J. Sci. Food Agric. 1979, 30, 999-1006

Lipid and Autoxidative Changes in Cold Stored Cod (Gadus mouhua)

Roy Hardy, Alister S. McGill and Frank D. Gunstonea

Ministry of Agriculture, Fisheries and Food, Torry Research Station, 135 Abbey Road, Aberdeen AB9 8DG and a Chemistry Department, St Andrew’s University, Scotland

(Manuscript received 28 June 1978)

A study of lipid changes during the frozen storage of cod confirms that the major change is that of lipolysis. Oxidation is extremely slow and occurs primarily in the phospholipid fraction. Nevertheless, sufficient oxidation takes place during cold storage to reduce the acceptability of the fish primarily through the production of hept-cis-4-enaI. The mechanism whereby hept-cis-4-enal occurs is discussed.

1. Introduction

During the frozen storage of cod, off-flavours develop primarily through the oxidation of the lipid components.1 Of the many compounds produced in this autoxidation unsaturated carbonyls are the strongest flavour potentiators and of these hept-cis-4-enal is the compound that is most closely associated with the off-flavour (see Addendum).z

In the work reported here we have examined the changes that occur in cod lipids during frozen storage and have attempted to identify both the lipids most susceptible to oxidation and the precursors of hept-cis-4-enal.

2. Experimental

Lipid analyses were carried out on the same samples of frozen stored cod described in detail in previous communications.lJ

Minced fish samples (50 g) were extracted by the following three methods: (i), the Bligh and Dyer technique in the presence of butylated hydroxytoluene (BHT : 10 mg)3; (ii), alkaline saponification. The mince was refluxed for 1 h with 10% sodium hydroxide in 95% ethanol (300 ml) plus propyl gallate (10 mg); (iii), a modified Bligh and Dyer extraction in the presence of BHT (10 mg) whereby the solution was kept monophasic and evaporated to dryness on a rotary evaporator. The lipids were recovered from the residue using chloroform. Extractions of lipids for the U.V. experiments were carried out in the absence of antioxidants. To minimise oxidation care was taken to exclude oxygen at all stages by careful use of nitrogen blanketing techniques.

For the gas liquid chromatographic (g.1.c.) analysis, the lipids obtained in the various extraction techniques were esterified by refluxing for 1 h in methanol containing 3 % HzS04, 5 % benzene and a crystal of BHT. In those experiments concerned with the analysis of hydrogenated fatty acids, the lipids were hydrogenated in ethyl acetate solution using 5 % palladium on charcoal. The hydrogenated esters were analysed by g.1.c. using a Pye 104 gas chromatograph fitted with a 2 m x 4 mm i.d. glass column packed with 20 % Diethylene glycol succinate on Chromosorb W HDMS 80-100 mesh whereas the non-hydrogenated esters were chromatographed by splitless injection on a Silar 5 CP 30 m x 0.3 mm i.d. glass capillary column fitted into a modified Pye 104.4

For the quantitative analyses (Tables 1 and 2) the fish was spiked with an accurately measured amount of isodocosanoic acid before extraction and the g.1.c. peaks measured by triangulation.

0022-5142/79/1000-0999 $02.00 0 1979 Society of Chemical Industry

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Table 1. Fatty acid recoveries from the flesh of fresh and cold stored cod (200 days at - 10°C) using different methods of extraction

Methyl esters recovered by g.1.c. (g 100 g-1 fish)

Method of extraction Fresh Cold stored

Bligh and Dyer 0.500

Monophasic Bligh and Dyer 0.500 Alkaline saponification 0.510

0.400 0.498 0.494

Table 2. Repetitive analysis by capillary g.1.c. of three of the fatty acid components of cod flesh lipid

Run 1 2 3 4 5 6 7 8 9 10 X U

Ci8:o 14.5 14.5 14.6 14.7 14.7 14.5 14.5 15.4 14.7 14.5 14.66 0.28 C Z O : ~ 32.9 31.0 31.8 30.9 32.6 31.9 24.5 32.7 29.5 32.5 31.03 2.52 cz%:6 103.5 99.8 97.6 97.2 98.5 108.1 105.0 97.4 95.7 94.5 99.70 4.39

Area of peaks in mm2 compared to a normalised standard of 100 mm2.

