + \
I C
H \ j o
~ o , C--C + C-- (33) / \ \
C-----C C _/ \ -,,_
Although generally catalyzed by acids, this type of cleavage is also known to occur thermaUy. 74'84 A mechanism suggested 45 for Hock-cleavage involves a dioxetane intermediate (34):
Although mechanisms based on dioxetane formation are controversial, TM these inter- mediates are still being invoked in the production of aldehydes from hydroper- oxides. 87'9° The thermal Hock-cleavage mechanism is related to that of Hawkins and Quin, 92 reactions (17) and (18), and that of Criegee 31'74 for the rearrangement of a bicyclic hydroperoxide via a hemiacetal intermediate. Both processes involve migration of groups to oxygen (35):
R2 CH~-----CH--CH--R1 --~
O--H
--~ R2CH2CHO + R1CHO.
(35)
Fragmentation schemes (33) and (35) account for the same aldehydes expected by reactions (25) and (26) and do not involve unstable vinylic radicals. The majority of aldehydes reported from the oxidation of unsaturated fatty esters can be explained by fragmentation reactions (24) and (35). However, the formation of alcohols, saturated hydrocarbons and lower fatty esters cannot be explained by Hock-cleavage reaction (35). Therefore, reactions of alkyl radicals (24) and vinyl radicals (25) with either hydroxyl or hydrogen radicals must also be considered in lipid oxidation (Sections llI and VI). 53'56
III. VOLATILE PRODUCTS FROM OXIDIZED LIPIDS
Most of the evidence available is based on the identification of stable volatile products that are most amenable to the separation and analytical techniques used. The assump- tion is also made that the volatile oxidation products are mainly derived from the
,~ E . N . F r anke l
breakdown of monohydroperoxides. However, the source of hydroperoxide decompo- sition products can be obscured by secondary reactions of unsaturated aldehydes. Furthermore, the secondary nonvolatile products of hydroperoxides can also. in turn, decompose and contribute significant amounts of volatile oxidation products. To unravel the complexity of the various volatile oxidation products, each unsaturated fatty ester will be considered first in terms of their primary hydroperoxide precursors, This dis- cussion will then be followed by the contributions from further reactions of unsaturated aldehydes and the hydroperoxides themselves (Section IV).
A. Ole~ttc
Early and recent studies of volatile decomposition products from oleate hydroper- oxides can best be explained on the basis of a mixture of the four positional 8-, 9-, 10-, and l l-hydroperoxides. Although it has been assumed that these isomers arc formed in about equal amounts by free radical autoxidation, there has been a wide variation in thc relative isomeric concentration reported by different workers . ' : Recent studies showed small but consistently higher amounts of the 8- and l 1-isomers than of the 9- and 10-isomers. 17'67'15° Only recently has the stereochemical composition of oleate hydro- peroxides been established. Free radical autoxidation produces a mixture of eight cis and tratls allylic hydroperoxides. 8~ Photosensitized oxidation produces a mixture of two trans
allylic 9- and 10-hydroperoxides. s" The relative proportion of cis-8- and l l-isomers decreased, whereas the cis-9- and 10-isomers increased with autoxidation temperature. The trans-8 and l l-isomers also increased with autoxidation temperatures while the corresponding trans-9- and 10-isomers showed no change, s~ A mechanism postulated 55 for oleate autoxidation involves formation of cis,trans radicals iC) and (D) that arc interconverted with cis,cis radicals (E) and (F) on one hand, and with trwl.,,tran.~ radicals (G) and (H) on the other hand (Fig. 1). The stereochemistry of oleate hydroperoxidc isomers can be explained by assuming the relative concentration of the allylic radicals to decrease in the order trans,trans > cis.trans > cis.ci.s. On this basis, autoxidation ten> peratures would be expected to influence the stereochemistry of volatile decomposition products, but this effect has not yet been investigated.
All the aldehydes expected from the decomposition schemes (24), (25), and {35) have been reported in studies with heated methyl oleate hydroperoxides. ~"'*s~'*'~' thermally oxidized oleate, ~'<l°s'lev'134.'sT'21~ and triolein lsu (Table 1). Major w~latile carbonyls include 2-undecenal, 2-decenal, octanal, nonanal, and decanal. Aldehyde esters, hydro- carbons, fatty esters, and H-alcohols expected from decomposition schemes 1241, (25), (2(~1, and (35) have also been identified: °' ~ :v, ~ ,~,~ 1 as.2 ~,~ as well as short-chain fatty acids- " ~:'' expected from reaction (30).
Shorter chain aldehyde products can be explained by reaction sequences (27) and (291
Other compounds Heptane 4.4 Octane 2.7 1-Heptanol 0.4 Me Heptanoate 1.5 1-Octanol 0.4 Me Octanoate 5.0 Me Nonanoate 1.5
5 0.9 6 1.9
ll 5.1 3 5 0.1 6 0.5 4 2.1
8.6 9.7 1.6
2.5
aNormalized. bOther minor volatiles included methyl ketones (0.8~/0), acids (1.2~), and gamma lactones (0.8~o), cValues for nonanal were divided by assuming that they come equally from 9-OOH and 10-OOH.
i n v o l v i n g p r i m a r y h y d r o p e r o x i d e s as i n t e rmed ia t e s . A l d e h y d e s o f s h o r t e r cha in t h a n
t hose p r e d i c t e d f r o m s c h e m e s (24) and (25) were a lso sugges t ed to ar ise f r o m bis-hydro- perox ides . 1°5 T h e s e s e c o n d a r y m i n o r a u t o x i d a t i o n p r o d u c t s w o u l d be f o r m e d by h y d r o -
gen a b s t r a c t i o n f r o m a m e t h y l e n e b e t a o r g a m m a to the h y d r o p e r o x i d e by r eac t i on
s e q u e n c e s (36) and (37).
C H 2 - C H 2 - - / /
C H 2 - - C H C H 2 - - C H / \ /
- - C H = C H - - C H H , - -CH~- - - - -CH- -CH H \ \ /
O O . O O
~H , - - C H ~ - - - - - C H - - - C H ~ C H 2 - - C H - - C H 2 - -
, -~CH~- - - - - -CH--CHO + O H C - - C H 2 - + " C H i
(36)
- - C H - - C H - - C H - - - C H z - - C H 2 - - C H - - C H 2
~ - - C H - - C H - - C H O + O H C - - C H 2 - - + " C H 2 - - C H i (37)
10 E.N. Frankel
Bis allylic hydroperoxides were also postulated by hydrogen abstraction (38) from the allylic hydroperoxide intermediates in reactions (36) and (37).
C H 2 - /
CH2--CH /
- - C H = C H - - C H \
\ OOH
-H--CH=-CH--CH--CH:CH--CH2-- (38)
Cleavage products expected from these bis-allyl hydroperoxides include lower, unsa- turated aldehydes. However, these bis-allyl hydroperoxides would tend to conjugate very readily ~5"~4 and a mixture of 4 pairs of conjugated diene hydroperoxides would be expected with OOH in the 8-12, 6-10, 7 11, and %13 positions. The absence of dienals expected from such diene hydroperoxides in oxidized oleate would seem to rule out a mechanism based on reaction (38). Dicarbonyls (Ca and C4) are other products that would form by cleavage reactions (36) and (37). Recent studies of secondary products of autoxidized methyl oleate °7'x51 showed indirect evidence for dihydroperoxides of the type suggested in reactions (36) and (37) based on identification of saturated and unsatu- rated dihydroxy esters after chemical reduction of the hydroperoxides. This evidence needs to be substantiated however by direct isolation of these products.
Other minor volatile products not predicted by schemes (24), (25), (26), and (35) include mono- and dibasic acids, 751v6 methyl ketonesJ 88'2~9 gamma lactones~ 188'216 benzene and o-xylene. 2~" 1,2-Dihydroperoxides in oxidized oleate have been suggested ~5 as a source of free acids. Oxidation of aldehyde esters would be an obvious route to dibasic acids. Other routes reviewed by Skellon and Wharry ~94 include double bond cleavage, hydrolysis of secondary hydroxy esters and oxidation of condensation products of alde- hyde esters. Methyl ketones in oxidized esters have been rationalized as products from the reaction between an acyl radical and an alkyl radical of c~-olefinJ 4~ or from an ester of oleic acid. 2~9 Gamma lactones may be derived from free oleic acid which is postu- lated 2~6 to produce 4- and 5-hydroperoxy octanoic acid radicals. Benzene may be an artifact but dehydrogenation of hexanal followed by dehydration has been suggested 219 as a possible route for its formation. Further oxidation of aldehydes and decomposition of secondary oxidation products are other important sources of volatile oxidation products (Section IV).
