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Nutritional content and bioactive properties of wild and farmed cod (Gadus morhua L.) subjected to food preparation
Journal of Food Composition and Analysis 31 (2013) 212–216
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Original Research Article
Nutritional content and bioactive properties of wild and farmed cod (Gadus morhua L.) subjected to food preparation Ida-Johanne Jensen a,*, Rune Larsen a, Turid Rustad b, Karl-Erik Eilertsen a a b
Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway Norwegian University of Technology and Science, Trondheim, Norway
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2012 Received in revised form 13 May 2013 Accepted 19 May 2013
Cod is a relatively new species in intensive aquaculture. The aim of this study was to compare the nutritional composition and bioactive properties of wild and farmed cod as well as investigate how cooking influenced these parameters. Wild cod contained significantly more docosahexaenoic acid, whereas the levels of linoleic acid, eicosapentaenoic acid, and docosapentaenoic acid were lower than in farmed cod. Only minor difference in amino acid profile were found between wild and farmed cod but protein content was higher in the latter. Baking resulted in lower loss of moisture and a corresponding lower loss of taurine compared to poaching. The angiotensin converting enzyme (ACE) inhibitory capacity did not differ between wild and farmed cod (IC50 values of 0.06 mg/mL) nor was it significantly affected by cooking. From this study, it is evident that intake of wild and farmed cod provides similar health-promoting effects that are maintained during cooking. ß 2013 Elsevier Inc. All rights reserved.
Keywords: Gadus morhua L. Atlantic cod Fatty acid composition Polyunsaturated fatty acids Fish consumption Angiotensin converting enzyme Food processing Home cooking and nutrient stability Nutrient bioavailability Wild fish Farmed fish Food analysis Food composition
1. Introduction Atlantic cod (Gadus morhua L.) is a developing species in Norwegian aquaculture. Although the commercial success have been limited, multiple aspects are continuously being researched in order to improve the sustainability and profitability of cod farming. In relation to fish quality, the research has mainly been focusing towards studying and improving the technical quality of the fillet, e.g. pH, water holding capacity, gaping, texture and proteolytic activities (Hultmann and Rustad, 2007; Kristoffersen et al., 2007; Morkore and Lilleholt, 2007; Digre et al., 2011; Rustad, 1992). Despite an increased focus on diet and health, the nutritional aspects of farmed cod and differences in nutritional quality and biochemical composition of farmed and wild cod have received less attention. Dietary recommendations of increased fish consumption are mainly ascribed to the marine polyunsaturated fatty acids EPA (eicosapentaenoic acid, 20:5n 3) and DHA (docosahexaenoic acid,
* Corresponding author. Tel.: +47 776 46 721; fax: +47 776 46 020. E-mail addresses: [email protected], [email protected] (I.-J. Jensen). 0889-1575/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2013.05.013
22:6n 3) (Kris-Etherton et al., 2002; Tsugane et al., 2006). Fish is also an excellent source of protein (Friedman, 1996) as well as the vitamins A, D and B12, and the minerals selenium and iodine (Lie et al., 1994). Substances in food that may possess bioactivities beyond the classical nutritional value are gaining increasing attention. Enzymatic degradation of proteins during fermentation, food processing or gastrointestinal (GI) digestion may release peptides and amino acids with biological activity such as antihypertensive activity (Kim and Mendis, 2006; Wijesekara and Kim, 2010; Shimizu and Ok Son, 2007). Hypertension is a major independent risk factor for cardiovascular disease (Harris et al., 1985) and an important health issue in both industrialised and developing countries (WHO, 2011). Angiotensin converting enzyme (ACE) plays an important role in the regulation of blood pressure, converting the inactive angiotensin 1 into the potent vasoconstrictor angiotensin 2 (Goodfriend et al., 1996). The enzyme ACE also inactivates the potent vasodilator bradykinin (Witherow et al., 2001). Synthetic ACE inhibitors are traditionally used to treat hypertension. Side effects such as cough, taste alterations, skin rashes and renal dysfunction associated with synthetic ACE inhibitors (Atkinson and Robertson, 1979) have resulted in an increased desire to find natural inhibitors
I.-J. Jensen et al. / Journal of Food Composition and Analysis 31 (2013) 212–216
and numerous studies have documented antihypertensive and ACE inhibitory effects of different food sources (Fujita et al., 2000; Wijesekara and Kim, 2010). Culinary processing results in important changes to the fish muscle, especially sensory characteristics, chemical components and nutritional composition. In addition to enhancement of the desired taste, thermal processing of food has advantages such as killing of pathogens, inactivation of anti-nutrient enzymes and augmenting digestibility and bioavailability. The changes are dependent on the preparation method, and time and temperature are the prevailing factors. Detrimental effects of such preparation on the nutritional value include browning reactions, racemisation, destruction of valuable components (Meade et al., 2005) and loss of water soluble – potentially bioactive – components (Larsen et al., 2007). The aim of this study was to evaluate differences in nutritional content and bioactive properties of wild and farmed cod. In addition, the effect of household cooking preparations and subsequent digestion on these parameters was studied. 2. Materials and methods
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2.3.2. Fatty acid composition The FA composition was determined after dissolving the extracted lipids (10 mg/mL) in dichloromethane/methanol (2:1, v/v) (Cequier-Sanchez et al., 2008) before methylation (Stoffel et al., 1959) with some modifications. Heptadecaenoic acid (Sigma Chemicals Co., St. Lous, MO, USA) was used as internal standard. The samples (100 mL) were mixed with 900 mL dichloromethane and 2 mL of 2% H2SO4 in methanol and boiled for 1 h. Thereafter, 3.5 mL of 5% NaCl and 3.5 mL of heptane were added, mixed, and the top phase was dried under nitrogen and redissolved in 100 mL of heptane. Gas chromatography was performed using an Agilent 6890N equipped with a 7683 B auto injection and a flame ionisation detector (FID) (Agilent Technologies Inc., Santa Clara, CA, USA). A Varian CP7419 capillary column (50 mm 250 mm nominal) (Varian Ing., Middelburg, The Netherlands) was used with helium as carrier gas. Injector and detector temperature programme was 50 8C for 2 min, then 10 8C/min to 150 8C followed by 2 8C/min to 205 8C and finally comparison to the FA standards PUFA no. 1, PUFA no. 2 and PUFA no. 3 (Sigma). The total amounts of FA were calculated on the basis of the internal standard and used to determine the amount of the individual FA in the samples.