Pure methyl linolenate was obtained from Lipid Supplies (St Andrews). Methyl linolenate hydroperoxides were prepared by the controlled oxidation (50 p m o l 0 ~ uptake) of pure methyl linolenate (1 g) at 0°C in a round bottomed flask (10 ml) fitted to a mercury manometer. Pure monohydroperoxides and polar hydroperoxides were isolated by silicic acid column chromato-

Volatile products from (a), the thermal decomposition (lOO°C) of methyl linolenate hydro- peroxides and (b), oxidised cod flesh lipids were collected in tapered flasks (% 500 p litre) by high vacuum distillation. The distillates were dissolved in pure pentane (200 p litre) and 30 p litre portions analysed by the two stage g.1.c. technique previously de~cribed.~

Methyl linolenate monohydroperoxides were reduced to the alcohols by sodium borohydride6 and the trimethylsilyl (TMS) derivatives prepared in the usual manner.7 G.1.c. of the TMS derivatives was carried out on a glass column (2 m x 2 mm i.d.1 packed with 15 % Carbowax 20 M on Universal Support 85-100 mesh and a 50 m x 0.25 mm i.d. Dexsil 300 GC metal capillary column.

Cod flesh lipids obtained using the extraction method (i) outlined earlier were fractionated according to the methods of Baron and Hannahan* for phospholipid and neutral components and of McCarthy and Duthieg for fractionation of the free fatty acids from neutral lipid.

Mass spectral analysis of methyl linolenate hydroperoxide and the TMS derivatives of the reduced hydroperoxides was carried out on an AEI MS 902 mass spectrometer. The monohydroperoxide structure was confirmed by using the direct insertion probe at a source temperature of 50°C above ambient. TMS derivatives were identified by connecting the Carbowax 20 M column via a Biemann Separator using a source and inlet line temperature of 200°C. All spectra were run at 70 eV. U.V. spectra were obtained using a Unicam SP 800 recording U.V. spectrophotometer.

g r a p h ~ . ~

3. Results and discussion

The lipid composition and content (as determined using the conventional Bligh and Dyer extraction procedures) of the fresh fish and after 200 days storage (three samples) is given in Table 3. Overall, some 30% of the lipid appeared to be lost during cold storage. More detailed analysis of the lipid classes showed that the loss was confined entirely to the phospholipids; the neutral lipid content remained unchanged and the free fatty acids increased. This result was not unexpected because others have shown that during the cold storage of cod, lipolysis of the phospholipids occurs10 and

Lipid and autoxidative changes in Gadus morhua 1001

Table 3. Analysis of lipid fractions from the flesh of fresh and cold stored cod (200 days at -10°C)

Fresh cod Cold stored cod samples from three different fish Lipid class (g 100 g-1 fish) (g 100 g-l fish)

Total 0.724 0.480 0.495 0.480 Neutral lipid 0.115 0.105 0.114 0.111 Free fatty acid 0.036 0.204 0.198 0.202 Phospholipid 0.573 0.171 0.183 0.167

with the loss of the phosphatidic acid moiety an overall decrease in the lipid content is to be expected. The decrease in the lipid content cannot however be explained entirely by such lipolytic activity and it must be assumed that either the phospholipid or free fatty acid was not recovered after cold storage of the fish because (a), it was reacting in some manner that made it inextractable or (b), the method of extraction was inefficient.

In the past we have noted that when the free fatty acid content of a lipid is high some loss of the acids to the methanol-water phase is experienced. This was examined in a little more detail in the present work.

The lipids were extracted by the three methods indicated in the experimental section, trans- esterified and the chromatographic results are shown in Table 1. These indicate that during cold storage of the fish no major loss of total fatty acid occurred and furthermore that the apparent loss of total lipid indicated in Table 3 is caused primarily by dissolution of fatty acid into the methanol-water phase which is discarded and not to the interaction of free fatty acid or phospho- lipid with the substrate or with oxygen. Supporting evidence for this was obtained by t.1.c. from which it was shown that the free fatty acids were the only components to increase on cold storage.

These analyses indicate that in the cold storage of frozen cod, loss of fatty acid by chemical reaction including autoxidation must be small. An attempt has been made to look at this in more detail by analysing the fatty acids in the fresh and frozen fish using capillary g.1.c. Direct quant- isation of fish polyenoic acids using this technique involves relatively large errors.ll In a series of ten repetitive analyses by capillary g.1.c. of cod lipid methyl esters using methyl isodocosanoate as an internal standard, it was shown that the coefficient of variation (CV) in analysis of the saturated esters was between 1-2%, the CV for the polyenoics was between 4 9 % ( Table 2). Thus although the reproducibility of results for the saturated esters was as good as or better than analyses reported elsewhere12 the methodology for the polyenoic components is inadequate to measure the small changes expected in the stored cod lipid samples.