Thermal decomposition of the hydroperoxides from photosensitized oxidized methyl oleate produced not only all the volatiles expected from the 9- and 10-isomers but also those expected from the 8- and 11-isomers. v° The evidence showed significant isomeriza- tion of the 9- and 10-isomers into a mixture of 8-, 9-, 10-, and l l-isomers under the conditions used for thermal decomposition. Thermal rearrangement of oleate hydroper- oxides by the same process suggested by Chan et al} 8 for linoleate (23), would involve the same allylic 3-carbon intermediates as those recognized for autoxidation 53'55 (39):
--CH--CH:CH-- ~---CH--CH--CH-- ~--CH:CH--CH-- 1
0 0 ' +
'OO"
(39)
Since more of the volatiles derived from the 9- and 10-hydroperoxides (octane, l-octanol, 2-decenal, and methyl octanoate) were identified in the hydroperoxides prepared by photosensitized oxidation, decomposition was suggested as a process competing with isomerization. ~°
Volatile lipid oxidation products I l
~ -H.
. 0 0 ~
(j) tj') (Ki
00. 00. + + +
00. 00. 00. OO.
FIG. 2. Mechanism I of linoleate autoxidation. 55
B. Linoleate
Because linoleate is the major unsaturated fatty acid in vegetable oils, it has received the most attention. Free radical autoxidation produces a mixture of cis, trans- and trans, trans-conjugated diene 9- and 13-hydroperoxides. 15,s 5,56,165 Photosensitized oxidation of linoleate produces the same cis,trans-conjugated hydroperoxides together with cis, trans-unconjugated 10- and 12-hydroperoxides. 72'2°7 The ratio of cis,trans/trans,trans- diene linoleate hydroperoxides decreases with temperature of oxi- dation, 12'S3,55,z64,a65'z68 and increases with the concentration of linoleic acid and with the addition of a hydrogen donor such as p-methoxyphenol. 165
In one mechanism postulated 55 for linoleate autoxidation, the initial pentadienyl radi- cal assumes 4 conformations before reaction with oxygen (Fig. 2). Pentadienyl radical (I) formed initially is converted to (J) and then to (K) at higher conversion and elevated temperatures. In another mechanism, supported by kinetic studies, z64"165 pentadienyl radical (J) is formed from cis, trans or trans,trans peroxy radicals by fl-scission (40) which competes with H abstraction (41) forming the corresponding hydroperoxides (Fig. 3).
On the basis of these studies, the autoxidation temperature would be expected to influence the stereochemistry of the unsaturated carbonyl products from the decompo- sition of linoleate hydroperoxides. Although little information is available on this aspect, there is indication that the autoxidation and decomposition temperatures may influence the relative importance of cleavage reactions (24) and (25). 126,200
As in oleate, aldehydes, aldehyde esters, hydrocarbons, n-alcohols, and lower fatty esters expected from decomposition schemes (24), (25), (26), and (35) have been reported in studies of the thermal decomposition of linoleate hydroperoxides, 19.,~,2.7o,2o6 and ther- mally oxidized linoleate, 4'4°'42'76'77'87'126,127,157,158,181.2o6 trilinolein~ 89,208 and lino-
(41)~L-H R2
R / " ~ ' ~ O 0 H
-,~(40) R ~ R2 ~(40)
"f +
-00-
• 00 ,R 2
(41 )~rL-H HO0 " R;~
164 165 FI~3. 3. Mechanism II of linoleate autoxidation. •
12 F.N. Frankcl
TABLI; 2. Volatile Decomposition Products from Heated Oxidizcd Linoleate and Its Isomeric Hydropcroxidcs
Relative pcrcenl tt~,droperoxidc Me Hydroperoxidcs Eth>l esters I rilmolcm
source Volatiles 70 Norm." 76 Nt~rm.' I ,~t)~, Norm.,~
Other ~fldetlydes Acetaldehyde 0.3 Acrolein Propanal Butanal Pentanal 0.S Heptanal Nonanal 2 3-Hexcnal 2-Heptcnal Trace 2-Octenal 2.7 2,4-Nonadienal 0.3 Me 8-Oxooctanoatc 1.3 Me 9-Oxononanoate 19 Me 10-Oxodecanoate 0.7 Me 10-Oxo-8-decenoate 4.9 4.5-Epoxx-2-decenal
OfJle'l" co~l{~otlllds Pentanc 9.9 Hexane ('71 I-Pentanol 1.3 I -Oc~cn- 3-ol Trace 2-Pentyl furan 2.4 Me Heptanoalc 1.0 Me Octanoate 15
2.9
C~.4 3.7
4.s
3.1~ x2
IS.~ 44
4.3
12.0 0.7 3.4 1.7 0.S
~'Normalized. bOther minor volatiles included acids (1.8'!,,). gamma lactone (0.5'I,,I, furan 10.4",1. and meth'd ketone {lraccl. "Determined as 2-isomer.
lea te-conta ining fats (Table 2). Major volatile carbonyls include hexanal, 2,4-decadienal,
and methyl 9 -oxononanoa te . Al though not expected from either 9- or 13-hydroperoxides of linoleate, 2-heptenal is also an impor t an t product reported in autoxidized linoleate esters and fats containing linoleate. <~°7< 1 0 3 . 1 2 ~ , , 1 5 8 , 1 8 9 . 2 0 ( ~ , 2 0 ( , . 2 0 8 When pure hydroper-
oxides were compared, only a trace of 2-heptenal was detected from autoxidized lmoleate bu t 9.9°0 from photosensi t ized oxidized linoleate. 7° Therefore, 2-heptenal arises probably from the 12-hydroperoxide that was identified in photosensi t ized oxidized linoleate as well as in m a n y vegetable oil esters at low levels of o x i d a t i o n ) s'"' '
The isomeric 9- and 13-hydroperoxides of methyl l inoleate were shown to be readily
in terconver ted 14,1s and both of these isomers gave the same volatile cleavage c ompounds
but in different amounts . ~ Evidently the isomerizat ion, 9 - l ino lea te -OOH ~_ 13-1inoleate- O O H , occurred rapidly and prior to thermal decomposi t ion. The volatiles reported in this study included only hexanal, methyl octanoate, 2,4-decadienal. and methyl 9-oxono- nanoate . The absence of 2-alkenals was explained ~'~ by a suggestion that these products came from low tempera ture react ions other than the basic cleavage reactions 124) and (25). This assumpt ion is not suppor ted by the work of Kimoto and Gaddis ~ z~, and by our f inding of 2-octenal and 2-nonenal by decomposing linoleate hydroperoxides at a higher tempera ture ( 2 1 0 C compared to 160 C). 7°
The format ion of 2-octenal was a t t r ibuted by early w o r k e r s < ~ ' ' 1 ~ ' ' 2 ° ° to the de- compos i t ion of a noncon juga ted l l -hydroperoxide by cleavage reaction ~24). However, this hydroperoxide has never been detected under a wide range of au toxida t ion con- ditions. ~s'Ss'~8"~s° More recently, evidence was reported that 2-octenal can or iginale
Volatile lipid oxidation products 13
from either the 9-hydroperoxide of linoleate or from 2,4-decadienal oxidized in the presence of tert-butoxy radicals. 181 Peroxy attack on the ~o-6 double bond was suggested for hexanal (42) and on the o)-8 double bond for 2-octenal formation (43), via allylic peroxy intermediates:
CH 3 (CH 2)4--C H~---CH--CH~----CH--CH--R 1
• O O R O O H
CH3(CH2)4--CH--CH--CH--CH--
l o. CH 3 (C H2)4--CH--C H--CH--C H--
L o. o. CH3(CH2)4CHO
CH3 (CH 2 h--CH~CH--CH~-----CH--CHO
' O O R
C H 3 (CH 2 h - - C H - - C H - - C H - - C H - - I
OOR
CH3 (CH2),~--CH~-CH--CH--CH--
I o.I OOR
CH 3 (CH2)4--CH=CH--CHO
(42)
(43)
Oxidation of linoleate favors the formation of hexanal under mild conditions and 2,4-decadienal at high temperatures.126"2°° Because these products are derived from both 9- and 13-hydroperoxides, this effect of temperature was explained by selective further oxidation of 2,4-decadienal. 2°° Another explanation is based on the relative importance of cleavage reactions (24) and (25). Because oxidation and decomposition of both oleate and linoleate favor the formation of alkanals, the carbon-carbon bond between a per- oxide group and a double bond was considered to be most easily cleaved [reaction (25)]. 116 On the other hand, enals and dienals produced by reaction (24) become more significant when oxidation and thermal decomposition are done at higher temperatures.