2.1. Fish samples Wild cod (n = 20) were purchased from a local fishing company and were caught with Danish seine off the coast of Troms, Norway, in December, 2010. Farmed cod (n = 20) were obtained from Leroy Aurora AS, Storfjord, Norway, in September 2010. The mean gutted weight for wild and farmed cod was 2.8 0.6 and 2.3 0.1 kg, respectively. The gutted fish were stored in ice and were manually filleted and skinned within 24 h after slaughter or landing. 2.2. Experimental design From each of the 20 left fillets of both wild and farmed cod, 3 adjoining pieces of approximately 100 g were transversally cut from the centre of the fillet, labelled and randomly distributed in three groups: raw, poached and baked. Raw pieces remained untreated and were used to evaluate nutrient differences between wild and farmed cod and to calculate true retention. Poaching was performed by placing pieces in 90–95 8C water with 0.5% NaCl until reaching a core temperature of 64 8C, approximately 10 min. For baking, pieces were wrapped in aluminium foil and heated at 175 8C for 20 min. The fillet pieces were weighed before and after heat treatment, minced and stored in plastic bags at 50 8C. All samples were analysed for proximate and fatty acids (FA) composition and ten samples from each group were subjected to analysis of total amino acids (TAA) and free amino acids (FAA). Antihypertensive activity was evaluated in ten samples using angiotensin converting enzyme (ACE) inhibitory activity assay following a simulated GI digestion. True retention (TR) of selected components was calculated according to Murphy et al. (1975) to evaluate compositional changes during processing. 2.3. Analytical methods 2.3.1. Proximate composition Water and ash content were determined using the AOAC 950.46b and AOAC 938.08 (Cunniff, 1995). Approximately 10 g of sample was dried at 105 8C until constant weight and water content was determined gravimetrically. The water-free sample was thereafter combusted at 500 8C for 12 h to determine ash content gravimetrically. Total lipids were extracted and determined gravimetrically (Folch et al., 1957) substituting chloroform with dichloromethane. Protein content was determined as TAAFAA following an amino acid analysis.
2.3.3. Amino acid analysis For analysis of TAA, approximately 200 mg sample, 100 mL 20 mM norleucine and 1200 mL HCl were hydrolysed at 110 8C for 24 h. An aliquot of the hydrolysates (100 mL) was dried under nitrogen and diluted with lithium citrate buffer (pH 2.2) to a suitable concentration before analysis. For analysis of FAA, 1 g sample was mixed with 1 mL 20 mM norleucine and 9 mL with water. An amount of 1 mL of 35% sulphosalicylic acid was added for removal of proteins and peptides. The mixtures were centrifuged and an aliquot of the supernatants was diluted with a lithium citrate buffer (pH 2.2) to a suitable concentration before analysis. All samples were analysed using a Biochrom 30 Amino Acid Analyzer (Biochrom Limited, Cambridge, UK) with a lithium citrate equilibrated column and post column derivatization with ninhydrine. The amino acids were quantified by using norleucine as an internal standard (Sigma Chemicals Co, St. Lous, MO, USA) and the signal was analysed with Chromeleon software (Dionex, Sunnyvale, CA, USA). The identification of the amino acids was made by comparison with an A9906 physiological amino acid standard. 2.3.4. In vitro gastrointestinal digestion For analysis of ACE inhibitory activity the in vitro GI model described by Dragnes et al. (2009) was used. After digestion, the digests were immediately frozen followed by freeze drying. The freeze dried samples were finely ground and kept at 55 8C until determination of ACE inhibitory effect. 2.3.5. Angiotensin-converting enzyme inhibitory assay Angiotensin converting enzyme inhibitory activity assay was performed based on a previous method (Cushman and Cheung, 1971) with modifications. The end product hippuric acid was measured after an enzymatic reaction between ACE (Sigma Chemical Co, St. Louis, MO) and the substrate hippuryl-histidylleucine (HHL) (Sigma). The sample (25 mL) was pre incubated with 100 mL of 2 mM HHL in 100 mM sodium borate buffer, pH 8.3, at 37 8C for ten min. A dilution series from 0.1 to 2 mg/mL was used for the samples. The enzymatic reaction was initiated by adding 50 mL of ACE (5 mU) and was carried out on a shaker at 37 8C for 30 min. The reaction was stopped by the addition of 215 mL 1 M HCl. Quantitative HPLC analyses were performed as previously described (Dragnes et al., 2009). The amount of ACE inhibitors acquired to inhibit 50% of ACE activity was defined as the IC50% value and presented as mg/mL.
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Table 1 Proximate composition, % of wet weight, of wild and farmed cod (Gadus morhua L.) (values are expressed as mean standard deviation of 20 parallels). Proximates
Wild cod
Farmed cod
Water Protein Fat Ash
80.9 1.0* 15.7 0.7* 0.9 0.2 1.3 0.1
78.0 0.4 18.6 1.4 1.0 0.2 1.3 0.1
*
Denotes a significant difference between wild and farmed cod (p 0.05).
2.4. Statistical analysis The results are presented as mean standard deviation. When distribution of samples was normal, an independent T-test was performed for determination of statistical significance. When distribution of samples was non-normal, a Mann Whitney U Test was performed. Values were considered significantly different at a level of p 0.05. 3. Results and discussion 3.1. Composition of wild and farmed cod fillets 3.1.1. Proximate composition The proximate compositions of wild and farmed cod are shown in Table 1. The water content (81 and 78%) and protein content (16 and 19%) were significantly different between wild and farmed cod, respectively. The water content was lower and protein content higher in farmed cod, which is consistent with an intensive feeding regime and a feed rich in protein. No major difference in fat content was observed between farmed and wild cod. Being a lean species which predominantly store lipids in the liver, intensive feeding is not found to substantially affect fat content in cod muscle. 3.1.2. Fatty acid composition Total FA of both wild and farmed cod were approximately distributed as 30% saturated FA, 14% monounsaturated FA and 50% polyunsaturated FA (Table 2). The individual FA composition, however, differed substantially between wild and farmed cod. The percentage of linoleic acid (LA, 18:2n 6) was four times higher in farmed cod (4%) than in wild cod (1%). The higher percentage of LA