In an attempt to overcome this the lipids were hydrogenated prior to conversion to the methyl esters and analysis by g.1.c. (Table 4). A slight but relatively consistent loss of the CZO and C Z ~ acids was detectable in the cold storage samples with a concomitant small relative increase in the c16

component. This is what one would expect if some oxidation of the C20 and C22 polyenoic acids had occurred or indeed if enzymatic interconversion of longer chain to shorter chain acids had occurred.

Analysis of the lipid fractions by this technique (Table 5 ) indicated that the neutral lipid tended to remain unchanged; the phospholipids showed an increase in the cl8 acids with a decrease in the c 1 6 and CZO components and in the free fatty acids the proportion of the C2z acids increased primarily at the expense of the CIS component. The change in the composition of the free fatty acids appears to be closely related to the liberation of phospholipid acid moieties. Thus using the lipid compositional figures quoted by Addison13 for cod phospholipids it is possible to calculate the free fatty acid composition on storage if the following assumptions are made: (i), the fatty acid compositions of the component phospholipids are identical; (ii), the measured loss of phospholipid is real and not a loss caused by solubilisation in the aqueous phase by the extraction procedure; (iii), there is no specific loss of fatty acid due to further reactions.

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Table 4. Composition of major flesh lipid fatty acids (after hydro- genation) from fresh and cold stored cod (200 days at - 10°C)

R. Hardy et al.

Fresh cod; samples from three different fish

Methyl ester % Composition 2 U

14:O 3.6 2 .4 2.6 2.9 0.64 16:O 19.0 21.2 21.2 20.5 1.27 18:O 16.6 16.8 16.4 16.6 0.20 20:o 18.6 17.6 18.1 18.0 0.50 22:o 42.2 42.0 41.7 42.0 0.25

Cold stored cod; samples from seven different fish, same batch as above

Methyl ester % Composition 2 U

14:O 3.3 2.9 3.3 3.6 2.8 3.0 3.2 3.2 0.28 16:O 25.5 25.7 23.3 25.5 22.5 23.7 23.9 24.3 1.26 18:O 17.7 17.0 16.1 17.1 18.3 16.7 17.1 17.1 0.70 20:o 15.5 17.0 16.3 16.6 17.7 15.5 17.3 16.6 0.85 22:o 38.0 37.4 41.0 37.2 38.7 41.1 38.5 38.8 1.60

Table 5. Composition (after hydrogenation) of the lipid fractions of fresh and cold stored cod (200 days at - 10°C). Each analysis was carried out on one fish

% Composition ________

Fresh Cold stored cod - _ _ _ ~ _ _ - ~ _ -

Methyl ester x u 2 u Calculated

Neutral lipid 14:O 3.0 2.8 2.9 2.9 0.1 3.0 2.5 2.7 2.7 0.25 16:O 22.0 22.3 22.1 22.1 0.15 23.0 22.2 22.5 22.6 0.40 18:O 29.0 30.1 31.0 30.0 1 .0 30.3 31.7 32.6 31.5 1.16 20:o 19.7 19.2 18.3 19.1 0.71 18.0 18.4 17.7 18.0 0.35 22:o 26.3 25.6 25.7 25.9 0.38 25.7 25.2 24.5 25.1 0.60

Free fatty acids 14:O 1 .0 1.0 0.9 1 .0 0.06 0.7 0.7 0.5 0.6 0.12 1.6 16:O 32.5 35.2 31.7 33.1 1.83 28.0 33.3 31.7 31.0 2.72 23.3 18:O 23.5 23.8 23.6 23.6 0.15 14.0 15.1 16.6 15.2 1.31 14.5 20:o 19.3 17.2 20.0 18.8 1.46 17.1 15.8 15.7 16.3 0.78 19.2 22:o 23.7 22.8 23.8 23.5 0 .55 40.2 35.1 35.5 36.9 2.84 41.4

Phospholipid 14:O 2.4 2.0 2.1 2.2 0.21 3.5 2.0 3.3 2.8 0.81 16:O 22.3 22.0 21.5 21.9 0.40 18.0 20.2 18.9 19.0 1.11 18:O 15.1 15.2 16.3 15.5 0.67 19.5 20.6 20.0 20.0 0.55 20:o 17.2 18.4 17.5 17.7 0.62 15.6 13.5 14.2 14.4 1.07 22:o 43.0 42.4 42.6 42.7 0.31 43.4 43.7 44.3 43.8 0.46

Lipid and autoxidative changes in Gudus morhua 1003

The calculated figures are given in Table 5 and taking into account the assumptions and the errors introduced in such calculations made here, the agreement between the calculated and experimental composition of the acids with the exception of the c16 components is good. The relatively small change that did occur in the phospholipids may possibly indicate that there is a slight preferential lypolysis of the CZO over the CIS acids whereas the small change of the CZZ acids is indicative of little oxidation. The error in the method of analysis is too great for one to say that no oxidation occurred, especially in the free fatty acid fraction.