Alkanals formed by cleavage reaction (25) were identified in much larger amounts among the products of thermal decomposition of oleate than of linoleate hydropero- xides.7O,lS 5 The much smaller amounts of alkanals from linoleate hydroperoxides (Table 2) may be due to other reactions of the conjugated diene radical. Reaction between diene (L) and hydroxy radicals would form a vinyl alcohol (M) which tautomerizes into 3-nonenal. This aldehyde is readily isomerized to the 2-isomer 7° (Fig. 4). Formation of the vinyl alcohol (M) provides another pathway for the production of 2-octenal and hexanal through an allylic radical (N) with formaldehyde and glyoxal as byproducts (Fig. 4). Formaldehyde formation was previously suggested in a mechanism to explain the production of lower aldehydes from primary hydroperoxide intermediates 7'32 [reaction (29)]. Glyoxal was also isolated from oxidized methyl linoleate. 27
The conjugated diene radical (L) can also provide a route for the formation of 2-alkyl furans 157 by reaction with oxygen to produce a vinyl hydroperoxide (O) that undergoes cyclization via the alkoxy radical (P) formed by loss of hydroxyl radical (Fig. 4). Another scheme suggested for 2-pentyl furan proceeds through 7-ketononanal by dehydration, z3
14 t!. N. Frankel
I | ! CH3--(CH2I~--K]H-'CH--CH--CH-~.-CH--B , I
CH3--ICH;I~--CH-'-CH--CH--CH. "OH/
J CH~--ICH~)a--CH--'CH--CH--CH--OH
. ~ 4M) .
CH?--(CH?Ia--CH~CH--CH2--CHO~I
A CH3--1CH2)a-- ! H~_CHzC_H + HCHO 2 Nonenal ", foo'.
CH3--ICH21a--CH--CH--CHO + CH3--ICH2Ia--CHO
20clenal Hexana[
+ OCH--CHO GIyoxal
[L) ~ + H.
CH~--(CH~h--CH--'CH--CH--CH--OOH (0) " ' ~ H
CH 3--(CH ?),~--CH--CH--CH--CHO. IP~ ~, .
CH3--(CH2)4-- ~ l
CH3--(CH2)4- ~
2.Pentyl Furan
FI(;. 4. Volatile products from decomposition of 9-1inoleate hydroperoxide.
It is not clear, however, how this dicarbonyl intermediate is formed from linoleate, nor is 7-2-nonenolactone also suggested as a source of 2-pentylfuran. 52
The configuration of 2,4-decadienal produced from fats containing linoleate was estab- lished by gas chromatography and infrared studies.~°~' l o4 A ratio of 28:72 was reported for the trans,cis:trans,tran~ isomers of decadienal. Assuming that trans-2,cis-4-decadienal comes from the 9-hydroperoxide of linoleate by cleavage (24), the same configuration was assigned to the hydroperoxide, i.e. 9-hydroperoxy-trans-lO,cis-12-octadecadienoate (44).
OOH /
~ - ~ / ~ ---* / / = ~ CHO (44)
C H 3 ( C H 2 J ¢ R C H 3 ( C H 2 ) 4
Although this assignment was correct, deduction of the hydroperoxide configuration based on that of the corresponding dienal cleavage product is hardly tenable because any ~fl-unsaturated aldehyde would have its double bond almost entirely ira the trans con- figuration. The trans,trans-2A-decadienal was regarded as derived from either the corre- sponding trans,trans-9-hydroperoxide or by isomerization of the cis,trans-dienal product.
A series of C1 to C5 hydrocarbons was identified from linoleate autoxidized at room temperature, with pentane representing 90yo of the total. I°6 Pentane was also the main hydrocarbon from thermal decomposition of either autoxidation derived hydroperoxides (9- + 13-OOH) or pure 13-1inoleate hydroperoxide prepared by soy lipoxygenase. 42 Pen- tane formation was explained by cleavage reaction (24) on the 13-hydroperoxide isomer followed by hydrogen abstraction by the resulting alkyl radical. 42'73 (45):
[ 13 C H 3 ( C H 2 ) 4 M- CH--CH~---CH--CH~---CH--R --~ C H 3 ( C H 2 ) 3 C H 2.
OOH I CH3(CH2)3CH3 + R" (45)
However, the 9-hydroperoxide that does not produce pentane in the autoxidation sample was ignored. 'L2 Apparently, thermal isomerization of 9-hydroperoxide to the 13-hydro- peroxide 14 would explain pentane formation as a major product in autoxidized linoleate. Pentane production correlated well with the peroxide value and flavor score of oxidized fats containing linoleate. 43
Volatile lipid oxidation products 15
Although the major part of the volatile oxidation products (about 85~o) 7° of linoleate are primarily attributable as decomposition products of the 9- and 13-hydroperoxides, a variety of minor products cannot be explained by the classical cleavage mechanism (24), (25), or (26). These products include esters, alcohols, substituted dioxolanes, ketones, acetals, acids, lactones, and aromatic compounds. 52'1°3'1°~'129,135.189.208 Some of these minor volatile oxidation products can be attributed to further products from aldehydes and secondary oxidation (Section IV).
Thermal decomposition of the hydroperoxides from photosensitized oxidized methyl linoleate produced volatiles expected from the 9-, 10-, 12-, and 13-isomers. 7° Unique products expected from the 10-hydroperoxide include 10-oxo-8-decenoate and l-octen-3-ol. This unsaturated alcohol is a rearrangement product from 2-octen-l-ol expected from the reaction of 2-octene radical with a hydroxy radical (46):
I
CH3 (CH2)4--CH~-----CH--CH2 + CH (O") R
1 ' CH 3 (CH2)4--CH~--------CH--CH 2.
'OH
CH3 (CH2)4--CH~----CH--CH2--OH
1 CH3(CH2)4--CH--CH=CH2 (46)
1-Octen-3-ol has also been identified from autoxidized linoleic acid and reverted soy- bean oil. 1°3'196 In one mechanism, rearrangement of a hemiacetal intermediate was postulated from an unstable C-11 free radical intermediate of linoleic acid.l°3 In another mechanism, cleavage (24) was suggested in a 10-hydroperoxide intermediate of lino- leate.196 The resulting 2-olefin is selectively oxidized to a 3-hydroperoxide that is con- verted to l-octen-3-ol. The 2-olefin would be expected, however, to give a mixture of isomeric hydroperoxides (Section II. B). Nevertheless, there is increasing evidence that the 10- and 12-hydroperoxides from singlet oxygenation are formed in significant amounts in linoleate-containing fats at low levels of oxidation. 65'66
In addition to l-octen-3-ol, 2-heptenal was previously mentioned as an important product from 12-hydroperoxide. Other volatile oxidation products common in both photooxidized and autoxidized linoleate may originate from hydroperoxy cyclic per- oxides 7° (Section IV. C).
C. Linolenate
Free radical autoxidation of this triunsaturated fatty ester produces a mixture of 8 isomers of cis,trans- and trans, trans-conjugated diene-triene 9-, 12-, 13- and 16-hydroper- oxides . 16'53'55'56'63'69 In addition to these isomers, photosensitized oxidation of linole- nate produces cis, trans-unconjugated triene 10- and 15-hydroperoxidesJ 3'28'66'15°'2°5 Under a wide range of conditions, the cis, trans-linolenate hydroperoxides remain as the main isomers, and their tendency to form trans,trans isomers is much smaller than linoleate hydroperoxides. 16'63 The preponderance of the external 9- and 16-hydroperox- ides in autoxidized linolenate has been known for a long time 63 and confirmed recently. 16'69 The relatively large concentration of the 9- and 16-isomers has now been shown to be explicable by the tendency of the internal 12- and 13-isomers to cyclize into hydroperoxy cyclic peroxides (Q) and (R) (Fig. 5).152
16 E .N . Frankel
OOH
00.-. Cw
0--0
o--o OOH
1o)
HO0
-"'l /.oo
0--0
O~H. HO0 0--0
R = ((]H?}TCOOCH 3
F'lt;. 5. 1,3-('vclization of 12- and 13-1inolenate hydroperoxides .~ :
Most of the products expected from decomposition reactions (24), (25t, (26), and (351 have been reported in studies of the thermal decomposition of linolenate hydroper- oxides,7 o thermally oxidized linolenate, 4°" ~ ~ 9. t 2¢,, ~ 27. t .s 7. ~ 84 trilinolenin,~ 87 and linolenate- containing oil (Table 3). '*° Major volatile carbonyls include acrolein, propanal, 3-hexena[, Z4-heptadienal, and 2,4,7-decatrienal. The unsaturated C~2, C~ 3, C~5, and C~6 aldehyde esters expected from the 12-, 13-, and 16-hydroperoxide esters have not been identified, ~'~ probably because they would be expected to undergo aldol condensation or polymeriz- ation. Methyl 9-undecenoate is another expected cleavage product s¢' of the 12-hydroper- oxide isomer that has not been reported. Minor volatile products reported from oxidized
TABLI 3. Volatile Decomposit ion Products from Heated-Oxidized Linolenate and Isomeric Hydroperoxides
Relative percent Hydroperoxide Me Hydroperoxides Methyl esters Trilinolenin
source Volatiles 70 N o r m ? 127 Norm/ ' 187" Norm."
