Effect of smoking on lipid stability in sardine (Sardina pilchardus W.)

Effect of smoking on lipid stability in sardine (Sardina pilchardus W.) Alfonso Beltrhn and Antonio Moral Instituto del Frio (CSIC), Ciudad Universitaria, E-28040 Madrid, Spain

Einflufl des Riiueherns auf die Lipidstabilitfit yon Sardinen (Sardina pilchardus, W.)

Zusammenfassung. Der Einflul3 des R/iucherns (2 h bei 30 °C und 45 min bei 75 °C) aufdie Lipide magerer Sardinen aus dem Mittelmeer, gefangen im M~irz, wurde untersucht. Als Mal3 der Ranzigkeit wurden ge- messen: Peroxidwert (POV), 2-Thiobarbiturs/iure- Wert (TBA), die freien Fetts/iuren. Ver/inderungen in der Fettsfiurezusammensetzung im Gesamtfett und verschiedene Fettfraktionen wurden ebenfalls gepriift. Nach dem R/iuchern hat die Ranzigkeit w/ihrend des Prozesses zugenommen. Diese Erscheinung ist be- zeichnend fiir die Lipidfraktion: Verringerung des Tri- glycerid- und Phospholipid-Anteils und die Erh/Shung des Anteils an freien Fetts/iuren. Ebenso ver/indert sich der Gehalt der Fetts/iuren; die polyungesfittigten Fettsfiuren mit langen Ketten (C2o und C22 )der n-3- Familie, die wichtig fiir die Ern/ihrung sind.

Summary. The effect of mixed smoking (2 h at 30 ° C and 45 min at 75 ° C) on lipids in lean sardine caught in the Mediterranean Sea in March was studied. The peroxide value, 2-thiobarbituric acid index, content of nonesterified and free fatty acid were used as measures of rancidity. Changes in the fatty acid composition in the total fat and various fat fractions were also exam- ined. Smoking resulted in increases in the peroxide value, the thiobarbituric acid index and fatty acid content because of rancidification taking place during processing. Such rancidification caused alterations in the relative percentage composition of the different lipid fractions, with significant decreases recorded in the triacylglycerols and phospholipids along with a concomitant increase in the nonesterified free fatty acids. Fatty acid composition also underwent changes, mainly a certain decrease in the polyunsaturated long-chain fatty acids (C2o and C22 ) of the family n-3, so important in nutrition.

Offprint requests to: A. Moral

Introduction

Smoking is a promising method of processing for small pelagic species like sardine, for which existing marketing channels are incapable of absorbing the large catches taken. Application of smoking to such species could increase consumption by making available attractive new products [1]. Smoking is one of the oldest methods of food preservation and combines the effects of brining, heating, and drying with that of the smoke itself [2]. All these factors directly affect the stability of the lipids in the fish, in particular the polyunsaturated long-chain fatty acids (C20 and C22 ) of the family n-3 [3]. Large amounts of such fatty acids, whose beneficial effects may help reduce the risk of cardiovascular disease, are present in small pelagic fish species [4]. Fish lipids are extremely vulnerable to oxidation precisely because of the high proportion of polyunsaturated long-chain fatty acids, and oxidative breakdown of fatty acids is one of the main problems faced when attempting to maintain the quality and food value of processed fish [5]. Few data are available in the literature on the effect of smoking on fatty acids in fish, and hence the object of this experiment was to study the changes taking place in sardine in the relative proportions of the different lipid fractions (triacylglycerols, phospholipids, and fatty acids) and in the fatty acid composition of the total lipids and each of the lipid fractions during smoking.

Materials and methods

Material

Sardines (Sardina pilchardus, W.) caught off Castel l rn de la Plana, Spain, during March were used. The fish was boxed in ice for trans- portation to the Institute, where it was processed and smoked within 30-32 h o f capture.

