Effects of feeding natural tocopherols and astaxanthin on Atlantic salmon (Salmo salar) fillet quality

Food Research International 27 (1994) 23-32

Effects of feeding natural tocopherols and astaxanthin on Atlantic salmon (Salvo salar) fillet quality S. Sigurgisladottir,ay* C. C. Parrish,aJBS. P. Lallb & R. G. AckmanaJ8 ‘Canadian Institute of Fisheries Technology, Technical University of Nova Scotia, PO Box 1000, Halifax, Nova Scotia, Canada, B3J 2X4 bDepartment of Fisheries and Oceans, PO Box 550, Halifax, Nova Scotia, Canada, B3J 2S7

Tocopherol has an important role in maintaining fish flesh quality, including the colour of salmon, by affecting the oxidative stability of lipids. The colour of salmon fillets is an important feature of salmon culture where salmon must be fed with carotenoid pigments in order to possess the characteristic pink or red colour. The objective of this study was to investigate the deposition of tocopherols and of astaxanthin in the muscle of Atlantic salmon. Their effects on fatty acid composition, taste/texture and effects on oxidative stability of salmon muscle were also examined. Tocopherols and astaxanthin were observed to not affect the lipid content or fatty acid composition. As expected, salmon muscle with the pink of astaxanthin was preferred by a sensory panel, but colour or tocopherol content did not affect the taste/texture of the flesh. Keywords: tocopherol, Atlantic salmon.

astaxanthin,

colour,

oxidative

stability,

taste/texture,

various biological processes such as ageing, cancer, arthritis and platelet aggregation (Packer & Landvik, 1989; Cerra et al., 1991; Murray et al., 1991; Fritsche et al., 1992). In addition to their antioxidant functions in live fish (Cowey et al., 1983; Watanabe, 1990), tocopherols are also believed to play an important role in maintaining the quality of salmonid flesh by providing postmortem oxidative stability (Frigg et al., 1990). Observations on frozen fillets of marine fish such as sole indicate that natural chemical and biochemical factors affect this role (Ackman, 1974; Ackman & Ratnayake, 1992). Carotenoids seemed to be likely co-factors in the post-mortem antioxidant process. Carotenoids are a group of fat-soluble pigments that contribute to the yellow, orange and red colours found in all families of the vegetable and animal kingdoms and are one of the most important natural marine pigment groups (Matsuno & Hirao, 1989). Carotenoids are also one of the main natural food colorants and their use is widespread. Animals are unable to perform de mvo synthesis

INTRODUCTION There are four quinone-based structures for tocopherols (a, trimethyl-; p and 7, dimethyl-; and S, monomethyl-). These are loosely called vitamin E, although often only a-tocopherol is meant by this usage. Vitamin E is an essential nutrient in the diet of salmonids (Watanabe et al., 1981a; Watanabe & Takeuchi, 1989). It has the same important role as a biological antioxidant in fish as in the human body (Poston et al., 1976). Tocopherols are generally classified as phenolic-type antioxidants which act as free radical acceptors, forming stable compounds that will not propagate further oxidation. Vitamin E has wide-ranging effects on *Present address: Technological Institute of Iceland, Keldnaholt, IS-l 12 Reykjavik, Iceland. tPresent address: Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada, AlC 5S7 §To whom correspondence should be addressed. Food Research International 0963-9969/94/$07.00 0 1994 Canadian Institute of Food Science and Technology 23

24

S. Sigurgisladottir, C. C. Parrish, S. P. Lall, R. G. Ackman

of carotenoid but some bacteria, algae, yeasts, moulds and the higher plants have this capacity (Liaaen-Jensen, 1978, 1991; Simpson, 1982). The consumer expects salmon flesh to be some shade of red (Skrede et al., 1989), but wild salmon and trout flesh is reddish only because they have consumed fish or crustaceans containing carotenoids; without these pigments salmonid muscle would be white. Salmon and trout raised in aquaculture are fed with carotenoid pigments, astaxanthin and canthaxanthin, to colour the muscle pink or red and thus to fulfil consumer expectations (No & Storebakken, 1992). Although astaxanthin is the most important natural red pigment found in salmon, lobster, crab, shrimp and red snapper, the synthetic carotenoid canthaxanthin has for some years been included in diets for salmon and trout (Torrissen et al., 1990; Storebakken & Choubert, 1991). Commercially available salmon diets contain 40-75 mg astaxanthin or canthaxanthin per kg. Once the salmon reaches 300400 g, this supplementation of carotenoid pigments increases feed-costs by 7-10% (Torrissen et al., 1989). The functions of carotenoids in living animal cells remain virtually unknown, as opposed to the profound knowledge of their chemistry (Karnaukhov, 1990), except for the role of carotene as a vitamin A precursor. Astaxanthin has been reported to have a positive effect on the growth rate of salmon during the initial feeding of young fish (Torrissen, 1984). During sexual maturation of salmon, large amounts of carotenoids are transferred to the skin and ovary from the muscle, suggesting that carotenoids have a function in reproduction (Torrissen et al., 1989). In a review on the functions of carotenoids, Tacon (1981) speculated that carotenoids can act as fertilisation hormones and enhance growth. However, any biological function of the carotenoids in fish remains unconfirmed (March et al., 1990). Besides pigmentation effects and the vitamin A activity of carotenoids, they may perform a biological role similar to that of a-tocopherol, i.e. protect delicate tissues from oxidative damage (Tacon, 1981; Bendich & Olson, 1989; Miki, 1991). Thus, p-carotene can readily undergo autoxidation (Burton & Ingold, 1984; Bendich & Olson, 1989; Burton, 1989; Chevolleau et al., 1992). Theoretically, all carotenoids with similar conjugated double bond systems should have chain breaking antioxidant capability and pro-oxidant characteristics (Burton, 1989). The colour of salmon fillets is an important