in the farmed cod may indicate that the feed was composed of a substantial amount of vegetable oils in contrast to the natural feed of the wild cod. Several studies have found that dietary lipid composition influence the muscle fatty acid profile of cod, particularly the LA (Jobling et al., 2008; Pickova and Morkore, 2007). This difference in LA content in farmed and wild fish is also documented in other species such as salmon (Jensen et al., 2012), sea bream (Lenas et al., 2011) and turbot (Martinez et al., 2010). The level of both EPA and DPA were significantly higher in farmed cod (16% and 2%) compared to wild cod (12% and 1%), whereas the level of DHA was highest in wild compared to farmed cod (32% and 26%, respectively). It is usually recommended to consume between 200 and 500 mg EPA and DHA per day (Harris et al., 2008; Kris-Etherton et al., 2009). The results show that 150 g of both wild and farmed cod would provide approximately 250 mg EPA+DHA and hence cover the recommended daily intake. The n 6/n 3 ratio of the current western diet has been estimated to be 15–17/1 (Simopoulos, 2008). Increasing dietary intake of long chain n 3 FA and lowering the ratio of n 6/n 3 FA within the diet has been suggested to be of benefit to human health (Harris and von Schacky, 2004; Simopoulos, 2008; Stanley et al., 2007). The n6/n3 ratio was 0.02 in wild cod and 0.09 in farmed cod. Although the n6/ n3 ratio of farmed cod was 4 times that of wild cod, the ratio is still very low and consumption of both wild and farmed cod could thus make a useful contribution to reducing the n 6/n 3 of the diet as a whole. 3.1.3. Total amino acids Analysis of TAA showed that, except for cysteine, the content of all amino acids was higher in farmed cod than in wild cod, and the difference was significant for 12 of the amino acids (Table 3). Glutamic acid was the most abundant amino acid in both wild and farmed cod with approximately 28–30 mg/g fillet. Aspartic acid, alanine, leucine and lysine were also abundant, significantly higher in farmed cod (15, 13, 16, 18 mg/g fillet) than in wild cod (12, 10, 14, 16 mg/g fillet). These differences are however primarily due to the higher protein content in wild cod. When evaluating the distribution of amino acids as mg/g protein, the differences between wild and farmed cod were reduced and only five amino acids (aspartic acid, threonine, glutamic acid, alanine and phenylalanine) were significantly higher in farmed cod. These differences in amino acid profile reflect a small variation in muscle
Table 2 Fatty acid (FA) composition (% of total FA), and amount of FA (mg/g fillet) in wild and farmed cod (Gadus morhua L) (values are expressed as mean standard deviation of 20 parallels). Fatty acids
14:0 16:0 16:1n 7 18:0 18:1n 9 18:1n 7 18:2n 6 18:4n 3 22:1n 11 20:5n 3 22:5n 3 22:6n 3 P SFA P MUFA P PUFA P n 3 n 6/n 3 *
Wild cod
Farmed cod
Composition (%)
Amount (mg/g ww)
Composition (%)
Amount (mg/g ww)
1.5 0.4 22.7 2.4 1.2 0.3 6.2 0.8 8.1 1.3 2.3 0.6 1.1 0.1* 2.9 0.7 2.1 0.6 11.9 1.2* 1.2 0.2* 32.3 2.9* 30.4 2.9 13.7 2.0 49.4 3.0 48.3 3.0 0.02 0.0*
0.1 0.0 0.8 0.1 0.1 0.0 0.2 0.1 0.3 0.1 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.4 0.1 0.0 0.0 1.1 0.3 1.0 0.1 0.5 0.1 1.7 0.4 1.7 0.4
1.2 0.3 22.7 2.0 1.3 0.2 6.6 0.5 8.4 1.6 2.4 0.2 4.0 0.4 1.9 0.3 2.1 0.2 16.0 1.4 2.0 0.2 25.7 2.8 30.5 2.6 14.1 1.7 49.6 3.5 45.6 3.7 0.09 0.0
0.1 0.0 1.0 0.1 0.1 0.0 0.3 0.0 0.4 0.1 0.1 0.0 0.2 0.0 0.1 0.0 0.1 0.0 0.7 0.1 0.1 0.0 1.2 0.2 1.4 0.1 0.6 0.1 2.2 0.3 2.1 0.3
Denotes a significant difference between wild and farmed cod (p 0.05).
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Table 3 Total amino acids (TAA) (mg/g fillet and mg/g protein) of wild and farmed cod (Gadus morhua L.) (values are expressed as mean standard deviation of 10 parallels). Amino acids
Wild cod mg/g fillet
Tau Asp Thr Ser Glu Pro Gly Ala Val Cys Met Ile Leu Tyr b-Ala Phe Lys 1-methis His Arg *
1.1 0.4 12.4 0.6* 7.4 0.3* 7.6 0.6* 27.9 1.1 5.8 0.6 7.4 1.5 10.0 0.7* 7.5 0.4* 1.1 0.4 5.4 0.3* 6.6 0.4* 14.0 0.8* 6.2 0.3* 0.3 0.1* 6.5 0.4* 16.0 0.7* 1.0 0.4 3.1 0.2* 11.3 0.7
Farmed cod mg/g protein
mg/g fillet
mg/g protein
1.2 0.1 15.1 1.2 9.0 0.7 9.4 0.7 30.8 2.6 6.7 0.8 9.8 0.6 13.1 0.9 8.7 0.6 0.8 0.2 6.5 0.6 7.8 0.7 16.2 1.3 7.2 0.6 1.6 0.1 8.3 0.6 18.2 1.3 1.3 0.1 3.8 0.3 12.1 0.9
79.2 1.3* 47.2 0.6* 48.6 2.1 178.3 1.5* 36.9 3.5 47.1 9.0 63.7 2.5* 48.0 1.5 7.2 2.3 29.6 1.5 42.2 1.5 89.1 2.9 39.7 1.5 2.1 0.8 41.7 1.7* 102.4 4.8 19.7 0.5 72.3 3.9
81.6 0.8 48.8 0.5 50.9 0.8 166.1 1.9 36.0 2.7 53.0 2.0 71.0 1.0 47.0 0.5 3.9 1.6 29.9 2.8 41.9 0.6 87.5 0.8 38.9 0.9 8.6 0.8 44.8 0.9 98.6 1.2 20.7 0.6 65.5 0.7
Denotes a significant difference between wild and farmed cod (p 0.05).
proteins, indicating that intensive feeding may slightly influence the muscle structure and composition of cod. The quality of protein depends on the presence of essential amino acids as well as the concentration of these (Friedman, 1996). Nine amino acids are considered essential to humans, i.e. they are not synthesised in the body and need to be supplied from the diet. Protein of both wild and farmed cod contained all essential amino acids in such amounts that it would cover the daily requirements for adults (FAO/WHO, 1991) and animal muscle protein is generally regarded to be of high quality. Isoleucine, leucine, lysine, and valine were found in higher amounts in protein of wild cod than farmed cod, whereas methionine, phenylalanine and threonine were more abundant in protein of farmed cod. The differences were, except for phenylalanine, not significant. 3.2. Changes in fillet composition after cooking Both cooking methods, poaching and baking, resulted in decreased water content and thus a relative increase in the content of protein and fat in the fillet. To evaluate compositional changes and the losses of components after poaching and baking, TR was calculated and is presented in Fig. 1. No substantial loss of protein or lipid was observed. Neither poaching nor baking had any effect on the FA composition (not shown). The only components
that were significantly affected by the cooking procedures were FAA. Taurine and other abundant free amino acids decreased significantly after poaching of fillets of both wild and farmed cod, but the loss was lower for farmed cod than for wild cod. The effect of baking was lower than that of poaching. Cooking methods involving immersion of muscle foods in water may render FAA more susceptible to leaching losses. 3.3. Angiotensin-converting enzyme inhibitory assay The GI digests from hydrolysed cod samples were subjected to in vitro ACE inhibitory screening and the IC50 values were determined (Table 4). There was no significant difference between the ACE inhibitory activity of wild and farmed cod, exhibiting IC50 values of 0.063 and 0.060 mg/mL, respectively. These results are in accordance with previous results obtained on cod (Dragnes et al., 2009). Katsuobushi oligopeptide (K.O.) is a dry powder, produced by Nippon Supplement Inc. which is on the market as a functional food to inhibit ACE and hypertension. This product has previously (Dragnes et al., 2009) been shown to exhibit an IC50 of 1.3 mg equivalent to 0.052 mg/mL. After poaching, the IC50 values were 0.055 mg/mL and 0.058 mg/mL for wild and farmed cod, respectively, corresponding to respective increases in ACE inhibitory capacity of 13% and 7%. The increase was, however, not significant. Similar non-significant effect was observed after baking, resulting in IC50 values of 0.054 and 0.057 mg/mL for wild and farmed cod, respectively. Naturally occurring ACE inhibitory peptides are small amino acid sequences of 2–12 amino acids (Pihlanto, 2006) incorporated in proteins. During heat treatments, such as poaching or baking, proteins are denatured which makes digestion easier. This could be a reason for a higher ACE inhibitory in the cooked Table 4 Angiotensin-converting enzyme (ACE) inhibitory activity (mg/ml) of digested raw and cooked fillets of wild and farmed cod (values are expressed as mean SD of 10 parallels). Wild cod Raw
Farmed cod Poached
Baked
Raw
Poached
Baked
IC50 0.06 0.01 0.06 0.01 0.05 0.01 0.06 0.01 0.06 0.01 0.06 0.01 Fig. 1. True retention of protein, lipid and ash after cooking of wild and farmed cod (Gadus morhua L.) (values are expressed as mean SD of 20 parallels).