Processing

The fish were washed and hand-filleted. The sardine fillets were im- mersed in brine (158 g salt/1 water) at a fish/brine ratio of 1:4 for 3 min at a temperature of 20 ° C. The brined fillets were then aired in a forced-air cold store for 12 h at 1 ° C to ensure uniform salt dis- tribution in the tissue. Smoking was carried out in a Afos Torry-

Z Lebensm Unters Forsch (1989) 189:317-321 © Springer-Verlag 1989

Origina| papers

type kiln. The fire box was charged with damp beech and holm oak sawdust. Air flow inside the kiln was constant (continuous ventila- tion) and relative humidity was approximately 60%. The sardine fillets were held at 30 ° C for 2 h, after which smoking was continued for a further 45 min, during which time the temperature of the kiln was raised to 75 ° C.

Methods of analysis

All measurements were taken from two replications on each of three separate sample groups. Samples within each group were homoge- nized before measurement. The lipid content in the sardine was determined according to AOAC method 24024 [6]. The peroxide value as in UNE standard 55-023-73 [7], the 2-thiobarbituric acid index according to the method described by Lemon [8], and nonesterified fatty acids, determined in accordance with the method recommended by AOCS [9], were used as measures of rancidity. Lipid extraction followed the method set out by Bligh and Dyer [10]. The lipid fractions (triacylglycerols, phospholipids, and fatty acids) were sepa- rated by thin-layer chromatography (TLC) as described by Acebal [11]. The fatty acids of the total lipids and the lipid fractions were then methylated as described by Morrison and Smith [12], an inter- nal standard (nonadecanoic acid) was added. The methyl esters of the fatty acids so obtained were analyzed by gas chromatography in a Perkin-Elmer 3920 gas chromatograph equipped with a flame ion- ization detector. A 25-m-long WCOT fused silica (Supelcowax 10) capillary column with an inner diameter of 0.25 mm was employed. Working conditions were: injector and detector temperatures, 250 ° C; initial heater temperature, 185°C for 32 rain, with a pro- grammed increase at a rate of 2 ° C/min up to 200 ° C, where it was maintained until completion of elution; carrier gas, helium; flow rate, 2 ml/min. Identification was accomplished by comparing the relative retention times for the methyl esters of the fatty acids from the samples with those for different standards. Quantitative determi- nation of the lipid fractions (triacylglycerols, phospholipids, and fatty acids) was effected according to the method of Christie et al. [13]. Statistical treatment consisted of analysis of variance using BMDP programs P1V and P2V.

Results and discussion

The sardine used in the present study has been termed "lean" sardine in reference to its lipid content (5.11%), in order to differentiate it from sardine caught in summer, whose lipid content is much higher, associated with the marked seasonal variability in chemical composition in such species.

Peroxide values rose significantly after smoking (Table 1), because of the transformation of some unsaturated fatty acids, particularly those at the surface,

Table 1. Effect of smoking on lipid oxidation and hydrolysis in lean sardine fillets

Fish Peroxide Thiobar- Nonesterified value bituric fatty acid (mmol/kg) acid index (% oleic acid)

(~tmol/100 g)

Unsmoked 1.94" 0.72* 0.95* Smoked 6.25" 1.60" 5.21 *

Asterisks indicate significant differences (P < 0.05) between numbers in the same column

into peroxides [3]. During smoking the fish is exposed to atmospheric oxygen and the applied temperature, which may accelerate oxidation and thereby lipid breakdown. The salt that penetrates into the muscle during salting or brining is another factor. Low salt concentrations may act as pro-oxidants [14]. Hobbs [15] reported that the antioxidant effect of smoke may be overcome by the pro-oxidant action of the salt; nev- ertheless, a number of authors [14] have suggested that, at certain concentrations, salt may have a protec- tive effect in preventing lipid oxidation. The temperature attained during smoking appears to affect lipids adversely [16]; however, certain compounds formed by the Maillard reactions taking place during cooking may exert antioxidant action [17, 18].

In the present study, heating and possibly certain other factors mentioned above intensified lipid oxidation. This same effect was noted by Woolfe [19], Baird et al. [20], and Bhuiyan et al. [3], who reported higher peroxide values in fish immediately after smoking than in the raw material (sardinella, herring, and At- lantic mackerel, respectively).

Like the peroxide values, the 2-thiobarbituric acid index values also increased after smoking (Table 1), as a result of lipid oxidation caused by several factors associated with processing. However, in this case it was the secondary oxidation products, rather than the initial oxidation products (peroxides), that were de- tected, that is, malondialdehyde and the other sub- stances that react with thiobarbituric acid. Other workers have reported higher thiobarbituric acid index values in smoked mackerel than in the raw material from which they were produced [3, 21].