marketing feature but whether the pigment also directly affects the taste/texture has not been investigated. The objective of this study was to investigate the deposition of a tocopherol mixture and astaxanthin in the muscle of Atlantic salmon (Salmo salar) and the effects of the combination on colour, lipid content and fatty acid composition, on taste/texture and on oxidative stability of salmon muscle. A novel consideration was to determine if there were any connections between the pigment stability and specific tocopherols and their contents in the edible muscle.

MATERIALS

AND METHODS

Four tanks of 25 Atlantic salmon smolts averaging 309 g each were acclimated to feed, aquaria and standardised environmental conditions (La11 & Bishop, 1977). Sandbed-filtered seawater was supplied at a flow rate of 8-10 litres/min to fibreglass tanks, 2 m in diameter and 2 m3 in volume. The fish were maintained at 6.5-14°C for 15 weeks with a photoperiod of 14 h of light per day. The fish were fed to satiation three times daily. A random sample of six to 10 fish from each tank was weighed once a week to obtain a mean weight for each group of fish. Four diets were formulated and steam-pelleted (Laboratory Pellet Mill, California Pellet Mill Co., San Francisco, CA, USA) in the Halifax Laboratory of the Department of Fisheries and Oceans (Tables 1 and 2; the lipid composition is given in Table 3). These four diets differed only in tocopherol and astaxanthin supplements; diet 1 (control diet) was supplemented with synthetic astaxanthin (Carophyll Pink, a product of Hoffman-La Roche & Co. Ltd, Basle, Switzerland) and a natural mixture of tocopherols (Covi-ox T-70; Henkel Corp., LaGrange, IL, USA, 70% total tocopherols, Table 2); diet 2 was supplemented with synthetic astaxanthin only; diet 3 was supplemented with the natural mixture of tocopherols only; diet 4 was not supplemented with either astaxanthin or tocopherols. The feed was stored at -20°C under nitrogen in 1 kg bags until used. Three fish were sacrificed from each tank after 0, 4, 7, 10 and 15 weeks of feeding. Fish were filleted, the muscle weighed and lipids extracted by the method of Bligh and Dyer (1959). Part of the lipid was saponified and the tocopherol content determined by high-performance liquid chromatography (HPLC). Astaxanthin and canthaxanthin in the lipid were also determined by

Effects of tocopherols and astaxanthin on salmon quality

HPLC. The lipids were also transesterified and the fatty acids analysed by capillary gas chromatography (GC) as methyl esters. Feed pellets were accurately weighed (20 g) and placed in a beaker with 70 ml of water and sonicated for 15 min. The lipid was then recovered using the method of Bligh and Dyer (1959) and analysed as described above.

Table 1. Composition of the experimental dieWb Percentage

Feed ingredients

42.5 4.2 10.0 4.0 3.0 10.1 8.0 2.0 2.0 0.2 13.8

Herring meal Feather meal, hydrolysed Soybean meal Brewers dried yeast Blood meal, spray dried Wheat middlings Whey, spray dried Vitamin mixtureC Mineral mixtured Choline chloride Herring oil

Separation of major lipid classes of fish muscle

100.0

Total

“The composition is the same for all four experimental diets except in respect to astaxanthin and tocopherol contents (Table 2). ‘Proximate analysis (% air dry basis); dry matter 91.8; crude protein (%N X 6.25), 44.3; lipid, 18.1; ash, 8.2. Vitamins added to supply the following levels (mg/kg unless stated otherwise); thiamin, 40; riboflavin, 50; D-CdCiUm pantothenate, 150; biotin, 0.08; folic acid, 15; vitamin B,,, 0.1; niacin, 200; pyridoxine HCl, 30; ascorbic acid, 1000; inositol, 400; ethoxyquin, 125; vitamin K, 30; vitamin A, 6000 IU; vitamin D,, 4000 IU. dMinerals added to supply at following levels (mg/kg); manganese, 50; iron, 60; zinc, 120; copper, 15.

Table 2. Astaxanthin and tocopherola contents of the experimental diets by analysis Diet no.

CY

1 2 3 4

84.2 88.6 0 0

236.0 9.8 212.0 6.7

P

Y

8

25.0 0.0 20.4 0.0

921.0 0.0 793.0 0.0

333.5 0.0 294.4 0.0

“A natural mix of tocopherols (Covi-ox T-70 natural antioxidant mixture from Henkel). bAll results are expressed on dry weight basis.