Data are presented as the concentration of sample needed for 50% inhibition of ACE in a 1 mU ACE-assay.
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samples. The results obtained from this work, indicate that ACE inhibition may be achieved by consumption of cod, regardless of whether it is farmed or wild and that poached or baked cod results in marginally more potent inhibition effects compared to raw fish muscle. 4. Conclusion Our results demonstrate that there were some differences in FA composition in wild and farmed cod, as farmed cod contains a higher percentage of LA, EPA and DPA and lower percentage of DHA. A 150 g portion of both wild and farmed cod nonetheless provide the recommended daily intake of marine n 3 polyunsaturated FA. The protein quality concerning essential amino acids was similar and high for both wild and farmed cod. No differences in bioactivity were observed between wild and farmed cod. Cooking of the fish did not appear to influence the proximate composition or the bioactive properties except for loss of taurine. Hence, both wild and farmed cod are good sources of potentially health-promoting food, also after poaching or baking. References Atkinson, A.B., Robertson, J.I.S., 1979. Captopril in the treatment of clinical hypertenstion and cardiac-failure. Lancet 2, 836–839. Cequier-Sanchez, E., Rodriguez, C., Ravelo, A.G., Zarate, R., 2008. Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. Journal of Agricultural and Food Chemistry 56, 4297–4303. Cunniff, P. (Ed.), 1995. Official Methods of Analysis of AOAC International. AOAC International, Gaithesburg. Cushman, D.W., Cheung, S.H., 1971. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology 20, 1637–1648. Digre, H., Erikson, U., Skaret, J., Lea, P., Gallart-Jornet, L., Misimi, E., 2011. Biochemical, physical and sensory quality of ice-stored Atlantic cod (Gadus morhua) as affected by pre-slaughter stress, percussion stunning and AQUI-S((TM)) anaesthesia. European Food Research and Technology 233, 447–456. Dragnes, B.T., Stormo, S.K., Larsen, R., Ernstsen, H.H., Elvevoll, E.O., 2009. Utilisation of fish industry residuals: screening the taurine concentration and angiotensin converting enzyme inhibition potential in cod and salmon. Journal of Food Composition and Analysis 22, 714–717. FAO/WHO, 1991. Protein Quality Evaluation. Food and Agricultural Organization of the United Nations, Rome, Italy. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry 226, 497–509. Friedman, M., 1996. Nutritional value of proteins from different food sources. A review. Journal of Agricultural and Food Chemistry 44, 6–29. Fujita, H., Yokoyama, K., Yoshikawa, M., 2000. Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. Journal of Food Science 65, 564–569. Goodfriend, T.L., Elliott, M.E., Catt, K.J., 1996. Drug therapy – Angiotensin receptors and their antagonists. New England Journal of Medicine 334, 1649–1654. Harris, T., Cook, E.F., Kannel, W., Schatzkin, A., Goldman, L., 1985. Blood-pressure experience and risk of cardiovascular-disease in the elderly. Hypertension 7, 118–124. Harris, W.S., Kris-Etherton, P.M., Harris, K.A., 2008. Intakes of long-chain omega-3 fatty acid associated with reduced risk for death from coronary heart disease in healthy adults. Current Atherosclerosis Reports 10, 503–509. Harris, W.S., von Schacky, C., 2004. The Omega-3 Index: a new risk factor for death from coronary heart disease? Preventive Medicine 39, 212–220.
Hultmann, L., Rustad, T., 2007. Effects of temperature abuse on textural proterties and proteolytic activities during post mortem iced storage of farmed Atlantic cod (Gadus morhua). Food Chemistry 104, 1687–1697. Jensen, I.J., Maehre, H.K., Tømmera˚s, S., Eilertsen, K.E., Elvevoll, E.O., Olsen, R.L., 2012. Farmed Atlantic salmon (Salmo salar.) is a good source of long-chain omega-3 fatty acids. Nutrition Bulletin 37, 25–29. Jobling, M., Leknes, O., Saether, B.S., Bendiksen, E.A., 2008. Lipid and fatty acid dynamics in Atlantic cod, Gadus morhua, tissues: influence of dietary lipid concentrations and feed oil sources. Aquaculture 281, 87–94. Kim, S.K., Mendis, E., 2006. Bioactive compounds from marine processing byproducts – a review. Food Research International 39, 383–393. Kris-Etherton, P.M., Grieger, J.A., Etherton, T.D., 2009. Dietary reference intakes for DHA and EPA. Prostaglandins Leukotrienes and Essential Fatty Acids 81, 99–104. Kris-Etherton, P.M., Harris, W.S., Appel, L.J., Comm, N., 2002. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106, 2747–2757. Kristoffersen, S., Vang, B., Larsen, R., Olsen, R.L., 2007. Pre-rigor filleting and drip loss from fillets of farmed Atlantic cod (Gadus morhua L.). Aquaculture Research 38, 1721–1731. Larsen, R., Stormo, S.K., Dragnes, B.T., Elvevoll, E.O., 2007. Losses of taurine, creatine, glycine and alanine from cod (Gadus morhua L.) fillet during processing. Journal of Food Composition and Analysis 20, 396–402. Lenas, D.S., Triantafillou, D.J., Chatziantoniou, S., Nathanailides, C., 2011. Fatty acid profile of wild and farmed gilthead sea bream (Sparus aurata). Journal Fur Verbraucherschutz Und Lebensmittelsicherheit: Journal of Consumer Protection and Food Safety 6, 435–440. Lie, Ø., Lied, E., Maage, A., Njaa, L.R., Sandnes, K., 1994. Nutrient content of fish and shellfish. Fisk Dir Skr Ser Ernæring 6, 83–104. Martinez, B., Miranda, J.M., Nebot, C., Rodriguez, J.L., Cepeda, A., Franco, C.M., 2010. Differentiation of farmed and wild turbot (Psetta maxima): proximate chemical composition, fatty acid profile, trace minerals and antimicrobial resistance of contaminant bacteria. Food Science and Technology International 16, 435–441. Meade, S.J., Reid, E.A., Gerrard, J.A., 2005. The impact of processing on the nutritional quality of food proteins. Journal of AOAC International 88, 904–922. Morkore, T., Lilleholt, R., 2007. Impact of freezing temperature on quality of farmed Atlantic cod (Gadus morhua L.). Journal of Texture Studies 38, 457–472. Murphy, E.W., Criner, P.E., Gray, B.C., 1975. Comparisons of methods for calculating retentions of nutrients in cooked foods. Journal of Agricultural and Food Chemistry 23, 1153–1157. Pickova, J., Morkore, T., 2007. Alternate oils in fish feeds. European Journal of Lipid Science and Technology 109, 256–263. Pihlanto, A., 2006. Antioxidative peptides derived from milk proteins. International Dairy Journal 16, 1306–1314. Rustad, T., 1992. Muscle chemistry and the quality of wild and farmed cod. In: Huss, H.H., Jakobsen, M., Liston, J. (Eds.), Quality Assurance in the Fish Industry. Elsevier Science Publisher B.V., Amsterdam, The Netherlands. Shimizu, M., Ok Son, D., 2007. Food-derived peptides and intestinal functions. Current Pharmaceutical Design 13, 885–895. Simopoulos, A.P., 2008. The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Experimental Biology and Medicine 233, 674–688. Stanley, J.C., El Som, R.L., Calder, P.C., Griffin, B.A., Harris, W.S., Jebb, S.A., Lovegrove, J.A., Moore, C.S., Riemersma, R.A., Sanders, T.A.B., 2007. UK Food Standards Agency Workshop Report: the effects of the dietary n 6:n 3 fatty acid ratio on cardiovascular health. British Journal of Nutrition 98, 1305–1310. Stoffel, W., Chu, F., Ahrens, E.H., 1959. Analysis of long-chain fatty acids by gas– liquid chromatography-micromethod for preparation of methyl esters. Analytical Chemistry 31, 307–308. Tsugane, S., Iso, H., Kobayashi, M., Ishihara, J., Sasaki, S., Okada, K., Kita, Y., Kokubo, Y., Grp, J.S., 2006. Intake of fish and n3 fatty acids and risk of coronary heart disease among Japanese – The Japan Public Health Center-based (JPHC) Study Cohort I. Circulation 113, 195–202. WHO, 2011. Hypertenstion Fact Sheet. Department of Sustainable Development and Healthy Environments. Wijesekara, I., Kim, S.K., 2010. Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: prospects in the pharmaceutical industry. Marine Drugs 8, 1080–1093. Witherow, F.N., Helmy, A., Webb, D.J., Fox, K.A.A., Newby, D.E., 2001. Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure. Circulation 104, 2177–2181.