Other uhh'hydes Ethanal 0.8 Butanal I). I 2-Butenal (1.5 2-Pentemd 1.6 2-Nonenal Me 8-Oxooctanoate 0.6 Me 9-Oxononanoate 13 Me 10-Oxodecanoate 1 Me lO-Oxo-8-decenoate 4.2 4,5-Epoxy-2-heptenal 0.2
Other compounds Ethane,,'ethene 10 Ethanol/furan 2-Butyl furan 0.5 Me Heptanoate 1.8 Me Octanoate 22 Me Nonanoate 0.7
32.9 100 84 100 53 100
6 2 3 3 9 6 4 1
T r a c e -)-)
1.4
;'Normalized. bOther minor volatiles included 2-pentene 10.8!..~,), 2-pentenyl furan (0.7y,,), ethyl furan (1 ?~,K and acids (1.5'~,,). "Values for 3-hexenal were divided by assuming that they come equally from 12-OOH and 13-OOH.
Volatile lipid oxidation products 17
linolenate include 3-hexen- 1,6-dial,119 ethyl and 2-pentenyl furan,~ 87 4,5-epoxy-2-hepte- nal,~ 87 2-pentenal,~ ~ 8.18 v and 4-heptenal.~ 84
3,5-Octadiene-2-one is a major volatile produced from linolenic avid oxidized in the presence of haemoglobin. 87 Although no mechanism was suggested for the formation of this ketone, the hydroperoxy cyclic peroxide (Q) identified ls2 from the 12-1inolenate, hydroperoxide (Fig. 5) is a likely source (Section IV. C). Similarly, 2-pentenal may be derived from the secondary oxidation of 3-hexenal or 2,4,7-decatrienal 5a (Section IV. A).
Thermal decomposition of the hydroperoxides from photosensitized oxidized methyl linolenate produced many of the same volatiles as the corresponding hydroperoxides from autoxidized linolenate but in different proportion. 7° Distinguishing products found in high proportions in the photosensitized oxidation hydroperoxides include 2-butenal (11~,) expected by cleavage (24) from the 15-hydroperoxide, 56 and methyl 10-oxo-8- decenoate (13%) expected by the same type of cleavage from the 10-hydroperoxide isomer. Oxidation of linolenic acid oxidized with singlet oxygen in aqueous methanol produced carbonyl compounds similar to those found by oxidation with haemoglobin, i.e. propanal, 2-hexenal, 2A-heptadienak and 2,6-nonadienal. 87 Surprisingly, 2-butenak previously found as a unique product of hydroperoxides from photosensitized oxidation of linolenate, 7° was not reported.
D. Soybean Oil
This oil has been studied extensively because it provides the most important source of vegetable food fat and has the tendency to produce a unique types of deterioration known as "flavor "reversion. ''29'53 This flavor problem has been reviewed recently s6,Sv and its origin is still being debated. 195
Soybean oil containing a mixture of oleate, linoleate, and linolenate can produce, on oxidation, 14 positional isomeric hydroperoxides, both by free radical autoxidation and by singlet oxygen from photosensitized oxidation, s6'66 Interaction between different fatty esters during oxidation of their mixtures has been demonstrated. For example, more linolenate than linoleate hydroperoxides were produced from a mixture of oleate, lino- leate, and linolenate when oxidized at low levels and more linoleate than linolenate hydroperoxides at higher levels of oxidation. 69 With soybean and other vegetable oil esters (cottonseed, safflower, and corn), an unexpectedly high concentration of the 12-hydroperoxide isomer was found at peroxide values below 50. 6s'e6 Because these oils do not contain linolenate, the likely source of the 12-hydroperoxide was suggested to be photosensitized oxidation of linoleate. This conclusion was supported by studies of the effect of singlet oxygen quenchers (//-carotene and a-tocopherol) and carbon black treat- ment to remove natural photosensitizers. It is not surprising therefore that the mechan- isms to explain the unusual odor and flavor deterioration of soybean oil have been difficult to unravel. Nevertheless, a large proportion of the volatiles identified by different workers s6 can be explained by the classical cleavage reactions (24), (25), and (26) of the hydroperoxides from oleate, linoleate, and linolenate. 51'52'1°2'118'186'196'198 Hydro- carbons (ethane, pentane, and 2-pentene) were the only volatiles identified in soybean oil oxidized at room temperature to a peroxide value of 0.8.186 A wider range of saturated and unsaturated hydrocarbons and aldehydes (C> C3, C5, C6) were the principal vola- tiles identified at peroxide values between 1 and 11. Since the initial formation of satu- rated hydrocarbons would require cleavage of type (24), the observations of Selke et al. 186 are not consistent with those of Kimoto and Gaddis, 126 who claimed that cleavage reaction (25) between a peroxide group and a double bond was favored under mild conditions. However, these authors limited their analyses to carbonyl compounds. The effect of temperature on the nature and relative proportion of volatile lipid oxidation products is not regarded as a simple phenomenon, x49 Factors contributing to this com- plicated problem include hydroperoxide structure, stability of decomposition products and competing secondary reactions such as carbon-oxygen scission, epoxidation, dihyd- roperoxidation, cyclization, and dimerization (Section IV). In the metal-catalyzed oxi-
J.I'.I.,R. 2 2 / I B
IS |!. N. F ranke l
dation of methyl linoleate, linolenate, and arachidonate, no hydrocarbons were detected during a 20-hr peroxidation period unless excess ascorbic acid was added) ~ Ascorbic acid apparently acts as a hydrogen donor in promoting the hydroperoxide decompo- sition catalyzed by Fe 3~ or Cu e ' .
Volatiles that are not expected as primary cleawtge products from unsaturated fat hydroperoxides include dialdehydes, ~ ~o ketones,2O,,~s. ~o2. ~ ,~,~ -~-~.,~,,, ethyl esters, C~, C~ 0, C~ hydrocarbons, 2-pentylfuran, 2-~ lactones, benzene, benzaldehyde, and acetophe- none. ~ ' A number of these volatile compounds may be derived from decomposition of secondary products (Section IVI. The possible origin of 2-pentyl furan from oxidized linoleate was discussed m Section III. B. A synthetic >pentenyl furan has also been attributed as a source of the flavor of reverted soybean oil. '~'~ Although it is not clear whether or not this furan is formed in oxidized soybean oil, its origin from 9-tinolenate hydroperoxide can be explained by the sequence in Fig. 4. Similarly, 5-(pentenyl)-2- furaldehyde has been detected m acid-treated oxidized soy phospholipids 1~2 and the 9-1inolenate hydroperoxide was postulated as its origin but no mechanism was proposed, Cyclic peroxides produced by singlet oxygen may be important precursors of substituted furans and furaldehyde tSection IVl.
Because flavor deterioration of soybean oil occurs at such low levels of oxidation, early workers considered this problem to be nonoxidutivc in origin. This issue has been recently reexamined and reviewed. 5~'~'5 Although offensive odors and flavors can be formed in soybean oils under nonoxidative conditions, there is much evidence that oxidatively derived precursors are inw~riably present that decompose into volatile flavor compounds. Such precursors include secondary oxidation products, oxidative polymers, and nonvolatile carbonyl compounds (Section IV). Volatile oxidation products with isolated {,~-3 double bond derived from linolenic acid are also considered to have an unusually low threshold value, ~ 22 thus contributing to flavor deterioration at relatively low peroxide values (Section VI.
E. Hydro.qenated Oils
Catalytic hydrogenation of unsaturated oils results in the positional and geometric isomerization of the double bonds throughout the C-18 fatty acid chain. >~'"~ By' selective hydrogenation of the polyunsaturated fatty acids in vegetable oils, autoxidation is retarded but flavor deterioration is still observed. 5"'5: Although an extremely compli- cated mixture of hydroperoxides would be expected to be formed by oxidizing hydroge- nated oils, much progress has been reported on the volatile oxidation products. The unusually potent flavor compounds formed in hydrogenated oils appear to be derived from non-conjugated unsaturated aldehydes.
Isomeric diene mixtures known as isolinoleic acid are formed by' hydrogenation of linolenate in soybean oil "~È'~s'~3~~:: By definition, these dienes have double bonds separated by more than one methylene group. Oxidation of the double bonds between C-14 and C-16 has been implicated in the flavor deterioration of hydrogenated oils. 34~23'~24 Volatiles from the oxidation of a 95°., concentrate of methyl isolinoleate included 2-hexenal, 2,6-decadienal, propanal, and acetone. 89 Volatile components con- centrated from hydrogenated linseed and soybean oils were separated and trans-6-none- nal was associated with the unpleasant "'hardening" flavor ! 24 (Section V). Studies of the oxidation of synthetic methyl cis,cis-9,15- and 8,15-octadecadienoates demonstrated that these dienes are the precursors of this flavor. ~e-~ The 10-hydroperoxide responsible for the production of 6-nonenal was assumed to be derived from either the 9,15-diene (with allylic rearrangement) or the 8,15-diene (without allylic rearrangement)(47).