Table 3. Fatty acid composition of the total lipid in the diets Fatty acid Saturated Monounsaturated C,,-polyunsaturated Cis-polyunsaturated 20 : 5n-3 22 : 5n-3 22 : 6n-3 Other polyunsaturated

Weight (%) 22.8 56.2 2.2 6.2 5.0 0.8 5.5 1.3

Phospholipid and triacylglyceride were separated by plate thin-layer chromatography (TLC) according to Christie (1987). TLC was performed on Adsorbosil 5 (Applied Science Laboratories, College Park, PA, USA) silica gel plates which were cleaned by development in ethyl acetate, activated at 100°C for 30 min and cooled in a desiccator. The plates were streaked, using a plate streaker (Applied Science Laboratories). The plates were developed in hexane/ethyl ether/acetic acid (80 : 20 : 1) for 45 min. The plates were air-dried, then sprayed with 0,2’/0 2’,7’-dichlorofluorescein in ethanol. The bands were visualised and marked in a UV light box and the separated bands were scraped into test tubes for extraction. The silica gel carrying the triacylglycerol bands was extracted with chloroformhexane (1: 1). The phospholipid bands were recovered with chloroform/methanol (2 : 1). Gas chromatographic analyses

Tocopherolb (mg/kg feed)

Astaxanthin (mg/kg feed)

25

All analyses of fatty acids from lipid samples were performed using gas chromatography after transesterification by the method of Morrison and Smith (1964). A Perkin-Elmer Model 900 gas chromatograph fitted with a SUPELCOWAX-10 (bonded polyethylene glycol) fused silica column, 30 m X 0.32 mm, with 0.2 pm phase (Supelco Canada, Oakville, ON) was used. Helium pressure was set at 76 kPa. The following temperature programme was used: isothermal at 195°C for 8 min, followed by an increase of 3”C/min to 24O”C, and finally, isothermal operation for 20 min. The weight percentage of each fatty acid was obtained by computer program (Ackman & Eaton, 1978). Peaks were identified by comparing retention times with those of a mixture of standard methyl esters. Analysis of lipid classes by Iatroscan TLUF’ID A Mark III Iatroscan TLC/FID (thin-layer chromatography with flame ionisation detection) was

26

S. Sigurgisladottir, C. C. Parrish, S. P. Lall, R. G. Ackman

used to measure lipid classes in the total lipid extracted from the flesh of the fish. The procedure followed that described by Parrish and Ackman (1983). Tocopherol determination Unsaponifiable matter was recovered by the standardised method of Pocklington and Dieffenbather (1988). Tocopherols were determined by HPLC according to O’Keefe and Ackman (1987), using a Model 6000A solvent delivery system (Waters Associates, Milford, MA, USA), a Model U6K septumless injector (Waters Associates), Model 420-AC fluorescence detector with excitation wavelength 290 nm and emission wavelength 330 nm (Waters Associates), and a laboratory computing integrator Model LCI-100 (Perkin Elmer Corp., Analytical Instruments, Norwalk, CT, USA). Chromatographic separations of the (Y-,p-, y-, and &tocopherols were carried out on a normal phase Partisil-5 silica column (12.5 cm X 4.6 mm, Chromatographic Specialties, Brockville, ON, Canada). Standard curves were made up using D-a-tocopherol (Kodak Laboratory Chem., Rochester, NY, USA, 6340, 99%), D--y-tocopherol (Kodak Laboratory Chem. 6685, 95%), D-y-tOCOpherol (Sigma Chemical Company, St. Louis, MO, USA, T-2028, 90%). Astaxanthin and canthaxanthin analyses Astaxanthin and canthaxanthin were analysed by HPLC according to Hoffman-La Roche Ltd (1987). Lipid extracted from salmon flesh was dissolved in hexane to approximately 20 pg/ml. The HPLC system included a Model 5000 solvent delivery system and a Model UV50 detector (Varian, Sunnyvale, CA, USA) set at 476 nm, with 0.1 absorbance units full scale (AUFS), a syringe loading sample injector Model 7125 (Varian) and a Model 5000 (Fisher Scientific Corp., Nepean, ON, Canada) chart recorder set at 1 mV full scale. Chromatographic separations were carried out on a normal phase silica column (PPorasil-5, 3.9 mm X 30 cm stainless-steel column, Millipore Corporation, Milford, MA, USA) and hexane/acetone (86 : 16) as a mobile phase with a flow rate of 1.5 ml/min. If the separation was insufficient, a solution of phosphoric acid in methanol (1 g/100 ml) was pumped through the column for 1 h at a flow rate of 1 ml/min. The mobile phase was then pumped through the column for at least 30 min to