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Original Research Article
Nutritional content and bioactive properties of wild and farmed cod (Gadus morhua L.) subjected to food preparation Ida-Johanne Jensen a,*, Rune Larsen a, Turid Rustad b, Karl-Erik Eilertsen a a b
Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway Norwegian University of Technology and Science, Trondheim, Norway
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2012 Received in revised form 13 May 2013 Accepted 19 May 2013
Cod is a relatively new species in intensive aquaculture. The aim of this study was to compare the nutritional composition and bioactive properties of wild and farmed cod as well as investigate how cooking influenced these parameters. Wild cod contained significantly more docosahexaenoic acid, whereas the levels of linoleic acid, eicosapentaenoic acid, and docosapentaenoic acid were lower than in farmed cod. Only minor difference in amino acid profile were found between wild and farmed cod but protein content was higher in the latter. Baking resulted in lower loss of moisture and a corresponding lower loss of taurine compared to poaching. The angiotensin converting enzyme (ACE) inhibitory capacity did not differ between wild and farmed cod (IC50 values of 0.06 mg/mL) nor was it significantly affected by cooking. From this study, it is evident that intake of wild and farmed cod provides similar health-promoting effects that are maintained during cooking. ß 2013 Elsevier Inc. All rights reserved.
Keywords: Gadus morhua L. Atlantic cod Fatty acid composition Polyunsaturated fatty acids Fish consumption Angiotensin converting enzyme Food processing Home cooking and nutrient stability Nutrient bioavailability Wild fish Farmed fish Food analysis Food composition
1. Introduction Atlantic cod (Gadus morhua L.) is a developing species in Norwegian aquaculture. Although the commercial success have been limited, multiple aspects are continuously being researched in order to improve the sustainability and profitability of cod farming. In relation to fish quality, the research has mainly been focusing towards studying and improving the technical quality of the fillet, e.g. pH, water holding capacity, gaping, texture and proteolytic activities (Hultmann and Rustad, 2007; Kristoffersen et al., 2007; Morkore and Lilleholt, 2007; Digre et al., 2011; Rustad, 1992). Despite an increased focus on diet and health, the nutritional aspects of farmed cod and differences in nutritional quality and biochemical composition of farmed and wild cod have received less attention. Dietary recommendations of increased fish consumption are mainly ascribed to the marine polyunsaturated fatty acids EPA (eicosapentaenoic acid, 20:5n 3) and DHA (docosahexaenoic acid,
* Corresponding author. Tel.: +47 776 46 721; fax: +47 776 46 020. E-mail addresses: [email protected], [email protected] (I.-J. Jensen). 0889-1575/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2013.05.013
22:6n 3) (Kris-Etherton et al., 2002; Tsugane et al., 2006). Fish is also an excellent source of protein (Friedman, 1996) as well as the vitamins A, D and B12, and the minerals selenium and iodine (Lie et al., 1994). Substances in food that may possess bioactivities beyond the classical nutritional value are gaining increasing attention. Enzymatic degradation of proteins during fermentation, food processing or gastrointestinal (GI) digestion may release peptides and amino acids with biological activity such as antihypertensive activity (Kim and Mendis, 2006; Wijesekara and Kim, 2010; Shimizu and Ok Son, 2007). Hypertension is a major independent risk factor for cardiovascular disease (Harris et al., 1985) and an important health issue in both industrialised and developing countries (WHO, 2011). Angiotensin converting enzyme (ACE) plays an important role in the regulation of blood pressure, converting the inactive angiotensin 1 into the potent vasoconstrictor angiotensin 2 (Goodfriend et al., 1996). The enzyme ACE also inactivates the potent vasodilator bradykinin (Witherow et al., 2001). Synthetic ACE inhibitors are traditionally used to treat hypertension. Side effects such as cough, taste alterations, skin rashes and renal dysfunction associated with synthetic ACE inhibitors (Atkinson and Robertson, 1979) have resulted in an increased desire to find natural inhibitors
I.-J. Jensen et al. / Journal of Food Composition and Analysis 31 (2013) 212–216
and numerous studies have documented antihypertensive and ACE inhibitory effects of different food sources (Fujita et al., 2000; Wijesekara and Kim, 2010). Culinary processing results in important changes to the fish muscle, especially sensory characteristics, chemical components and nutritional composition. In addition to enhancement of the desired taste, thermal processing of food has advantages such as killing of pathogens, inactivation of anti-nutrient enzymes and augmenting digestibility and bioavailability. The changes are dependent on the preparation method, and time and temperature are the prevailing factors. Detrimental effects of such preparation on the nutritional value include browning reactions, racemisation, destruction of valuable components (Meade et al., 2005) and loss of water soluble – potentially bioactive – components (Larsen et al., 2007). The aim of this study was to evaluate differences in nutritional content and bioactive properties of wild and farmed cod. In addition, the effect of household cooking preparations and subsequent digestion on these parameters was studied. 2. Materials and methods
213
2.3.2. Fatty acid composition The FA composition was determined after dissolving the extracted lipids (10 mg/mL) in dichloromethane/methanol (2:1, v/v) (Cequier-Sanchez et al., 2008) before methylation (Stoffel et al., 1959) with some modifications. Heptadecaenoic acid (Sigma Chemicals Co., St. Lous, MO, USA) was used as internal standard. The samples (100 mL) were mixed with 900 mL dichloromethane and 2 mL of 2% H2SO4 in methanol and boiled for 1 h. Thereafter, 3.5 mL of 5% NaCl and 3.5 mL of heptane were added, mixed, and the top phase was dried under nitrogen and redissolved in 100 mL of heptane. Gas chromatography was performed using an Agilent 6890N equipped with a 7683 B auto injection and a flame ionisation detector (FID) (Agilent Technologies Inc., Santa Clara, CA, USA). A Varian CP7419 capillary column (50 mm 250 mm nominal) (Varian Ing., Middelburg, The Netherlands) was used with helium as carrier gas. Injector and detector temperature programme was 50 8C for 2 min, then 10 8C/min to 150 8C followed by 2 8C/min to 205 8C and finally comparison to the FA standards PUFA no. 1, PUFA no. 2 and PUFA no. 3 (Sigma). The total amounts of FA were calculated on the basis of the internal standard and used to determine the amount of the individual FA in the samples.