U.V. examination of the lipids and the lipid class fractions (Table 6) shows that little conjugation of lipid occurred during cold storage which again suggests that the extent of oxidation in the fish was small. In fact an increase in conjugation of the lipids could only be observed with any certainty in fish stored for a relatively long period (- 4 years). In these fish only the phospholipids appeared to oxidise and these only slightly.

Table 6. U.V. absorption measurements of flesh lipids from fresh and cold stored cod

U.V. absorption (Eleml%)a 232 nm

Cold stored

Lipid class Fresh (28 weeks -20°C) (4years -15°C)

Neutral/free fatty acid 0.59 0.60 0.58 0.60 Phospholipid 0.48 0.56 1.28 3.20 Total 0.40 0.50 0.81 0.92

~~ ~ ~

a El cml%= absorption of 1 % solution in a 1 cm cell.

These results are in agreement with previous studies in which it was observed that autoxidation as measured by increasing peroxide values appeared to be very small.14 Taken together these observations are strongly indicative that little autoxidation occurs in white fish lipids during cold storage. Superficially, white fish lipids which have a high content of polyenoic fatty acids (ca 60 %), should be susceptible to oxidation and indeed when extracted they oxidise very rapidly. This suggests that some inhibiting influence is operable within the tissue.

Substances are present in fish such as tocopherols, ubiquinones and amino acids which are antioxidants and thus chemical inhibition is probably involved. But physical effects may be important also. In white fish such as cod, the lipid is bound mainly to the substrate in a diffuse manner (i.e. there are little or no lipid pools as often found in fatty fish). The concentration of the lipid at any reacting site is low and consequently the reaction rate will be reduced. In addition, in the autoxidation of tissue phospholipids (PL), radicals (X') and oxygen attack result in the formation of peroxy radicals which are bound to the substrate and are relatively immobile.

X' + PL + PL' + HX PL' f 0 2 + PL02'

The normal liquid phase propagation of the autoxidation reaction will not occur therefore unless: (a), the alignment of the lipid in the phospholipid protein complex is ordered in such a manner that the reactive methylene groups of surrounding polyunsaturated lipids are sufficiently close to the peroxy radical for hydrogen abstraction to occur; (b), lipids are released from the substrate, perhaps by lypolysis, and become free to oxidise, diffuse and interact with other lipid molecules; or (c), the radical is transferred by an exchange mechanism perhaps through the inter-

1004 R. Hardy el al.

action of water or another lipid molecule and the radical so formed propagates the autoxidative reaction.

PL02'fHzO +PLOOH+OH' OH' + PL + PL' + HzO

With the possible exception of (a), intermediary reactions of this nature will reduce the autoxidation rate. The likelihood that the phospholipid fatty acids are aligned as in (a) is improbable and as little or no oxidation was observed in the neutral lipid or free fatty acids, then propagation by (b)-type mechanisms must also be unimportant. The dominant controlling mechanism in the propagation reaction is most probably that described in (c), which may also be the rate controlling step in the overall oxidation reaction too.

Although gross oxidative changes do not occur in white fish lipids previous work has shown that during low temperature storage sufficient amounts of volatile lipid oxidation products are produced to detract from the acceptability of the fish.l1 These products are carbonylic in nature, the most important being hept-cis-Cenal. With the exception of this aldehyde most of the carbonyls present can be accounted for by simple breakdown of known hydroperoxide products of 11-3 and 72-6 methylene interrupted (MI) polyenoic acids. Production of hept-cis-4-enal is not so readily explained.

F I D I x 10' I n teg ro to r 311

F I D I ~ I O ~ I n t e g r o t o r 506

I D 2Ox I ntegrator 6;

- u c a, I cf I vi

Figure 1. G.1.c. traces showing the relative amounts of hept-cis-4-enal and hepta-trans-2-cis-4-dienal produced on heating (a), methyl linolenate monohydroperoxide; (b), methyl linolenate polar hydroperoxide.

Lipid and autoxidative changes in Gadus morhua 1005

This compound has been identified in other foodstuffs and autoxidising lipids and mechanisms of production have been postulated. Thus it has been suggested that autoxidation of non-methylene interrupted (NMI) fatty acid dienes such as n-3,7 or n-3,8 would produce this a1deh~de.l~ NMI acids, although not n-3,7 and n-3,8, have been reported in marine biotal6J7 but we could find no evidence in cod for any that would produce hept-cis-4-enal by autoxidation. Only the n-3 polyenoic acids present in cod lipids produced hept-cis-4-enal and since pure methyl linolenate obtained from linseed oil also produced hept-cis-4-enal it is concluded that it is a product of the oxidation of n-3 MI polyenoic acids.