equilibrate the system. Astaxanthin and canthaxanthin reference standards (99O/ pure) were obtained from Hoffman-La Roche Ltd, Basle, Switzerland. The contents of the standard solutions were determined by measuring the absorbance in a spectrophotometer (Model PU 8600 UV-Vis, Pye Unicam) at 476 nm using 1 cm cuvettes. Extinction coefficients of 191 for astaxanthin and 220 for canthaxanthin were used. Determination of colour of salmon fillets Colour cards for salmonids (Hoffman-La Roche Ltd) were used to determine the colour difference between the fillets from Atlantic salmon fed on four different diets. The colour score ranges from very light (score 11) to dark pink (score 18). The colour matching was performed according to the procedure recommended by Hoffman-La Roche Ltd. Sensory evaluations A triangle test with 10 panellists (difference test) was used to determine if there were a detectable difference between taste/texture and colour (Larmond, 1977) of salmon fillets from Atlantic salmon fed the four different diets. For the taste/texture difference the room was darkened and the panellists instructed to wear sunglasses, to mask the colour difference between the samples. The triangle test was performed on combinations of all four groups; diet l-diet 2; diet ldiet 3; diet l-diet 4; diet 2-diet 3; diet 2-diet 4; diet 3diet 4. Analysis of the results of the triangle test was based on the probability that if there were no detectable difference, the odd sample would be selected by chance one-third of the time. The results of a triangle test indicate whether or not there was a detectable difference (significant at P < 0.05) between two samples. The second part of the triangle test was to choose the more acceptable sample. The results from the judges who correctly identified the odd sample were then considered for preference of the two samples. Oxidative stability Oxidative stability was determined by a forced oxidation of the salmon muscle, according to Frigg et al. (1990). Salmon muscle pieces (c, 1 g) taken from all parts of the fillet (10 g in total) were stored in a beaker (500 ml) covered with

27

Effects of tocopherols and astaxanthin on salmon quality

parafilm at 35°C for 60 min. The thiobarbituric acid value was then measured to determine the oxidative stability, according to Woyewoda et al. (1986). Statistical analyses

Data sets for each group were analysed by twosample t-tests to determine whether sample means were different, using pooled standard deviation. The difference was found to be significant if PC 0.05. The arcsin transformation (Armitage & Berry, 1988) was used for proportion data if statistical comparisons were performed.

RESULTS

AND DISCUSSION

Fish growth

During the experimental period, while the temperature of the seawater increased slowly from 6.5 to 14”C, the fish doubled their weight from a mean of 308 to 560-630 g (Fig. 1). Fish fed diet 4, which contained neither astaxanthin nor tocopherols, showed less appetite and gained less weight than those fed the other diets, except for the two last sets of data points, where the mean weight was similar to that of the other groups. Sensory panels were performed in the week prior to the weighing and large numbers of fish were therefore removed as samples. This may have skewed the last two sets of data for fish fed diet 4 and the last set of data for fish fed the control diet. Neither astaxanthin nor vitamin E can be separately considered as

4

0

Sampling

Fig. 1. A comparjson

7

period

IO

limiting growth factors, since diets 2 and 3 were lacking these compounds individually and the weight of the fish was comparable to those on the control diet (diet 1) which had both. The diets lacking tocopherols and astaxanthin both may have led to enough stress that the fish fed diet 4 did not grow as well as those in the other groups. The mean of the feed conversions over the feeding period was similar for the four groups, control, 0.72; diet 2, 0.83; diet 3, 0.73; diet 4, 0.79. Lipid content and composition of salmon muscle

The lipid content of the muscle increased with growth during the feeding trial from 2.6 to 3.6% in fish fed diet 1 (Fig. 2). With deposition of additional fat in the muscle, the degree of unsaturation of the fatty acids was lowered in total lipid; the level of eicosapentaenoic acid (EPA) (Fig. 3) and docosahexaenoic acid (DHA) (not shown) decreased significantly (P < 0.05) during the 15 weeks of the feeding trial in all the diets. The accumulation of

0

4 Sampling

Fig. 2. Lipid

content of course of the experiment described for Fig. 1. Data three fish. No significant between

10

7 period

I5

(weeks)

salmon muscle through the time when fed the four different diets are the mean *SD of samples from difference (P> 0.05) was observed any two groups.

1.5

(weeks)

of growth of Atlantic salmon fed the four diets. Diet 1 contains astaxanthin and tocopherols; diet 2 contains astaxanthin but no tocopherols; diet 3 contains tocopherols, but no astaxanthin; diet 4 contains no astaxanthin and no tocopherols. Data points are the mean &SD of the weight of at least six fish. No significant differences were observed among the groups (P > 0.05).

Total

lipid

Triglyceride

Phospholipid

Fig. 3. Changes in content of eicosapentaenoic acid of fatty acids in total lipid, triacylglycerides and phospholipids (w/w %) of muscle of salmon fed the control (Number 1) diet described for Fig. 1 for 15 weeks.