2.1. Fish samples Wild cod (n = 20) were purchased from a local fishing company and were caught with Danish seine off the coast of Troms, Norway, in December, 2010. Farmed cod (n = 20) were obtained from Leroy Aurora AS, Storfjord, Norway, in September 2010. The mean gutted weight for wild and farmed cod was 2.8 0.6 and 2.3 0.1 kg, respectively. The gutted fish were stored in ice and were manually filleted and skinned within 24 h after slaughter or landing. 2.2. Experimental design From each of the 20 left fillets of both wild and farmed cod, 3 adjoining pieces of approximately 100 g were transversally cut from the centre of the fillet, labelled and randomly distributed in three groups: raw, poached and baked. Raw pieces remained untreated and were used to evaluate nutrient differences between wild and farmed cod and to calculate true retention. Poaching was performed by placing pieces in 90–95 8C water with 0.5% NaCl until reaching a core temperature of 64 8C, approximately 10 min. For baking, pieces were wrapped in aluminium foil and heated at 175 8C for 20 min. The fillet pieces were weighed before and after heat treatment, minced and stored in plastic bags at 50 8C. All samples were analysed for proximate and fatty acids (FA) composition and ten samples from each group were subjected to analysis of total amino acids (TAA) and free amino acids (FAA). Antihypertensive activity was evaluated in ten samples using angiotensin converting enzyme (ACE) inhibitory activity assay following a simulated GI digestion. True retention (TR) of selected components was calculated according to Murphy et al. (1975) to evaluate compositional changes during processing. 2.3. Analytical methods 2.3.1. Proximate composition Water and ash content were determined using the AOAC 950.46b and AOAC 938.08 (Cunniff, 1995). Approximately 10 g of sample was dried at 105 8C until constant weight and water content was determined gravimetrically. The water-free sample was thereafter combusted at 500 8C for 12 h to determine ash content gravimetrically. Total lipids were extracted and determined gravimetrically (Folch et al., 1957) substituting chloroform with dichloromethane. Protein content was determined as TAAFAA following an amino acid analysis.
2.3.3. Amino acid analysis For analysis of TAA, approximately 200 mg sample, 100 mL 20 mM norleucine and 1200 mL HCl were hydrolysed at 110 8C for 24 h. An aliquot of the hydrolysates (100 mL) was dried under nitrogen and diluted with lithium citrate buffer (pH 2.2) to a suitable concentration before analysis. For analysis of FAA, 1 g sample was mixed with 1 mL 20 mM norleucine and 9 mL with water. An amount of 1 mL of 35% sulphosalicylic acid was added for removal of proteins and peptides. The mixtures were centrifuged and an aliquot of the supernatants was diluted with a lithium citrate buffer (pH 2.2) to a suitable concentration before analysis. All samples were analysed using a Biochrom 30 Amino Acid Analyzer (Biochrom Limited, Cambridge, UK) with a lithium citrate equilibrated column and post column derivatization with ninhydrine. The amino acids were quantified by using norleucine as an internal standard (Sigma Chemicals Co, St. Lous, MO, USA) and the signal was analysed with Chromeleon software (Dionex, Sunnyvale, CA, USA). The identification of the amino acids was made by comparison with an A9906 physiological amino acid standard. 2.3.4. In vitro gastrointestinal digestion For analysis of ACE inhibitory activity the in vitro GI model described by Dragnes et al. (2009) was used. After digestion, the digests were immediately frozen followed by freeze drying. The freeze dried samples were finely ground and kept at 55 8C until determination of ACE inhibitory effect. 2.3.5. Angiotensin-converting enzyme inhibitory assay Angiotensin converting enzyme inhibitory activity assay was performed based on a previous method (Cushman and Cheung, 1971) with modifications. The end product hippuric acid was measured after an enzymatic reaction between ACE (Sigma Chemical Co, St. Louis, MO) and the substrate hippuryl-histidylleucine (HHL) (Sigma). The sample (25 mL) was pre incubated with 100 mL of 2 mM HHL in 100 mM sodium borate buffer, pH 8.3, at 37 8C for ten min. A dilution series from 0.1 to 2 mg/mL was used for the samples. The enzymatic reaction was initiated by adding 50 mL of ACE (5 mU) and was carried out on a shaker at 37 8C for 30 min. The reaction was stopped by the addition of 215 mL 1 M HCl. Quantitative HPLC analyses were performed as previously described (Dragnes et al., 2009). The amount of ACE inhibitors acquired to inhibit 50% of ACE activity was defined as the IC50% value and presented as mg/mL.
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Table 1 Proximate composition, % of wet weight, of wild and farmed cod (Gadus morhua L.) (values are expressed as mean standard deviation of 20 parallels). Proximates
Wild cod
Farmed cod
Water Protein Fat Ash
80.9 1.0* 15.7 0.7* 0.9 0.2 1.3 0.1
78.0 0.4 18.6 1.4 1.0 0.2 1.3 0.1
*
Denotes a significant difference between wild and farmed cod (p 0.05).