Table 1 clearly shows the effect of smoking on lipid hydrolysis. In all cases the various factors involved in smoking resulted in higher content of nonesterified fatty acid. In this respect, heating the fillets at 30 ° C for 2 h seemed to accelerate the hydrolysis reactions and activate the enzyme systems. Increased fatty acid production in smoked fish has been recorded in a number of papers [21-24]. However, the fatty acids produced by hydrolysis should not contribute to the appearance of rancid odours and tastes, because they are derived from long-chain, and hence high-molecu- lar mass, fatty acid esters of low volatility and solubil- ity.

Fractionation of the lipids from the sardine showed that triacylglycerols, phospholipids, and nonesterified fatty acids made up 99% of the total lipids, and consequently for calculation purposes these three fractions were regarded as making up 100%. The other fractions (monoacylglycerols, di- acylglycerols, cholesterol, and cholesterol esters) thus accounted for approximately 1% of the total, as has been reported by other workers, who recorded similar

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Table 2. Effect of smoking on the composition of triacylglycerols, phospholipids and nonesterified fatty acids in lean sardine

Sample Lipid Triacyl- Phospho- None- content glycerol lipid sterified (% dry (%) (%) fatty acid mass) (%)

Unsmoked 19.40 85.87* 13.16' 0.97* Smoked 19.24 85.32* 9.96* 4.72*

Asterisks indicate significant differences (P<0.05) between numbers in the same column

proportions of triacylglycerols, phospholipids and fatty acids in sardine with a 6.2% lipid content [25, 26]. The total lipid content in sardine has a direct in- fluence on the proportion of these three fractions, with triacylglycerol concentrations lower the leaner the fish [26].

Table 2 reflects the increase in the free fatty acid fraction after smoking, as reported by other authors [22, 23, 27]. The percentage composition of the phospholipid fraction decreased, and the share of the triacylglycerol fraction also declined slightly after smoking, though to a lesser extent than the phospholipids. However, because triacylglycerols are the major fraction, small decreases in their proportion are translated into distinct increases in the nonesterified fatty acid fraction. Decreases in the triacylglycerol and phospholipid fractions, particularly the latter, have been reported in smoked fish by some investigators [22, 28]. Other workers, however, failed to record any appre- ciable variations in these contents before and after hot smoking [3].

Some papers have considered the effect of heating on fish lipids. Bastavizi and Smirnova [29] and Koizumi et al. [30] reported decreases in the proportion of phospholipids as a result of the temperatures attained during smoking. Takiguchi [31] studied the effect of temperatures of around 30 ° C in the drying of typical Japanese anchovy-based products and reported an increase in the nonesterified fatty acid fraction accompanied by a decrease in the triacylglycerol fraction, with variable changes in the phospholipid fraction over the drying period.

Both the increase in the fatty acids and the reduc- tions in the triacylglycerols and, particularly, the phospholipids have often been ascribed to hydrolysis and oxidation of fish lipids subjected to heating pro- cesses like smoking [22, 27, 31]. This would appear to be the effect in the present instance, in which the phospholipid fraction underwent the greatest variation in response to oxidation and hydrolysis, with concomitant formation of nonesterified fatty acids.

The changes brought about by smoking in the percentage composition of fatty acids in the total lipids

Table 3. Fatty acid composition in the total lipids before and after smoking in lean sardine (caught in March)

Fatty acid Amount (% by mass) in fish

Unsmoked Smoked

14:0 7.13 6.93 16:0 16.40" 17.16" 16:1 (n--7) 9.12 9.07 16:2 (n--4) 1.13 0.98 16:4 (n-3) 0.85 0.84 18:0 2.65 2.58 18:1 (n-9) 4.50 4.32 18:1 (n-7) 2.19 2.06 18:3 (n-3) 0.69 0.78 18:4 (n-3) 1.82" 1.53" 20:1 (n-9) 7.10 7.18 20:4 (n-3) 0.90* 0.69* 20:5 (n-3) 13.19" 11.67" 22:1 ( n - 11) 11.52" 12.70" 22:5 (n-3) 1.07 0.83 22:6 (n--3) 12.64" 11.01 *

Asterisks indicate significant differences (P<0.05) between values for smoked and unsmoked fish

and in the various lipid fractions in sardine are re- fleeted in Tables 3-6. Of the 42 fatty acids identified, the 16 present in the largest amounts were selected for statistical analysis. The share of the fatty acids in the raw material was similar to that reported in other studies using sardine with similar lipid contents [32].