28

S. Sigurgisladottir, C. C. Parrish, S. P. Lull, R. G. Ackman

new depot fat in the muscle was in the form of triacylglycerol (depot lipid). Thus, the phospholipid remained at approximately 1.6% of muscle weight while triacylglycerol increased from 1 to 2% (i.e. the triacylglyceride: phospholipid ratio increased from 39.3 : 60.7% at time 0 to 54.5 : 45.5% over 15 weeks of feeding). The fatty acid composition of the respective phospholipids and triacylglycerols did not change appreciably during the feeding trial (see EPA in Fig. 3 as an example). The small changes in fatty acid composition in total lipid of the muscle over the course of the experiment (Table 4) reflect the increase in the triacylglycerol portion. DHA was -40% of total fatty acids in the Table 4. Fatty acid composition of total lipid from salmon muscle after feeding for 15 weeks on 4 diets” Fatty

acid

14:o IS0 15 :0 15:o 16:0 16 : ln-9 16 : ln-7 16 : ln-5 16:2n-4 16: 3n-4 16:4n-1 17:o 18:0 18 : ln-9 18 : ln-7 18 : ln-5 18:2n-6 18 : 3n-6 18:3n-4 18 : 3n-3 18:4n-3 18:4n-1 20:o 20 : ln-9 20 : ln-7 20 : 2n-6 20 : 4n-6 20 : 4n-3 20 : 5n-3 21: 5%3 22: In-11 22 : ln-9 22 : ln-7 22 : 5n-6 22 : 5n-3 22 : 6n-3 24 : ln-9 Other ‘Data are significant (P < 0.05). bDiet 2 ‘Diet 3 dDiet 4 -

Diet 1

Diet 2’

5.9 f 0.9 0.2 f 0.2 0.3 * 0 13.6f0.2 0.1 * 0.09 6.9 + 0.2 0.2f0.01 0.6 + 0.3 0.4 * 0.2 0.4 f 0.09 @; ; ;.y

6.2 + 0.5 0.06 + 0.01 0.3 + 0 13.5,0.2 0.1 f 0.01 7.3 + 0.6 0.2f0.01 0.6 f 0.04 0.4 * 0.03 0.4 * 0.03 0.1 AZ0.01 1.8 * 0.08 10.4kO.3 2.4 + 0.06 0.4 f 0.01 3.2 If:0.0 0.2 * 0.09 0.2 + 0.01 0.8 * 0.4 1.4fO.l 0.3 + 0.01 0.1 f 0.01 14.2kO.6 0.5f0.01 0.3 If:0.02 0.3 f 0.04 0.6 f 0.02 4.0 + 0.5 0.2 f 0.01 14.6kO.8 1.1 * 0.05 0.2 + 0.01 0.1 + 0.08 1.3+0.08 10.1 f 1.0 0.2 + 0.06 1.9

10.6f0.7 2.0 + 0.5 0.4 * 0.09 3.4 + 0.5 0.1 f 0.01 0.2 + 0.02 0.8 * 0.08 1.3 * 0.1 0.3 + 0.05 0.1 * 0.09 13.7 * 0.2 0.5+0.02 ;I; f 8’;” 0.7 + 0.09 4.6 + 0.4 0.2fO.l 13.2 f 1.3 1.0 YC0.05 0.5 + 0.2 0.3 + 0.2 1.5 z!z0.2 12-3 + 1.6 @; IL 0.03

Diet 3”

Diet 4d

6.0 L 0.1 5.8 * 0.1 0.07 + 0.03 0.08 + 0.03 0.3 * 0.02 0.3 f 0.01 13.8kO.6 13.6kO.3 0.1 + 0.01 0.1 * 0.01 7.1 + 0.1 7.1 + 0.4 0.2+0.02 0.2+0.01 0.6 f 0.01 0.6 f 0.02 0.4 * 0.02 0.4 * 0.02 0.3 f 0.19 0.4 * 0.02 0.2 * 0.01 0.1 f 0.01 1.7 * 0.08 1.7 * 0.1 10.7 ?c 0.2 10.7 + 0.6 2.4 + 0.06 2.3 + 0.1 0.4 f 0.01 0.4 f 0.01 3.4 & 0.5 3.3kO.3 0.1 f 0.01 0.1 * 0.01 0.2 + 0.03 0.2 + 0.02 0.5 f 0.4 0.8 f 0.05 1.4f0.06 1.3f0.3 0.3 + 0.04 0.2 f 0.03 0.1 f 0.03 0.1 f 0.0 13.9f0.4 13.9+0.4 0.5f0.02 0.5kO.l 0.3 + 0.04 0.2 + 0.02 0.3 f 0.02 0.3 + 0.03 0.6 f 0.1 0.6 * 0.01 4.6 f 0.3 4.7 * 0.3 0.2 f 0.03 0.2 + 0.03 13.8+ 1.1 14.4+0.4 0.9 f 0.2 1.1 * 0.07 0.2 ?C0.04 0.2 * 0.01 0.1 + 0.02 0.1 + O-05 1.4kO.2 1.4 z!z0.07 11.6+ 1.0 11.1 + l-2 0.2 + 0.07 0.3 f O-09 1.0 0.9

the mean +SD of samples from three fish. No difference were observed among the groups astaxanthin but no tocopherol mix. tocopherol mix but no astaxanthin. no astaxanthin and no tocopherol mix.