2.4. Statistical analysis The results are presented as mean standard deviation. When distribution of samples was normal, an independent T-test was performed for determination of statistical significance. When distribution of samples was non-normal, a Mann Whitney U Test was performed. Values were considered significantly different at a level of p 0.05. 3. Results and discussion 3.1. Composition of wild and farmed cod fillets 3.1.1. Proximate composition The proximate compositions of wild and farmed cod are shown in Table 1. The water content (81 and 78%) and protein content (16 and 19%) were significantly different between wild and farmed cod, respectively. The water content was lower and protein content higher in farmed cod, which is consistent with an intensive feeding regime and a feed rich in protein. No major difference in fat content was observed between farmed and wild cod. Being a lean species which predominantly store lipids in the liver, intensive feeding is not found to substantially affect fat content in cod muscle. 3.1.2. Fatty acid composition Total FA of both wild and farmed cod were approximately distributed as 30% saturated FA, 14% monounsaturated FA and 50% polyunsaturated FA (Table 2). The individual FA composition, however, differed substantially between wild and farmed cod. The percentage of linoleic acid (LA, 18:2n 6) was four times higher in farmed cod (4%) than in wild cod (1%). The higher percentage of LA
in the farmed cod may indicate that the feed was composed of a substantial amount of vegetable oils in contrast to the natural feed of the wild cod. Several studies have found that dietary lipid composition influence the muscle fatty acid profile of cod, particularly the LA (Jobling et al., 2008; Pickova and Morkore, 2007). This difference in LA content in farmed and wild fish is also documented in other species such as salmon (Jensen et al., 2012), sea bream (Lenas et al., 2011) and turbot (Martinez et al., 2010). The level of both EPA and DPA were significantly higher in farmed cod (16% and 2%) compared to wild cod (12% and 1%), whereas the level of DHA was highest in wild compared to farmed cod (32% and 26%, respectively). It is usually recommended to consume between 200 and 500 mg EPA and DHA per day (Harris et al., 2008; Kris-Etherton et al., 2009). The results show that 150 g of both wild and farmed cod would provide approximately 250 mg EPA+DHA and hence cover the recommended daily intake. The n 6/n 3 ratio of the current western diet has been estimated to be 15–17/1 (Simopoulos, 2008). Increasing dietary intake of long chain n 3 FA and lowering the ratio of n 6/n 3 FA within the diet has been suggested to be of benefit to human health (Harris and von Schacky, 2004; Simopoulos, 2008; Stanley et al., 2007). The n6/n3 ratio was 0.02 in wild cod and 0.09 in farmed cod. Although the n6/ n3 ratio of farmed cod was 4 times that of wild cod, the ratio is still very low and consumption of both wild and farmed cod could thus make a useful contribution to reducing the n 6/n 3 of the diet as a whole. 3.1.3. Total amino acids Analysis of TAA showed that, except for cysteine, the content of all amino acids was higher in farmed cod than in wild cod, and the difference was significant for 12 of the amino acids (Table 3). Glutamic acid was the most abundant amino acid in both wild and farmed cod with approximately 28–30 mg/g fillet. Aspartic acid, alanine, leucine and lysine were also abundant, significantly higher in farmed cod (15, 13, 16, 18 mg/g fillet) than in wild cod (12, 10, 14, 16 mg/g fillet). These differences are however primarily due to the higher protein content in wild cod. When evaluating the distribution of amino acids as mg/g protein, the differences between wild and farmed cod were reduced and only five amino acids (aspartic acid, threonine, glutamic acid, alanine and phenylalanine) were significantly higher in farmed cod. These differences in amino acid profile reflect a small variation in muscle
Table 2 Fatty acid (FA) composition (% of total FA), and amount of FA (mg/g fillet) in wild and farmed cod (Gadus morhua L) (values are expressed as mean standard deviation of 20 parallels). Fatty acids
14:0 16:0 16:1n 7 18:0 18:1n 9 18:1n 7 18:2n 6 18:4n 3 22:1n 11 20:5n 3 22:5n 3 22:6n 3 P SFA P MUFA P PUFA P n 3 n 6/n 3 *
Wild cod
Farmed cod
Composition (%)
Amount (mg/g ww)
Composition (%)
Amount (mg/g ww)
1.5 0.4 22.7 2.4 1.2 0.3 6.2 0.8 8.1 1.3 2.3 0.6 1.1 0.1* 2.9 0.7 2.1 0.6 11.9 1.2* 1.2 0.2* 32.3 2.9* 30.4 2.9 13.7 2.0 49.4 3.0 48.3 3.0 0.02 0.0*
0.1 0.0 0.8 0.1 0.1 0.0 0.2 0.1 0.3 0.1 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.4 0.1 0.0 0.0 1.1 0.3 1.0 0.1 0.5 0.1 1.7 0.4 1.7 0.4
1.2 0.3 22.7 2.0 1.3 0.2 6.6 0.5 8.4 1.6 2.4 0.2 4.0 0.4 1.9 0.3 2.1 0.2 16.0 1.4 2.0 0.2 25.7 2.8 30.5 2.6 14.1 1.7 49.6 3.5 45.6 3.7 0.09 0.0
0.1 0.0 1.0 0.1 0.1 0.0 0.3 0.0 0.4 0.1 0.1 0.0 0.2 0.0 0.1 0.0 0.1 0.0 0.7 0.1 0.1 0.0 1.2 0.2 1.4 0.1 0.6 0.1 2.2 0.3 2.1 0.3
Denotes a significant difference between wild and farmed cod (p 0.05).
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Table 3 Total amino acids (TAA) (mg/g fillet and mg/g protein) of wild and farmed cod (Gadus morhua L.) (values are expressed as mean standard deviation of 10 parallels). Amino acids
Wild cod mg/g fillet
Tau Asp Thr Ser Glu Pro Gly Ala Val Cys Met Ile Leu Tyr b-Ala Phe Lys 1-methis His Arg *
1.1 0.4 12.4 0.6* 7.4 0.3* 7.6 0.6* 27.9 1.1 5.8 0.6 7.4 1.5 10.0 0.7* 7.5 0.4* 1.1 0.4 5.4 0.3* 6.6 0.4* 14.0 0.8* 6.2 0.3* 0.3 0.1* 6.5 0.4* 16.0 0.7* 1.0 0.4 3.1 0.2* 11.3 0.7
Farmed cod mg/g protein
mg/g fillet
mg/g protein
1.2 0.1 15.1 1.2 9.0 0.7 9.4 0.7 30.8 2.6 6.7 0.8 9.8 0.6 13.1 0.9 8.7 0.6 0.8 0.2 6.5 0.6 7.8 0.7 16.2 1.3 7.2 0.6 1.6 0.1 8.3 0.6 18.2 1.3 1.3 0.1 3.8 0.3 12.1 0.9
79.2 1.3* 47.2 0.6* 48.6 2.1 178.3 1.5* 36.9 3.5 47.1 9.0 63.7 2.5* 48.0 1.5 7.2 2.3 29.6 1.5 42.2 1.5 89.1 2.9 39.7 1.5 2.1 0.8 41.7 1.7* 102.4 4.8 19.7 0.5 72.3 3.9
81.6 0.8 48.8 0.5 50.9 0.8 166.1 1.9 36.0 2.7 53.0 2.0 71.0 1.0 47.0 0.5 3.9 1.6 29.9 2.8 41.9 0.6 87.5 0.8 38.9 0.9 8.6 0.8 44.8 0.9 98.6 1.2 20.7 0.6 65.5 0.7
Denotes a significant difference between wild and farmed cod (p 0.05).