I
9,15-diene lo i 8,15-diene --+ C H 3 - - C H z - - C H = C H - - ( C H z ) , , - - C H ( O O H ) -}- C H = C H - - R (47)
r I
CH 3 - - C H x - - C H = C H - - ( C H 2 ) 4 - - C H O
Volatile lipid oxidation products 19
The formation of trans-2-nonenal was also demonstrated in hydrogenated peanut oil and oxidation of 10- and 11-octadecenoic acid was attributed as the source of the 10-hydro- peroxide precursor. 46
Of the 48 compounds identified in hydrogenated soybean oil aged at 85°C, trans, trans-2,6-octadienal, higher alcohols, and lactones were considered to play an important part of the "hydrogenation" flavor.221 Trans, trans- 12,16-octadecadienoic acid was sug- gested as the precursor of 2,6-octadienal, but a 10-hydroperoxide was mistakenly formu- lated instead of the l l-hydroperoxide required for its formation. In another study, 12° trans-6-nonenal was identified in hydrogenated linseed oil but not in hydrogenated soy- bean oil after aging at 130°C.
Eight isomeric hydroperoxides were recently identified from oxidized 9,15-diene and two conjugated hydroperoxides from oxidized 12,15-diene. 6° Assuming the same mech- anism for free radical oxidation as methyl oleate, oxidation of the A15 double bond of 9,15-diene produces a mixture of allylic 14-, 15-, 16-, and 17-hydroperoxides with an isolated A9 double bond. Oxidation of the A9 double bond produces a mixture of allylic 8-, 9-, 10-, and 11-hydroperoxides with an isolated AI5 double bond. Significantly higher concentration of the 16- (15~o) and 17- (22~o) hydroperoxides were found than the other isomers. 6° The aldehydes expected by cleavage type (24) from the 8-, 9-, 10-, and 11-hyd- roperoxides are, respectively: 2,8-undecadienal, 2,7-decadienal, 6-nonenal, and 5-octe- nal. 56 From the 14-, 15-, 16-, and 17-hydroperoxides, the aldehydes expected by the same cleavage reaction are, respectively: 2-pentenal, 2-butenal, propanal, and acetaldehyde. As with linoleate, free radical oxidation of 12,15-diene produces the two conjugated 12- and 16-hydroperoxides. Aldehydes expected from these hydroperoxides by cleavage (24) are 2,4-heptadienal and propanal. Additional carbonyl compounds, hydrocarbons, and alco- hol would be expected from both 9,15- and 12,15-dienes by cleavage reactions (25), (26), or (35). Decomposition of dihydroperoxides identified in oxidized 9,15-diene would con- tribute further to the complex volatile products influencing the flavors and odors of hydrogenated oils (Section IV). The similarity between many of these volatile products with those formed by oxidation of linolenate (Table 3) explains why hydrogenation of soybean oil, with conventional catalysts producing isolinoleate, has not completely solved the problem of flavor reversion in this oil . 56 '57 '148
Although a great multitude of the volatiles expected from the ten hydroperoxides of 9,15- and 12,15-dienes have not yet been identified, nonenal is reportedly the only alde- hyde responsible for the unpleasant flavor of hydrogenated oils. 46'123'124 The melon or cucumber odor of this aldehyde, which has one of the lowest threshold values reported (0.005 ppm for odor and 0.0003 ppm for taste), appears to overwhelm the contribution of any other volatile oxidation products in hydrogenated vegetable oils (Section V).
IV. DECOMPOSITION OF SECONDARY PRODUCTS
Complications from secondary volatile oxidation products have been alluded to throughout Section III because of the multitude of compounds that cannot be accounted for by the basic cleavage mechanisms involving monohydroperoxides. Unsaturated alde- hydes and ketones formed as primary decomposition products from hydroperoxides are obvious sources of additional volatile products because of their susceptibility to further oxidation. Significant advances have been made recently on the identification of many nonvolatile secondary products from lipid oxidation, 56'5s including hydroperoxy epox- ides, hydroperoxy cyclic peroxides, and dihydroperoxides. Any secondary oxidation products with one or more hydroperoxide function would be expected to decompose and contribute further volatile lipid oxidation products. Monohydroperoxides can also form dimers and polymers and can react with unsaturated lipid substrates. Although many of these secondary products have not been well identified, there is much evidence that they can also contribute important volatile oxidation products that affect the overall flavor quality of lipid-containing foods.
20 E.N. Frankcl
Double bond isomerization in oxidation products is another important change affect- ing the flavor of lipid-containing foods. The conversion of cis,trans- to trans, trans-conju- gated hydroperoxides has already been discussed in Section II1. B. The configuration of the resulting unsaturated aldehydes affects flavor properties. Although the double bond in aft-unsaturated aldehydes is essentially always in the stable trans contiguration, the nonconjugated aldehydes are usually in the natural cis configuration. This cis double bond is readily isomerized on thermal oxidation to trans and in conjugation with the carbonyl group. These positional and geometric isomerizations of double bonds have marked effects on both the quantitative and qualitative flavor response of volatile oxi- dation products (Section Vt.
A. Ahtd~ydes
Many, of the volatile compounds found in oxidized lipids cannot be explained b\ cleavage of monohydroperoxides and are believed to originate from further oxidation of unsaturated aldehydes. The oxidation of unsaturated aldehydes was first suggested as a source of additional aldehydes from the decomposition of linolenate hydroperoxides. 5~ The formation of allylic intermediates in 2,4,7-decatrienal and 3-hexenal was postulated with formation of hydroperoxy aldehydes cleaving into lower aldehydes (2-pentenal and propanal) and dialdehydes (2,4-hexadiene-l,6-dial, glyoxal, 2,4,6-octatriene-l,8-dial, and 2-butene-l,4-dial). These suggestions were later confirmed experimentally with other unsaturated aldehydes.
Oxidation of 2-nonenal at 45 C produced C2, C> C> C8 alkanals, glyoxal, and a mixture of C~. C a. and C9 a-keto aldehydes. 133 The oxidation products of 2,4-heptadie- nal included C2, C3, ('4 alkanals, glyoxal, Cs-C9 a-keto aldehydes, cis-2-butene-l,4-diat and malonaldehyde. When 0.5 mole of oxygen was absorbed, 2-nonenal produced the corresponding acid and 2A-heptadienal polymerized. The cleavage mechanism postu- lated for the oxidation of 2-nonenal involves hydroperoxide formation of carbons 2, 3, 4, 5, and 6 with production of lower chain aldehydes and dialdehydes (48):
R - - C H 2 I C H = C H - - C H O o , R__CH2__C.H__CH(OO.I__CHO
2RH , R__CHz__CH2__CH(OOH)CHO
, R--CH2CHz--CHO + 2R. (48)
a-Hydroperoxidation of 2,4-heptadienal was the route suggested for ~-keto aldehyde formation (49):
CH 3--CH 2- -CH=CH-vCH2--CH (OOH }--CHO
II O
The C, and Ca a-keto aldehydes were presumed to come from dihydroperoxides. Both 2-nonenal and 2,4-heptadienal were more rapidly oxidized than either methyl linoleate or linolenate, whereas n-nonanal was much less oxidized than any of the unsaturated com- pounds. Therefore, during oxidation of unsaturated lipids, saturated aldehydes would be expected to accumulate, whereas the unsaturated aldehydes would further oxidize to lower aldehydes and dialdehydes at the more advanced stages.
A mechanism for oxidative degradation of aldehydes postulated, without direct experi- mental evidence, proceeds through a peracid intermediate ~ 35 (50).
R__CH2__CH O o, , R--CHz--CO3H --+ R--CH2COOH ~ RCHO + HCOOH. J ~ 50~
Volatile lipid oxidation products 21
This mechanism was extended to explain the formation of lower aldehydes, alcohols, alkyl formates and hydrocarbons by oxidation of n-nonanal. 48' 134 Cleavage of a peroxy acid intermediate would produce hydroxy and alkoxy radicals that lead to hydrocarbons and alcohols (51).
Autoxidation of purified 2,4-decadienal at ambient conditions produced a complex mixture of volatiles including pentane, furan, ethanol, hexanal, acrolein, butenal, 2-hepte- nal, 2-octenal, benzaldehyde, glyoxal, trans-2-buten-l,4-dial, acetic, hexanoic, 2-octenoic and 2,4-decadienoic acids as well as benzene. 136 The absence of pentanal was considered to rule out allylic oxidation (on C-6). Olefinic attack was supported to explain formation of 2-octenal and glyoxal (A2 double bond), and hexanal and 2-buten-l,4-dial (A4 double bond).
When 14C-labeled hexanal was oxidized in soybean oil, hexanoic acid was produced at 50°C, but no lower aldehydes were produced. 145 Similarly labeled 2,4-decadienal was converted to the corresponding acid at room temperature, as were also heptanal, 2-octe- nal, 2-nonenal, glyoxal, and malonaldehyde. The same sequence (48) was used to explain these oxidation products. 14C-labeled 1-octen-3-ol was converted to the corresponding ketone at room temperature.