phospholipid versus -6% in the triacylglycerols, comparable to results shown by Polvi et al. (1991). In muscle lipids the DHA content of total fatty acids therefore fell from 18% of total fatty acids to 13% (not shown) through the increase in the proportion of triglycerol. The presence or absence of tocopherols and astaxanthin in the diet did not affect lipid content (Fig. 2) or the fatty acid composition of the salmon muscle (Table 4). This is in agreement with results from Frigg et al. (1990) who observed that different levels of vitamin E in the diets did not affect the fatty acid composition in trout muscle. However, Runge et al. (1987) found that levels of n-3 fatty acids in carp were not totally independent of vitamin E content in the feed. The present results suggest that a diet deficient in vitamin E needs to be fed to Atlantic salmon for more than 15 weeks to be able to deplete the tocopherol enough to cause clinical signs of vitamin E deficiency (La11 & Olivier, 1993). Muscle tocopherols

The a-tocopherol contents of muscle from fish fed diets 2 and 4, which were not supplemented with any tocopherol, decreased gradually through the experiment (Fig. 4) and after 7 weeks of feeding, the a-tocopherol in muscle from fish fed the diet not supplemented with tocopherols (diet 2) was observed to be significantly lower (P < 0.05) than in muscle from fish fed the diets supplemented with the natural mixture of tocopherols.

si

3

? ?Diet1

0

7

4

Sampling

period

10

15

(weeks)

Fig. 4. c+Tocopherol content of salmon muscle over the course of the experiment, for the four different diets. Diet 1 contains astaxanthin and tocopherol mix, diet 2 contains astaxanthin but no tocopherol mix, diet 3 contains tocopherol mix but no astaxanthin, diet 4 contains no astaxanthin and no tocopherol mix. Data points are the mean f SD from three fish. “Significantly different from time 0 (PC 0.05); bsignificantly different from control diet (P < 0.05); and ‘significantly different from control and diet 3 (P < 0.05).

29

Eflects of tocopherols and astaxanthin on salmon quality

Subsequent to the conclusion of this study Cabrini et al. (1992) reported that the Bligh and Dyer (1959) lipid extraction method did not recover all of the tocopherol present in fish tissues. Apparently, it was also inferior, compared to hexane/ethanol, for recovery of retinol palmitate. If correct, we feel that this deficiency in respect to tocopherol and pigments is less critical in a time course study with a control group than in, for examong different species. ample, comparisons Cabrini et al. (1992) also report that these neutral materials in fish muscle homogenates were recovered with little difference among three methods, whereas liver homogenates differed significantly. Muscle carotenoids

The measured level of astaxanthin increased in the muscle (Fig. 5) with the growth of salmon when diets supplemented with astaxanthin (control diet and diet 2) were fed. Fish from those groups increased in colour from light pink, 12-l 3 according to the Hoffman-La Roche Ltd colour card (-1.5 mg/g muscle) to a strong pink colour of 1617 (-2.5 mg/g muscle) in 15 weeks (Fig. 5). An acceptable carotenoid content for market-size Atlantic salmon is approximately 24 mg astaxanthin or canthaxanthin per kg (wet weight basis) (Torrissen et al., 1989). The deposition of astaxanthin in the muscle was not affected by the presence of vitamin E in the diet, as can be seen in Fig. 5. The astaxanthin content of muscle from fish fed a diet supplemented with Covi-ox T-70 (diet 1), and from fish fed the diet not supplemented with Covi-ox T-70 (diet 2), was not different (P > 0.05). 3

“u

cs Y

4 W Diet 3

These results are in agreement with results from Torrissen (1985) for rainbow trout. He found that the quantity of astaxanthin laid down in the muscle of rainbow trout was not reduced if vitamin E were excluded from the diet. However, Pozo et al. (1988) observed that canthaxanthin was deposited at a higher level in the muscle of trout fed a diet supplemented with vitamin E at levels of 50 mg DL-(w-tocopherol/lOO g feed (dry weight basis), compared with feeding a diet containing only 2.6 mg/lOO g feed. The astaxanthin level decreased slowly in muscle from fish fed diets not fortified with astaxanthin (diets 3 and 4). A significant (PcO.05) drop in the astaxanthin level in the muscle was observed after four weeks of feeding (Fig. 5). The colour changed over 15 weeks from the level of light pink, or 12-l 3 according to colour cards (-1.5 E_Llg muscle), to a very light pink or nearly colourless flesh at 11-12 (-0.4 ,&g muscle in fish fed diet 4). None of the diets contained any amount of canthaxanthin, but at time 0, when the experiment started, the fish contained 0.45 ,ug/g muscle canthaxanthin from stock commercial feeds. The depletion of canthaxanthin in the muscle was very slow over the course of the experiment and after 15 weeks a mean of 0.11 pg/g muscle was observed (diet 1) (Fig. 6). It has to be taken into account that the fish almost doubled their weight during the experimental feeding period. Astaxanthin was depleted at a significantly (PcO.05) faster rate than canthaxanthin. When the amount of astaxanthin was calculated as ,ug/g muscle the decrease was linear and correlated with time (r = 0.92); from a mean of 1.5 pg/g muscle for astaxanthin to a mean of 0.4 pglg muscle (diet 4)

1

Diet 2

1

,M

.::

2 3

2

2

1

OfJ 0.5 ‘M 2 0.4

2 ; 1

0 0

4

Sampling

Fig. 5. Astaxanthin

I

period

10

15

(weeks)

content of muscle of salmon, fed the four diets described for Fig. 4 over the course of the experiment. Data points are the mean f SD from three fish. “Significantly different from time 0 (PC 0.05); ‘significantly different from control diet (P < 0.05); “significantly different from control and diet 2 (P < 0.05); and dsignificantly different from diet 3 (P < 0.05).