proteins, indicating that intensive feeding may slightly influence the muscle structure and composition of cod. The quality of protein depends on the presence of essential amino acids as well as the concentration of these (Friedman, 1996). Nine amino acids are considered essential to humans, i.e. they are not synthesised in the body and need to be supplied from the diet. Protein of both wild and farmed cod contained all essential amino acids in such amounts that it would cover the daily requirements for adults (FAO/WHO, 1991) and animal muscle protein is generally regarded to be of high quality. Isoleucine, leucine, lysine, and valine were found in higher amounts in protein of wild cod than farmed cod, whereas methionine, phenylalanine and threonine were more abundant in protein of farmed cod. The differences were, except for phenylalanine, not significant. 3.2. Changes in fillet composition after cooking Both cooking methods, poaching and baking, resulted in decreased water content and thus a relative increase in the content of protein and fat in the fillet. To evaluate compositional changes and the losses of components after poaching and baking, TR was calculated and is presented in Fig. 1. No substantial loss of protein or lipid was observed. Neither poaching nor baking had any effect on the FA composition (not shown). The only components
that were significantly affected by the cooking procedures were FAA. Taurine and other abundant free amino acids decreased significantly after poaching of fillets of both wild and farmed cod, but the loss was lower for farmed cod than for wild cod. The effect of baking was lower than that of poaching. Cooking methods involving immersion of muscle foods in water may render FAA more susceptible to leaching losses. 3.3. Angiotensin-converting enzyme inhibitory assay The GI digests from hydrolysed cod samples were subjected to in vitro ACE inhibitory screening and the IC50 values were determined (Table 4). There was no significant difference between the ACE inhibitory activity of wild and farmed cod, exhibiting IC50 values of 0.063 and 0.060 mg/mL, respectively. These results are in accordance with previous results obtained on cod (Dragnes et al., 2009). Katsuobushi oligopeptide (K.O.) is a dry powder, produced by Nippon Supplement Inc. which is on the market as a functional food to inhibit ACE and hypertension. This product has previously (Dragnes et al., 2009) been shown to exhibit an IC50 of 1.3 mg equivalent to 0.052 mg/mL. After poaching, the IC50 values were 0.055 mg/mL and 0.058 mg/mL for wild and farmed cod, respectively, corresponding to respective increases in ACE inhibitory capacity of 13% and 7%. The increase was, however, not significant. Similar non-significant effect was observed after baking, resulting in IC50 values of 0.054 and 0.057 mg/mL for wild and farmed cod, respectively. Naturally occurring ACE inhibitory peptides are small amino acid sequences of 2–12 amino acids (Pihlanto, 2006) incorporated in proteins. During heat treatments, such as poaching or baking, proteins are denatured which makes digestion easier. This could be a reason for a higher ACE inhibitory in the cooked Table 4 Angiotensin-converting enzyme (ACE) inhibitory activity (mg/ml) of digested raw and cooked fillets of wild and farmed cod (values are expressed as mean SD of 10 parallels). Wild cod Raw
Farmed cod Poached
Baked
Raw
Poached
Baked
IC50 0.06 0.01 0.06 0.01 0.05 0.01 0.06 0.01 0.06 0.01 0.06 0.01 Fig. 1. True retention of protein, lipid and ash after cooking of wild and farmed cod (Gadus morhua L.) (values are expressed as mean SD of 20 parallels).
Data are presented as the concentration of sample needed for 50% inhibition of ACE in a 1 mU ACE-assay.
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samples. The results obtained from this work, indicate that ACE inhibition may be achieved by consumption of cod, regardless of whether it is farmed or wild and that poached or baked cod results in marginally more potent inhibition effects compared to raw fish muscle. 4. Conclusion Our results demonstrate that there were some differences in FA composition in wild and farmed cod, as farmed cod contains a higher percentage of LA, EPA and DPA and lower percentage of DHA. A 150 g portion of both wild and farmed cod nonetheless provide the recommended daily intake of marine n 3 polyunsaturated FA. The protein quality concerning essential amino acids was similar and high for both wild and farmed cod. No differences in bioactivity were observed between wild and farmed cod. Cooking of the fish did not appear to influence the proximate composition or the bioactive properties except for loss of taurine. Hence, both wild and farmed cod are good sources of potentially health-promoting food, also after poaching or baking. References Atkinson, A.B., Robertson, J.I.S., 1979. Captopril in the treatment of clinical hypertenstion and cardiac-failure. Lancet 2, 836–839. Cequier-Sanchez, E., Rodriguez, C., Ravelo, A.G., Zarate, R., 2008. Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. Journal of Agricultural and Food Chemistry 56, 4297–4303. Cunniff, P. (Ed.), 1995. Official Methods of Analysis of AOAC International. AOAC International, Gaithesburg. Cushman, D.W., Cheung, S.H., 1971. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology 20, 1637–1648. Digre, H., Erikson, U., Skaret, J., Lea, P., Gallart-Jornet, L., Misimi, E., 2011. Biochemical, physical and sensory quality of ice-stored Atlantic cod (Gadus morhua) as affected by pre-slaughter stress, percussion stunning and AQUI-S((TM)) anaesthesia. European Food Research and Technology 233, 447–456. Dragnes, B.T., Stormo, S.K., Larsen, R., Ernstsen, H.H., Elvevoll, E.O., 2009. Utilisation of fish industry residuals: screening the taurine concentration and angiotensin converting enzyme inhibition potential in cod and salmon. Journal of Food Composition and Analysis 22, 714–717. FAO/WHO, 1991. Protein Quality Evaluation. Food and Agricultural Organization of the United Nations, Rome, Italy. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry 226, 497–509. Friedman, M., 1996. Nutritional value of proteins from different food sources. A review. Journal of Agricultural and Food Chemistry 44, 6–29. Fujita, H., Yokoyama, K., Yoshikawa, M., 2000. Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. Journal of Food Science 65, 564–569. Goodfriend, T.L., Elliott, M.E., Catt, K.J., 1996. Drug therapy – Angiotensin receptors and their antagonists. New England Journal of Medicine 334, 1649–1654. Harris, T., Cook, E.F., Kannel, W., Schatzkin, A., Goldman, L., 1985. Blood-pressure experience and risk of cardiovascular-disease in the elderly. Hypertension 7, 118–124. Harris, W.S., Kris-Etherton, P.M., Harris, K.A., 2008. Intakes of long-chain omega-3 fatty acid associated with reduced risk for death from coronary heart disease in healthy adults. Current Atherosclerosis Reports 10, 503–509. Harris, W.S., von Schacky, C., 2004. The Omega-3 Index: a new risk factor for death from coronary heart disease? Preventive Medicine 39, 212–220.
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