Table 3 presents the alterations in fatty acid composition in the total lipids brought about by smoking. The concentration levels of the different fatty acids re- vealed three trends, namely: constant, decreasing, and increasing. The percentage share of most of the fatty acids remained constant after smoking, as in the cases of the acids 14:0, 16:1 (n-7), 16:2 (n-4), 16:4 (n-3), 18:0, 18:1 (n-9), 18:1 (n-7), 18:3 (n-3), 20:1 (n-9), and 22:5 (n-3). The percentage share of acids 16:0 and 22:1 (n-I 1) underwent significant increases during smoking, while that of such other acids as 18 : 4 (n- 3), 20:4 (n-3), 20 : 5 (n-3), and 22 : 6 (n-3) fell.

The behaviour of the fatty acids in the triacylglycerol fraction during smoking (Table 4) was similar to that of the fatty acids in the total lipids, as might be expected, since the fraction accounted for 85% of the total lipids.

Marked changes were observed in the percentage composition of the fatty acids in the phospholipid fraction (Table 5)~ in line with the fact that this fraction underwent the greatest decrease during smoking (Table 2; see above). Accordingly, after smoking, the most pronounced losses in fatty acids in any of the fractions studied (11.5% and 19.7%, respectively) were recorded for the acids 20 : 5 (n-3) and 22 : 6 (n-3). The proportion of other acids like 22 : 5 (n-3), 20 : 4 (n-

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Table 4. Fatty acid composition in the triacylglycerol fraction before and after smoking in lean sardine (caught in March)


Unsmoked Smoked

14:0 7.01" 7.74* 16:0 16.10 16.27 16:1 ( n - 7 ) 9.10 9.44 16:2 ( n - 4 ) 1.17 1.14 16:4 ( n - 3 ) 0.80 0.94 18:0 2.11 2.28 18:1 ( n - 9 ) 4.74 4.78 18:1 ( n - 7 ) 2.34 2.55 18:3 ( n - 3 ) 0.74 0.68 18:4 ( n - 3 ) 1.90" 1.47" 20:1 ( n - 9 ) 7.53 8.05 20:4 ( n - 3 ) 0.61 0.73 20:5 ( n - 3 ) 12.56" 10.90" 22:1 ( n - l l ) 11.77" 12.99" 22:5 ( n - 3 ) 1.20 1.17 22: 6 (n - 3) 8.23 * 7.29 *

Asterisks indicate significant differences (P < 0.05) between values for smoked and unsmoked fish

Table 5. Fatty acid composition in the phospholipid fraction before and after smoking in lean sardine (caught in March)


Unsmoked Smoked

14:0 0.76* 1.13" 16:0 17.01 * 20.71 * 16:1 ( n - 7 ) 1.00" 2.77* 16:2 ( n - 4 ) 0.16 0.22 16:4 ( n - 3 ) 0.14 0.18 18:0 3.86 3.63 18:1 ( n - 9 ) 2.66 2.68 18:1 ( n - 7 ) 0.99 1.10 18:3 ( n - 3 ) 0.59* 0.24* 18:4 ( n - 3 ) 0.76* 0.46* 20:1 ( n - 9 ) 0.38* 0.66* 20:4 ( n - 3 ) 0.90* 0.54* 20:5 ( n - 3 ) 13.77" 12.19" 22:1 ( n - l l ) 0.59* 0.94* 22:5 ( n - 3 ) 0.80* 0.52* 22:6 ( n - 3 ) 58.75* 47.16"


3), 18:4 (n-3), and 18:3 (n-3) also dropped. In contrast, the concentrations of the acids 14: 0, 16 : 0, 16 : 1 (n-7), 20:1 (n-9), and 22:1 (n-ll) increased during smoking. The five remaining acids underwent no change.