H

0.6 TTTT

3

0.3

zs x da

0.1

5

0.0

Diet2 Diet 3

? ?Diet4

0.2

0

4

Sampling

7

period

10

15

(weeks)

Fig. 6. Canthaxanthin content of muscle of salmon fed the four different diets described for Fig. 4 over the course of the experiment. Data points are the mean &SD from three fish. “Significantly different from time 0 (P 4 0.05).

30

S. Sigurgisladottir, C. C. Parrish, S. P. Lall, R. G, Ackman

(Fig. 7). For canthaxanthin the decrease was also linear and correlated with time (r=0.88); from a mean of 0.45 pg/g muscle to a mean of 0.11 pg/g muscle (Fig. 7). The ‘dilution’ of the pigment in the muscle can be taken into account by calculating on a ‘per fish’ basis. The decreases in the pigment level per fish for diet 4 were small (Fig. 8). Astaxanthin decreased from a mean of 191 f47 pg in muscle per fish in the beginning (time 0) to 117 + 24 pg after 15 weeks time (time 4, diet 4), with correlation r = 0.74. The decrease was even less for canthaxanthin, from 56.9+ 19 pg in muscle per fish (time 0) to 42f 10 pg after 15 weeks (time 4, diet 4) with a low correlation (r = 0.55) (Fig. 7). The depletion of canthaxanthin was therefore a very slow process and the decrease per g over 15 weeks was primarily due to the weight gain of the fish. The faster depletion of astaxanthin may be because it can be excreted by the glucuronic acid pathway, or because astaxanthin is chemically less stable. The de-

2

6

Sampling

10

period

14

18

(weeks)

Fig. 7. Depletion of astaxanthin and canthaxanthin from muscle of individual salmon &g/g) over the course of the experiment in fish fed diet 4. Data are the mean +SD of three samples.

300 a, 3 ; c 250 .Y

T

??

Astaxanthin

??

Canthaxanthin

R=0.74 R=0.55

pletions of both astaxanthin and canthaxanthin were more complete at the end of the experiment in muscle from fish fed diets not supplemented with astaxanthin or vitamin E, than in muscle from fish fed diets supplemented with the mixed tocopherols. This difference was significant for astaxanthin (P < 0.05). This indicates that tocopherols had some effects on retention of colour in salmon muscle. Oxidative stability of fillets Accelerated oxidation under standardised conditions was used instead of oxidation under ambient conditions. The TBA (thiobarbituric acid reactive compounds) method required less sample than peroxide values. It was preferred as it also indicates the potential for further oxidation due to changes in anti- or pro-oxidants, an objective of this study. TBA values gave very reproducible results and a significant (P < 0.05) interrelationship with the tocopherol level in the muscle (Table 5). This agrees with Frigg et al. (1990) who suggested an effect of dietary vitamin E levels on oxidative stability of trout fillets. Foote et al. (1974), Fahrenholtz et al. (1974), and McCay and King (1980) have suggested that tocopherol may play a role in the prevention of rancidity in fish tissues during frozen storage and marketing. The relative depositions of different tocopherols in fish are important and will be reviewed elsewhere (Sigurgisladottir et al., 1993). Many authors have tested only the alpha tocopherol, but gamma in particular is taken up by fish as well (cf. Watanabe et al., 1981b; Erickson, 1992) and warrants further study. The effects of astaxanthin as an antioxidant in fish muscle could not be measured and no effects as an antioxidant were observed. One possible Table 5. Thiobarbituric acid reactive compounds in salmon f?llets from 15 weeks of feeding, in an accelerated oxidation test expressed as pmols malonaldehyde/kg muscle, versus diets with and without tocopherols’ Diet no.

i

-2

2

Sampling

6

period

10

14

18

(weeks)

Fig. 8. Depletion of astaxanthin and canthaxanthin from content of salmon muscle of individual fish as total pg fed (diet 4) over the course of the experiment. Data are the mean &SD of three samples.

1 2 3 4

TBA value (pmol/kg muscle)

6.2+0.8a 9.5&O-5b 5.5 +0.4a 8.9+0.lb

Tocopherols

@g/g muscle)

(Y

Y

6

17++1,0a 0 13.Oir l.Ob 0

3.0+0.2a 0 1.8+0.3b 0

%1+1,2a 4.1+0.6b 7.6f0.6a 2.9fl.Ob

“In each column values followed by a different letter are significantly different (P < 0.05).