In further studies of the oxidation of 2-nonenal, the formation of equal amounts of glyoxal and heptadecanal was explained by double bond cleavage resulting from alkyl peroxy radical attack on C-3 followed by ~-hydroperoxidation 49 (52):
R--CH~-----CH--CHO Ro~, R--CH(OOR)---C.H--CHO
o2 ~ R--CH(OOR)--CH(OOH)---CHO RH
RCHO + RO"
OHC--CHO + .OH (52)
Hexanal formation was explained by hydroperoxidation on C-4. Several acidic components have been identified in autoxidized linoleate 1°7 and veg-
etable oils. 189'196 Monobasic acids are apparently further oxidation products of the corresponding primary aldehydes. Dibasic acids are probably derived from oxidation of the aldehyde esters. 2°9
Although pure saturated aldehydes can be oxidized into a multitude of lower alde- hydes, hydrocarbons, alcohol, and acidic p r o d u c t s , 48'49'133-135'z45'156 they are much more stable than the corresponding enals and dienals. On the basis of the work of Lillard and Day 133 in a mixture of both saturated and unsaturated aldehydes, the saturated aldehydes would not be expected to further oxidize to any significant extent. Recent studies TM of autoxidation of mixtures of octanal and 2,4-decadienal revealed that satu- rated aldehydes are protected to a certain extent in the presence of unsaturated alde- hydes. Formation of 2-octenal from 2,4-decadienal in the presence of alkyl radicals was postulated to occur by a mechanism involving a 3-carbon allylic radical undergoing successive alkoxy and oxygen attack on C-2 and C-3 (53).
Formation of heptanal from 2-octenal was considered to proceed through the peracid with loss of CO2 and tautomerization of a vinylic alcohol (54):
R - - C H = C H - - C H O - - - 2 E - ~ H R - - C H ~ C H - - C = O ( O O . t
u , R--CH~-------CH--C~-O(OOH)
(o : , R - - C H - - C H O H ~ R - - C H 2 - - C H O 1541
To generalize previous studies, enals give on oxidation mainly the next lower saturated aldehyde, and 2,4-dienals give both the next lower enal and a saturated aldehyde of four carbons less than the parent aldehyde, together with dialdehydes. In the presence of any unsaturated substrate or oxidation products, saturated aldehydes would not be expected to contribute to the lower volatile lipid products.
B. High-Molecular-Wei,qht Materials
Monohydroperoxides undergo further oxidation and condensation to produce a very complex mixture of monomeric and polymeric polar and nonpolar materials. Early studies with autoxidized linoleate and linolenate showed the formation of significant amounts of polar and polymeric materials. 22"22° Purified hydroperoxides were later de- composed under various conditions and dimeric/polymeric materials proved to be the main products. 1 ~'1~'~ Although the evidence indicated that oxidative polymers of lino- leate and linolenate could readily form further volatile oxidation products, early workers were actually dealing with a mixture of oxygenated monomeric and polymeric products that gave an average molecular weight corresponding to dimers and trimers. One of the early theories advanced for the cause of flavor reversion in soybean oil was the decompo- sition of high-molecular weight secondary products from oxidized linolenate. 5~'~ '~s
When "'polymeric" fractions (tool. wt 536 1268) from autoxidized linolenate were further oxidized, the volatile products formed included acetaldehyde, propanal, 2-pentc- nal, and methyl ethyl ketone. ~ ~'~ Because these aldehydes were also identified in reverted soybean oil, oxidative polymers were suggested as precursors of the flavor reversion compounds. The volatile products from the polymeric fractions (tool. wt. 865 10701 from autoxidized linoleate included propanal, pentanal, and hexanal. 21 These oxidative pols- mers of linoleate were also implicated as precursors of favor reversion compounds.
Many studies have now been published on the nature of dimeric and high-molecular- weight materials that are formed by thermal decomposition of oxidized lipids. '*'*'47"~'2'154"'1~'1'1'm'21~ This subject has also been reviewed. 2'1~° These oxidative polymers and polar materials are lnown to decrease the flavor and oxidative stability of soybean oil. 33''~'~5 However, these materials are extremely difficult to characterize because of their complex composition. Although the structure of individual molecular species is still largely unknown, some general structures may bc considered in order to predict the kind of volatile products that might be expected.
The dimeric compounds with carbon carbon linkage identified from the decompo- sition of linoleate hydroperoxides had double bonds scattered between carbon-~; and carbon-10; dimers from oleate hydroperoxides had double bonds between carbon-6 and carbon-10) 2 Hydroperoxidation of these double bonds followed by scission would be expected to produce the same kind of volatile materials as the monohydroperoxide precursors. However, the relative position of the double bonds with respect to the car- bon carbon intermolecular bond may have steric effects on oxidative susceptibility. Pen- tane was the main hydrocarbon produced by thermal decomposition of the oxidative 'Mimer" fraction isolated chromatographically from linoleic acid oxidized with soy lipoxy- genase. '~2 However, this chromatographic fraction contained trimers, higher polymers. and polar products. Different volatile scission products may be expected from dirners
Volatile lipid oxidation products 23
having either an ether or a tetrahydrofuran linkage. 154 Clearly more information is needed by further studies with pure dimers.
High-molecular-weight aldehydoglycerides detected in oxidized fats 33'1°5 are expected by the same cleavage producing aldehyde esters from hydroperoxide esters, v3 Many of the unsaturated aldehyde esters expected from linoleate and linolenate hydro- peroxides have not been detected by thermal decomposition, T° probably because of their tendency to undergo aldol condensation and to polymerize. Similarly, glycerides contain- ing many of the same functional groups as the volatile products discussed in Section III are also present in oxidized fats, but these materials have not been reported. The effect of these high-molecular-weight materials on the formation of further volatile oxidation products affecting flavor is unknown and remans to be investigated.
C. Hydroperoxy Epoxy, Cyclic Peroxides and Other Oxygenated Compounds
A large number of secondary oxidation products have recently been identified from the decomposition of oleate, linoleate, and linolenate hydroperoxides. 56'58 Any hydroperoxy oxygenated fatty derivatives would be expected to decompose by the basic cleavage mechanisms discussed in Section II. C to generate new oxygenated volatile compounds. Only recently has progress been made possible in this area by advances in separation by high-pressure liquid chromatography and identification by gas chromatography-mass spectrometry (GC-MS).58
From oxidized oleate, the following monomeric oxygenated products were ident- ified :6~,132,151 epoxystearate, hydroxy and ketomonoenes, ketoepoxy, hydroxyepoxy and hydroperoxyepoxy, dihydroxymonoenes, and dihydroxystearates. Since some identifica- tions were based on reduced or hydrogenated products, many of the allylic hydroxy compounds may have been derived from the corresponding hydroperoxy or keto com- pounds. Therefore, possible precursors for these secondary products may include dihyd- roperoxides, hydroxyhydroperoxides or ketohydroperoxides.
From oxidized linoleate, a large number of secondary monomeric oxygenated products have been identified from both enzymatic 78's°'85'86'93"222 and nonenzymatic oxi- dations. 68'182'2°4 This subject was reviewed by Gardner. v9 Nonenzymatic secondary products include: keto and hydroxydienes, ketohydroxymonoenes, ketoepoxymonoenes, hydroxyepoxymonoenes, hydroperoxyepoxymonoenes, dihydroxymonoenes, dihydroxy- ketomonoenes, and trihydroxymonoenes. Possible precursors of dihydroxy esters include dihydroperoxides, hydroxyhydroperoxides, or ketohydroperoxides. Allylic hydroperoxy- epoxides identified among the products of linoleic 13-hydroperoxides, decomposed with cysteine-FeC13 or a soybean extract, 8° are the likely precursors of the allylic hydroxy- epoxides identified in autoxidized linoleate. 6s'ls2
Trans-2,3-epoxyoctanoic acid was identified as an important volatile product in autox- idized methyl linoleate, i o8 This epoxy acid may be derived from the trans- 12,13-epoxy-9- hydroperoxy-trans-lO-enoic acid, 8° by double bond cleavage (55) (Fig. 6). Trans-4,5- epoxy-2-heptenal was identified in oxidized butterfat 2°2 and trans- 15,16-epoxy- 12-hydro- peroxy-cis-9,trans-13-dienoic acid, expected from the 16-hydroperoxide of linolenic acid, was suggested as the precursor. This assumption is supported by the identification of 4,5-epoxy-2-heptenal as a minor volatile product from thermally decomposed methyl linolenate hydroperoxides. 7° 4,5-Epoxy-2-decenal was identified in thermally oxidized trilinolein,189 and the same allylic hydroperoxy epoxide precursor as shown in reaction (56) (Fig. 6) was suggested.
On the basis of these results, a large number of oxygenated aldehydes can be postu- lated as cleavage products from dihydroperoxides (57), hydroxyhydroperoxides (58), and ketohydroperoxides (59) (Fig. 6). Without further evidence, however, these reactions are only speculative.