The alterations in the composition of the nonesterified fatty acid fraction during smoking were greater (Table 6) than those taking place in the triacylglycerol fraction. Increases were recorded for the same acids as in the triacylglycerol fraction (see above) plus

Tible 6. Composition of the unesterified fatty acid fraction before and after smoking in lean sardine (caught in March)


Unsmoked Smoked

14:0 5.82* 6.46* 16:0 15.36" 18.30" 16:1 ( n - 7 ) 8.79 8.87 16:2 ( n - 4 ) 1.16 1.27 16:4 ( n - 3 ) 1.43 1.33 18:0 1.91 * 2.37* 18:1 ( n - 9 ) 4.19 4.11 18:1 ( n - 7 ) 3.25 3.30 18:3 ( n - 3 ) 1.28 1.18 18:4 ( n - 3 ) 2.15' 1.75" 20:1 ( n - 9 ) 4.13 3.96 20:4 ( n - 3 ) 1.28" 0.86* 20:5 (n--3) 13.64' 11.62" 22:1 (n-- 11) 6.45* 7.19" 22:5 (n--3) 1.49' 1.13" 22:6 (n--3) 13.89" 11.81 *


acids 14:0 and 18:0. Of the acids that decreased, both 20: 5 (n-3) and 22 : 6 (n-3) showed higher losses than in the case of the triacylglycerol fraction, that is, 14.8% and 14.9% in the nonesterified fatty acid fraction as opposed to declines of 11.4% and 13.2% in the triacylglycerol fraction. The concentrations of acids 18 : 4 (n- 3), 20:4 (n-3), and 22:5 (n-3) also fell during smoking.

Summing up, the saturated acids (chiefly acid 16:0) tended to increase during smoking, as did the monounsaturated acids [particularly acid 22:1 (n-I 1)]. In contrast, the percentage compositions of the polyunsaturated acids of the family n-3, principally acids 20:5 (n-3) and 22:6 (n-3), exhibited significant decreases during smoking. These results agree with those for fatty acid composition in smoked herring set out by Meizies and Reichwald [33], who reported an increase in acid 22 : 1 and a decrease in acid 22 : 6 as com- pared to levels in the raw material. Other authors have also reported decreases in the percentage concentrations of polyunsaturated fatty acids in response to smoking [22, 23, 34-36]. Conversely, some researchers have indicated that smoking exerted no practical effect on the fatty acid composition of fresh fish [3, 37].

A number of workers have related decreases in the polyunsaturated fatty acid content with lipid autoxidation [38-40]. Such acids are extremely vulnerable to oxidation, since, according to Enser [41], the relation- ship between the relative oxidation rate and the number of double bonds is nearly geometric in unsaturated acids, with the acid 22:6 (n-3) accounting for up to 70-80% of oxidation.

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Conclusions

It can be concluded from the results that oxidation and hydrolysis of lipids during smoking brings about a reduction in the content of the polyunsaturated fatty acids of the family n-3, particularly in the major acids 20:5 (n-3) and 22:6 (n-3). This is related to the observed increases in the peroxide value and 2-thiobarbituric acid index (autoxidation) as well as to the increase in the titratable fatty acids (hydrolysis) brought about by the various factors involved in the smoking process (temperature, salt, exposure to 02, etc.). Oxi- dation and hydrolysis were more evident for the phospholipid fraction, no doubt because of this fraction's extreme vulnerability to rancidification. Con- sequently, the greatest alterations in fatty acid composition took place in this fraction. Oxidation of the fatty acids in the triacylglycerol fraction was less than in the nonesterified fatty acid fraction, because the free form of fatty acids is more vulnerable to oxidation than the esterified form.