In the autoxidation of these latter acids, hydroperoxides are produced which when isolated and heated yield hept-cis-Cenal. It was noticeable that hept-cis-4-enal was produced, along with other carbonylsl from both the monohydroperoxide and also a more polar hydroperoxide isolated from the autoxidised methyl linolenate (Figure l), although the latter peroxide produced less hept-cis-4- enal relative to one of the major autoxidative products hepta-trans-2-cis-4-dienal.

Analysis of the TMS derivatives of the reduced monohydroperoxides from methyl linolenate by g.1.cJm.s. using a packed column showed the presence of at least four major components derived from the 9, 12, 13 and 16 monohydroperoxides of methyl linolenate [Figure 2(a)]. Analysis using capillary g.1.c. indicated the presence of a greater number of hydroperoxides [Figure 2(b)] but unfortunately the separation was incomplete and we have not been able to assign structures to all

Figure 2. G.1.c. of the TMS derivatives of reduced methyl linolenate monohydroperoxide on: (a), Carbowax 20 M packed column (2 m x 2 mm i.d.) at 230°C 30 ml min-l. Separation of the TMS derivatives of the 9, 12, 13 and 16-hydroxy-methyl-octa-decatrienoates; (b), Dexsil 300 GC metal capillary column (50 m) at 250°C indicating a more complex mixture.

1006 R. Hardy ef al.

the various components. Because of this, it is not possible to state which is the most likely precursor of hept-cis-4-enal although it seems highly improbable that simple scission of the four major peroxides (i.e. 9,12, 13 and 16) found and predicted by theoryL8 would give rise to this component.

References 1. McGill, A. S . ; Hardy, R.; Gunstone, F. D. J. Sci. Food Agric. 1977, 28, 200. 2. McGill, A. S.; Hardy, R.; Burt, J. R.; Gunstone, F. D. J. Sci. Food Agric. 1974, 25, 1477. 3. Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911. 4. McGill, A. S.; Parsons, E.; Smith, A. Chemy Ind. 1977, 456. 5 . Gardner, H. W.; Weisleder, D. Lipids 1970, 5, 678. 6. Frankel, E. N.; Evans, C. D.; McConnell, D. G.; Selke, E.; Dutton, H. J. J. Org. Chem. 1961, 26, 4663. 7. Freedman, G. J . Am. Oil Chem. SOC. 1967,44, 113. 8. Barron, E. J.; Hannahan, D. J. J. Biol. Chem. 1958, 231, 493. 9. McCarthy, R. D.; Duthie, A. H. J . Lipid Res. 1962, 3, 117.

10. Olley, J.; Lovern, J. A. J . Sci. Food Agric. 1960, 11, 644. 11. Ackmann, R. G.; Sipos, J. C. ; Jangaard, P. M. Lipids 1967, 2, 25 I . 12. Ackmann, R. G.; BarIow, S . M.; Duthie, I. F. J. Chromatog. Sci. 1977, 15, 290. 13. Addison, R. F. ; Ackmann, R. G.; Hingley, J. J . Fish. Res. Bd Can. 1968, 25, 2083. 14. Hardy, R.; McGill, A. S . Torry Research Station, Internal Report. 15. De Jong, K.; Van Der Wel, H. Nature (London) 1964, 202, 553. 16. Ackmann, R. G.; Hooper, S . N. Comp. Biochem. Physiol. 1973,46(B), 153. 17. Paradis, M.; Ackmann, R. G. Lipids 1977, 12, 170. 18. Farmer, E.; Sundralingham, A. J. J. Chem. SOC. 1942, 121. 19. Swoboda, P. A. T.; Peers, K. E. J. Sci. Food Agric. 1977, 28, 1010.

Addendum Some confusion regarding the flavour of this compound in fish has a r i~en .1~ During our prelim- inary threshold experiments with pure synthetic hept-cis-4-enal only two out of 30 untrained participants used the descriptive term ‘fishy’. The panel, however overwhelmingly recognise the flavour and odour as being ‘cardboardy’ at ten-fold its threshold concentration in water, while at higher concentrations it becomes unmistakably ‘painty’, ‘putty’ or ‘linseed oil-like’ in character. In cod it detracts from the ‘fishy’ flavour producing one of the dominant cold storage off-flavour notes (namely cardboardy).