72 204 Hydroperoxy cyclic peroxides 72'146 and dihydroperoxides . were recently ident- ified as secondary oxidation products of photosensitized oxidized methyl linoleate. The cyclic peroxides were suggested as important precursors of some of the common volatile
FK;. 6. I)ecomposition of secondary oxidation products of linolcatc.
products generated from linoleate hydroperoxides obtained either by autoxidation or photosensitized oxidation. 7° This suggestion has now been confirmed by thermal de- composition of the pure hydroperoxy cyclic peroxides from linoleate. ~ -~ The 13-hydroper- oxy-10,12-epidioxy-trans-8-enoic acid produced hexanal and methyl 10-oxo-8-decenoatc as major volatiles. The 9-hydroperoxy-10,12-epidioxy-trans-13-enoic acid generated 2-heptenal and methyl 9-oxo-nonanoate (Fig. 7). Other minor volatiles were the same as those from monohydroperoxides, except for 2-heptanone (fi'om the 13-hydroperoxy- 10,12-cyclic peroxide) and 3-octene-2-one (from the 9-hydroperoxy- 10,12-cyclic peroxidel, which appear to be unique volatile products.
The same cleavage schemes as discussed for monohydroperoxides (Section II.C) explain the formation of pentane and hexanal from the 13-hydroperoxy-10,12-cyclic per- oxide and the formation of methyl octanoate and methyl 9-oxononanoate from the 9-hydroperoxy-10,12-cyclic peroxide (Fig. 7). Another important cleawlge of the perox) ring and carbon-carbon bond [:~ to the trans double bond produced major amounts of methyl 10-oxo-8-decenoate and 2-heptenal from these respective cyclic peroxides. Less favorable fragmentation occurs between the double bond and the peroxy ring and clea-
i HO+-O 0 - ~ 0 I I I I I
CH3--(C H2)3 ~ ~ ~ \ I Ii {C H2)6--COOM e
~.,2 ,, I', ~ o . I i I
(2'4%)CH3--(CH2)3--CH3/ i ~ OHC--(CH2)7--COOMe (3"8%)
(2.5%) CH3--(CH2),--CHO / H ~ ~,H3--(CH2)6--COOMe 15.0%)
CH~--ICH~I~--CH=CH--CHO~ OHC--ICH21~--COOM, (39%) (27%1 / - u 2 , OHC--CH=CH--ICH 2)6--C OOMe C H3--(C H 2 )3 -C H'--C H--C--C H3 (2.6%) (4.9%)
F[(i. 7. Mechanism of thermal cleaw~ge of hydroperox~ cyclic peroxides from mcflL~,l ImoLcatc oxidized with ~O2. "a
Volatile lipid oxidation products 25
vage of the peroxy ring and carbon-carbon bonds fl to the hydroperoxy group. Malonal- dehyde postulated by cleavage on either side of the cyclic peroxides 174 was not identified under the GC-MS conditions used.
Secondary products identified from oxidized methyl linolenate included hydroperoxy- cyclic peroxides, 3 o,91,152 epoxyhydroxy dienes, and dihydroperoxides. ~ 52 Five-membered cyclic peroxides were identified from the enzymatic oxidation of linolenate. ~79 Autoxida- tion of enzymatically produced linolenate hydroperoxides also produced monocyclic peroxides 3° and bicycloendoperoxides. ~53 On the basis of the fragmentation schemes shown in Fig. 7, the hydroperoxy cyclic peroxides from linolenate would contribute the same type of volatile cleavage products as the monohydroperoxides before cyclization. Recent studies 7~ on the thermal decomposition of pure hydroperoxy cyclic peroxides from autoxidized linolenate have now confirmed the formation of the volatiles expected from the cleavage reactions in Fig. 7. The significant amount of 3,5-octadiene-2-0ne produced by oxidation of linolenic acid in the presence of haemoglobin a7 may be explained by the cleavage of the hydroperoxy cyclic peroxide (Q) identified in autoxi- dized linolenate 152 (60)(Fig. 5).
A recent report indicated the elimination of hydroperoxy cyclic peroxides from autoxi- dized methyl linolenate when 5~ ~-tocopherol was added. 159 However, this level of tocopherol is more than 50 times larger than that present in soybean oil and other vegetable oils, and at this concentration, tocopherol acts as a prooxidant. 59 At the normal concentrations of 0.05-0.1~o tocopherol, significant amounts of hydroperoxy cyc- lic peroxides remained in autoxidized methyl linolenate. Hydroperoxy cyclic peroxides are, therefore, probably important sources of volatile decomposition products of photooxidized fats as well as of fats such as soybean oil containing linolenate.
Six-membered peroxide rings can be formed by the reaction of substituted conjugated dienes with singlet oxygen, and these products are readily converted to alkyl furan, zl° Therefore, cyclization of the conjugated diene system in the 9-hydroperoxide of methyl linoleate by singlet oxygen can produce a hydroperoxy cyclic peroxide that can be decomposed into pentyl furaldehyde and then to pentyl furan 23'196 by loss of HCHO (Fig. 8). Although pentyl furaldehyde has not been reported, pentenyl furaldehyde ident- ified in oxidized soy phospholipid 192 (Section III. D) can be derived from the corre-
CH 3--(C1"12)4~ CH--(CH217C O0 Me I
102 ~ OOH
CH3--(CH~)4 0 ' ~ ~ 0 ~H--(CH2)TCOOMe OOH
CH3--1CH2)4~CHO • CH3--(CH2)4 ~ FIG. 8. Mechanism for pentyl furaldehyde and pentyl furan formation.
FIe;. 9. Mechanism of dihydroperoxide formation from autoxidized methyl linolenate. 152
sponding 9-hydroperoxide of linolenate. Therefore, cyclic peroxides produced by singlet oxygen may be important precursors of substituted furans and furaldehydes. The hyd- roxy and keto aldehyde from reactions (58) and (59) (Fig. 6) are also possible candidates for cyclization into pentyl furaldehyde and then to pentyl furan (Section III. B) by loss of HCHO.
The formation of dihydroperoxides from linolenate hydroperoxides was first suggested by hydrogen abstraction at the active methylene sites leading to structures containing both conjugated diene and triene systems. 53 This suggestion was later confirmed with the finding of conjugated dienoic and trienoic dihydroperoxides in autoxidized methyl linole- nate and the 9- and 16-hydroperoxides were indicated as precursors 1s2 (Fig. 9). These dihydroperoxides can serve as precursors of volatile compounds by the same cleavage mechanisms discussed for monohydroperoxides (Section II. C). Cleavage around the hyd- roperoxide group at the end of the fatty chain would produce the same volatile com- pounds as the monohydroperoxides. Cleavage around the hydroperoxide group on the ester end of the fatty chain would produce hydroperoxy aldehydes that can either lose the hydroperoxide (by elimination of H202) to give a conjugated aldehydes or cleave again to give dialdehydes. The formation of dialdehydes has already been reported in the oxidation of unsaturated aldehydes (Section IV. A). These suggested mechanisms for decomposition of dihydroperoxides are now only speculative and need to be confirmed experimentally.
V. FLAVOR SIGNIFICANCE OF VOLATILES
In studying volatile oxidation products, the chemist's job has been generally to isolate, identify, and classify a large number of substances. Much information has been accumu- lated on the identity of many compounds, but these volatiles have often only been assumed to have an impact on flavor. The type of flavor developed from a number of volatiles can depend on their complex interactions, concentration ranges, and the medium in which they are tested. The great complexity of flavors and the need for reliable panel testing have been main obstacles to advances in this field, Many studies of threshold and minimum detectable levels for a number of volatile substances have been reported, but the problem of interactions of many compounds in combination and per- mutation can be staggering.
The threshold values of a large number of volatile compounds reported in lipids and foods have been systematically compiled. ~'5's2'~22'l~°'14°'ls~'193 A wide variety of flavor descriptions have been reported for these compounds, and some controversy persists as to what compound(s) cause what particular flavor in fats and oils. 6'5°'88'98'141'1.7"184 Since different people use different terms to describe the same odor and flavor, it is often very difficult to reach agreement. To evaluate the importance of these volatile com- pounds, it is not only necessary to know their individual threshold values but also the
Volatile lipid oxidation products 27
quantities present in samples under different conditions. Although this task may appear staggering, certain individual compounds present in trace quantities may have such a strong intensive odor that they are significant in the overall odor and flavor of the food lipid. For example, when we were studying the hydroperoxides produced by oxidizing methyl 9,15-octadecadienoate, 6° we detected a very powerful cucumber-melon odor prevalent in the entire laboratory area. A flavor reminiscent of green melon was pre- viously ascribed to a mixture of cis- and trans-6-nonenal and suggested to come from the decomposition of the 10-hydroperoxide in oxidized 9,15-octadecadienoate. 124 Although this hydroperoxide isomer was one found in relatively small amounts (11%), 6° its de- composition produces an aldehyde that overpowers the effect of the other volatiles derived from the other hydroperoxides identified in oxidized 9,15-octadecadienoate. The trans-6-nonenal has indeed one of the smallest reported flavor threshold values of 0.0003 ppm in oi1.123 Other examples of potent aroma compounds include cis-4-heptenal from lipids in cod muscle with a flavor threshold of 0.0005-0.0016 ppm in oil, 139 and 1,cis-5-octadiene-3-one from butterfat with a flavor threshold of 0.00002 ppm. 2°1 No satisfactory mechanisms have been suggested for the origin of these volatile compounds.