Efsects of tocopherols and astaxanthin on salmon quality

reason was that carotenoids are not only conventional chain-breaking antioxidants, but astaxanthin in particular is superior to a-tocopherol as a reagent for reducing the concentration of the chain-carrying peroxyl radicals as well as quenching singlet oxygen (Miki, 1991). It has been shown that carotenoids have an antioxidant effect in low oxygen partial pressure such as found in tissues under physiological conditions (Burton & Ingold, 1984; Terao, 1989). In the 1989 review by Bendich and Olson (1989) it was stated that carotenoids enhanced immune response, inhibited mutagenesis, and reduced photo-induced nuclear damage in cells, tissues and whole animals. Retention of astaxanthin is thus not only a point of sale feature of salmon for eye-appeal preference but may attract health-conscious consumers as well. Sensory evaluations

Triangle tests by 10 panellists were performed after 9 weeks of feeding and again after 15 weeks of feeding. In appearance significant differences (PC 0.05) in colour were observed at both times between salmon fed the control diet 1 and diet 3, diets 1 and 4, diets 2 and 3 and diets 2 and 4. The salmon muscle with the higher content of astaxanthin was preferred (P < O.OS), presumably because of the natural red pigmentation (No & Storebakken, 1992). Significant differences (P < 0.05) in taste and texture were obtained between diets 1 and 3, and diets 2 and 3, on the first occasion (9 weeks) and between diets 1 and 4 and diets 2 and 4 on the second occasion (15 weeks). The sensorypanel results were not as reproducible; however, there is some indication in these results that astaxanthin levels in the feed may affect fillet taste and texture. Tocopherol levels did not have any pronounced effect on the taste and texture of the samples. Frigg et al. (1990) observed taste/texture difference of trout flesh with different levels of vitamin E, and further work on this subject is indicated as necessary. Tocopherols and astaxanthin were observed not to affect the lipid content or fatty acid composition of Atlantic salmon. However, tocopherols enhanced retention of astaxanthin and did have oxidative stability effects on the salmon muscle in an accelerated oxidation study. As expected, a pinker-coloured salmon muscle was preferred by the sensory panel, but the natural tocopherol mixture had no immediate effect on flavor.

31

ACKNOWLEDGEMENTS

Financial support provided by the Natural Sciences and Engineering Research Council of Canada is acknowledged. Donations of Covi-ox T-70 tocopherol mixture by the Henkel Corporation, La Grange, IL, USA, and of astaxanthin and canthaxanthin by Hoffman-La Roche, Basle, Switzerland, are much appreciated. The assistance of Mr I. Wilson in conducting feeding trials is also acknowledged. REFERENCES Ackman, R. G. (1974). Marine lipids and fatty acids in human nutrition. In Fishery Products, ed. R. Kreuzer. Fishing News Books, Oxford, UK, pp. 112-31. Ackman, R. G. & Eaton, C. A. (1978). Some contemporary applications of open tubular gas liquid chromatography in analyses of methyl esters of longer chain fatty acids. Fette Seifen Anstrichmittel, 80, 21-37. A&man, R. G. & Ratnayake, W. M. N. (1992). Non-enzymatic oxidation of seafood lipids. In Advances in Seafood Biochemistry_Composition and Quality, ed. G. J. Flick & R. E. Martin. Technomic Publishing Co., Inc., Lancaster, PA, pp. 245-67. Armitage, P. & Berry, G. (1988). Statistica Methods in Medical Research (2nd edn). Blackwell Scientific Publ., London, UK, pp. 358-70. Bendich, A. & Olson, J. A. (1989). Biological actions of carotenoids. Fed. Am. Sot. Exper. Biochem., 3, 1927-32. Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37,911-17. Burton, G. W. (1989). Antioxidant action of carotenoids. J. Nutr., 119, 109-11. Burton, G. W. & Ingold, K. T-J. (1984). p-carotene: An unusual type of lipid antioxidant. Science, 224, 269-73. Cabrini, L., Landi, L., Stefanelli, C., Barzanti, V. & Sechi, A. M. (1992). Extraction of lipids and lipophilic antioxidants from fish tissues: A comparison among different methods. Comp. Biochem. Physiol., lOlB, 383-6. Cerra, F. B., Holman, R. T., Bankey, P. E., Mazuski, J. E. & LiCari, J. J. (1991). w3-Polyunsaturated fatty acids as modulators of cellular function in the critically ill. Pharmacotherapy, 11, 71-6. Chevolleau, S., Debal, A. & Ucciani, E. (1992). Determination of the antioxidant activity of plant extracts. Rev. Fraq. Corps Gras, 39, 3-8. Christie, W. W. (1987). In Lipid Analysis (2nd edn). Pergamon Press, Oxford, UK, pp. 22-63. Cowey, C. B., Adron, J. W. & Youngson, A. (1983). The vitamin E requirement of rainbow trout (Salmo gairdnerz] given diets containing polyunsaturated fatty acids derived from fish oil. Aquaculture, 30, 85-93. Erickson, M. C. (1992). Lipid and tocopherol composition of farm-raised striped and hybrid striped bass. Comp. Biochem. Physiol., lOlA, 171-6. Fahrenholtz, S. R., Doleiden, F. H., Trozzolo, A. M. & Lamola, A. A. (1974). On the quenching of singlet oxygen by (Ytocopherol. Photochem. Photobiol., 20, 505-9. Foote, C. S., Ching, T. & Geller, G. G. (1974). Chemistry of singlet oxygen-XVIII. Rates of reaction and quenching of a-tocopherol and singlet oxygen. Photochem. Photobiol., 20,511-13.

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(Received 20 March 1993; accepted 25 May 1993)