References

1. Beltr~m A (1988) Ph.D. Thesis. Universidad Complutense de Madrid, Facultad de Veterinaria

2. Toth L, Potthast K (1984) In: Advances in food research, CO Chicheter (ed). Academic Press, New York, p 87

3. Bhuiyan AKMA, Ratnayake WMN, Ackman RG (1986) J Am Oil Chem Soc 63:324

4. Driss F, Darcet P (1988) Rev Fr Corps Gras 1:7 5. Suzuki M, Wade S, Hayakawa S, Tamura S (1985) J Food Sci

50:358 6. AOAC Official Methods of Analysis (1975) 12th edn Anal

Chem. Washington, DC 7. UNE Norma 55-023-73 (1973) Materias grasas. Indice de

per6xidos 8. Lemon DW (1975) Environment Canada. Fisheries and Marine

Service Halifax Laboratory. New Series Circular 51 9. AOCS Official and Tentative Methods of the American Oil

Chemists' Society (1955) Free Fatty Acids Ca 5a-40. VC Mehlenbacher (ed), 2nd edn

10. Bligh EG, Dyer WJ (1959) Can J Biochem Phys 37:911 11. Acebal C (1975) Ph.D. Thesis. Universidad Complutense de

Madrid, Facultad de Ciencias, Secci6n Quimicas 12. Morrison WR, Smith LM (1964) J Lipid Res 5:600

13. Christie WW, Noble RC, Moore JH (1970) Analyst (Lond.) 95:940

14. Nambudiry DD (1980) J Food Sci Technol 17:176 15. Hobbs G (1982) In: Fish handling and processing. Mackie IM,

Merritt JH, Windsor ML (eds). Torry Research Station. Minis- try of Agriculture Fisheries Food, p 27

16. Aitken A, Connell JJ (1979) In: Effects of heating on foodstuffs. Priestly RI (ed.). Appl Sci Publ, London, p. 219

17. Koizumi C, Wada S, Toshiaki O (1987) Nippon Suisan Gak- kashi 53:2003

18. Gray JI, Pearson AM (1987) In: Advances in meat research VIII. Pearson AM, Dutson TR (eds.). AVI, Wesport, Con- necticut, p 221

19. Woolfe ML (1975) J Food Technol 10:515 20. Baird PD, Bennett R, Hamilton M (1985) Storage life of chilled

and frozen fish and fish products. IIF, Paris 21. Hanumanthappa B, Chandrasekhar C (1987) Fish Technol

24:112 22. Lapshin II, Shevchenko MG (1973) Rybnoe-Khozyaistvo 6:73 23. Shevchenko MG, Lapshin II (1975) Rybnoe-Khozyaistvo

11:74 24. Khammadi KA, Goncharov AM (1979) Rybnoe-Khozyaistvo

1:57 25. Melva PM, Tsukuda N, Okada M (1982) Bull Tokai Reg Fish

Res Lab 106:89 26. Vicetti R, Palma J (1984) Boletin de Investigacirn. Inst Tecnol

Pesq 2:45 27. Tsubouchi T, Matsui K, Kusaka H, Ohta S, Kaminura S (1985)

J Jpn Oil Chem Soc 34:563 28. Markina EV, Lapshin II (1981) Tovarovedenie 13:39 29. Bastavizi MA, Smirnova GA (1972) Investiya Vysshikh Ucheb-

nykh Zavademi Pishchevaya Technol 4:33 30. Koizumi C, Takada M, Ohshima T, Wada S (1986) S Bull Jpn

Soc Fish 52:1095 31. Takiguchi A (1987) Nippon Suisan Gakkaishi 53:14 32. Nair PGV, Antony PD, Gopakumar K (1979) J Food Sci Tech-

nol 16:151 33. Meizies A, Reichwald I (1973) Z Ern/ihrungswiss 12:248 34. Renon P, Cantoni C, Comi G (1978) Ind Alimentari 17:509 35. Goncharov AM, Khammadi KA (1979) Rybnoe-Khozyaistvo

3:54 36. Ostyakova EB, Skupova IP (1983) Rybnoe-Khozyaistvo 8:69 37. Afolabi OA, Osmola A, Arawomo A, Olusegun LO (1985) J

Food Technol 19:333 38. Shono T, Toyomizu M (1971) Bull Jpn Soc Sci Fish 37:912 39. Kim IS, Park YH (1984) Bull Korean Fish Soc 17:313 40. Murata H, Yamauchi K, Goto I, Nasu T (1987) Nippon Suisan

Gakkaishi 53:1835 41. Enser MB (1974) Meat freezing: why and how. Meat Research

Institute Symposium 3, Bristol

Received February 10, 1989

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Effect of smoking on lipid stability in sardine (Sardina pilchardus W.)