Attempts to relate molecular structure with flavor intensity have not led to straightfor- ward generalizations. 122'14° With the exception of nonanal, 2-alkenals have a higher threshold value than the corresponding alkanals of same chain length.14° For 2-alkenals and trans,cis-alkadienals, homologous series of uneven carbon numbers have lower threshold values than those of even carbon numbers. The reverse trend is observed for the trans,trans-alkadienal series. Alkenals with an isolated double bond are more in- tensely flavored than the corresponding 2-alkenals. The effect of double bond configur- ation in the isolated alkenals is not consistent. ~39'14° On the one hand, cis-3-hexenal (0,11 ppm) and cis-4-heptenal (0.0005-0.0016 ppm) are much more intense than the corre- sponding trans-3-hexenal (1.2ppm) and trans-4-heptenal (0.1 0.32ppm). On the other hand, cis-6-nonenal has a higher threshold value (0.002 ppm) than the corresponding trans-6-nonenal (0.0003 ppm).123,124
The detection of rancidity off-flavors characterizing linoleate-containing fats requires a much higher level of oxidation than reversion off-flavors characterizing linolenate-con- taining fats. 56 The reason for this difference appears to be due to the low threshold value of the volatile aldehydes produced from oxidized linolenate and fatty acids with an to-3 double bond.122 Unsaturated aldehydes with an e)-3 double bond have particularly low threshold values, such as 3,6-nonadienal (0.0015 ppm), 2,6-nonadienal (0.002 ppm), 2-pen- tenal (0.046 ppm), 2,4-heptadienal (0.055 ppm), 3-hexenal (0.09 ppm), and 2,4,7-decatrienal (0.15 ppm). Hydrogenated soybean oil also produces volatiles of particularly low threshold values because they are derived from the oxidation of isolinoleate (Section III. E), producing aldehydes with remote double bonds such as cis-6-nonenal (0.002 ppm), cis-7-nonenal (0.0003 ppm), 2,6-nonadienal (0.002 ppm), and 2,7-decadienal (0.02 ppm).
Much attention has been given recently to the correlation between volatile analyses by gas chromatography and flavor evaluations. 3v'l12,113`16v'z15'2~8 Although this infor- mation has been useful in providing an objective approach to sensory evaluations of food lipids, little progress has yet been made in relating flavor descriptions with individual volatile compounds. No reliable method is yet available to predict the flavor stability of edible oils and lipid-containing foods. There is much variation on the description of individual compounds by different investigators, and this only denotes the subjective nature of panel testing. Further complications arise from additive and antagonistic inter- actions between mixtures of volatile compounds. ~ 4o,193
VI. V O L A T I L E S F R O M I N VIVO O X I D A T I O N
Much attention has been given to the problems of measuring lipid oxidation in vivo, and this subject was reviewed well recently. 2°3 One of the most sensitive methods that has been used extensively in the last few years consists of measuring respiratory hydro- carbons formed by decomposition of lipid hydroperoxides. This remote detection of in
28 E.N. Frankel
z,il~o lipid peroxidation is based on observations that chemical agents causing prooxidant stress, such as ozone, carbon tetrachloride, ethanol, nitrogen dioxide, and iron com- pounds, increase the levels of respiratory ethane and pentane in animals. Antioxidants such as vitamin E, and selenium decrease these respiratory hydrocarbons.
Ethane was reported as the major hydrocarbon produced by thermolysis of autoxi- dized linolenic acid. 42 Ethane and pentane were formed respectively by the iron-cata- lyzed decomposition of linolenic and linoleic acid hydroperoxides) ~' Ethane is derived from homolytic cleavage of the 16-hydroperoxide of linolenate by reaction (25) lSection II. C). Pentane is similarly produced from the 13-hydroperoxidc of linolcate. =° In ad- dition to these hydrocarbons, a large multitude of aldehydes and othcr oxygenated products would be expected to be formed by the cleawtge reactions discussed in Section III. s" Whether or not there is biological selectivity for hydrocarbon-producing cleavages in l,iro is also not known. However, if there is no such selectivity, quantitatively these hydrocarbons would represent a very minor fraction of the total xolatile decomposition products expected from lipid hydroperoxides. 7~ The fate and effects of these other hydro- peroxide decomposition products remain to be determined, and they may be of consider- able biological importance.
In ripo lipid peroxidation is known to be inhibited by selenium-glutathione peroxidase, an enzyme that reduces hydroperoxides into the corresponding hydroxy derivatives. 2°~ Chemically reduced hydroperoxides are also known not to produce volatile cleavage products by thermolysis under gas chromatographic conditions. ~ Ho~vever, there is evidence that a certain amount of in ~:iro formed hydroperoxides survive the enzyme systems responsible for protecting the body by reducing these hydroperoxides. The de- generative process of aging has also been related to slow steady-statc lipid peroxidation reactions. 2°~ From the absence of hydroxy fatty acids produced by' incubating phospho- lipid hydroperoxides from microsomal and mitochondrial membrane x~ith glutathione peroxidase, it was concluded that this enzyme system would not reduce hydropcroxides in the membrane. ~ 3s However, the glutathione peroxidase system is known to effectivelx reduce hydroperoxides in the gastrointestinal tract s and the liver, e" Animals have an apparent ability to var~ the activity of the glutathione peroxidasc system to protect the body, depending on lhe extent of oxidative damage by exposure to air pollutants such as ozone and nitrogen dioxide or rancid f o o d s . 2°'~
Most of the biological research has focused on the effect of monohydroperoxides and related organic peroxides. We still do not know how effective the glutathionc peroxidase and other protective enzyme systems are in reducing the multitude of secondary oxi- dation products discussed in Section IV. The formation of small amounts of respiratory hydrocarbons in animals adequately protected by vitamin E in thcir diet would seem to indicate that a certain amount of in ~,il,o lipid peroxidation may be a normal process. Recent experiments with swimming rats suggested that lipid peroxidation, as measured by respiratory hydrocarbons, is moderately increased after exhaustive exercises, s: Does this result imply that exercise may cause peroxidative damage, and that supplemenlary vitamin E in the diet is needed'? Another report suggests that respiratory, pentane in rats does not originate from membrane lipid peroxidation but rather from tile action of intestinal bacteria on linoleate hydroperoxides, a3 This subject is becoming controversial, and only further research can resolve some of these questions of rather important bio- logical consequences.
VII . ( ' O N ( ' L U S I O N S
Free radical mechanisms recognized for hydroperoxide decomposition tire based oll model alkyl hydroperoxides that are relatively stable and may not simulate the real events in lipid oxidation. A better mechanistic understanding is needed of the mode of decomposition of lipid oxidation products to provide a basis for predicting the course of the reactions that produce flavor problems. Identification of reaction intermediates and
Volatile lipid oxidation products 29
reaction pathways may provide the basis for preventing or directing the decomposition of hydroperoxides and secondary oxidation products into innocuous materials.
The oxidation of unsaturated lipids can produce an appallingly complex mixture of volatiles that can significantly affect the organoleptic properties of foods in extremely small quantities. Despite the progress made in understanding the origin of many of the volatile oxidation products, we still do not know how to eliminate undesirable flavors in foods containing polyunsaturated lipids. Flavor problems are complicated by a lack of understanding of odor and flavor development. Attempts to simulate familiar flavors by known combinations of volatile compounds have been mostly unsuccessful. With advances in analytical methodology, we have accumulated a mass of information on volatile flavor compounds that has been difficult to relate to practical flavor problems in foods. More selective studies are needed to prevent or eliminate the formation of undesir- able flavor compounds in lipid foods. It will be necessary to determine the quantities of selected volatile oxidation products present in samples under different conditions. A better understanding is needed of the mechanism of formation of volatile oxidation products that are organoleptically important. The joint efforts of flavor and organic chemists are needed with fresh approaches to better understand the relation between flavor and food acceptance. A multitude of volatile compounds can be derived from further oxidation of secondary products of the type discussed in Section IV. Although many of these secondary products have not been well identified, they apparently have an important impact on both flavor and nutritional quality of lipid-containing foods.
The effects of hydroperoxide decomposition products in biological systems are vital problems of major consequence. This area of biological lipid oxidation is continuing to attract the attention of many investigators and remains controversial. The biological effects of the multitude of complex products of hydroperoxide decomposition remain largely unexplored and offer an important challenge for further research.
(Received 8 March 1982)
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