Comparative Biochemistry and Physiology, Part B 146 (2007) 395 – 401 www.elsevier.com/locate/cbpb
Chemical composition, mineral content and amino acid and lipid profiles in bones from various fish species Jogeir Toppe ⁎, Sissel Albrektsen, Britt Hope, Anders Aksnes Norwegian Institute of Fisheries and Aquaculture Research, N-5141 Fyllingsdalen, Bergen, Norway Received 10 July 2006; received in revised form 20 November 2006; accepted 24 November 2006 Available online 29 November 2006
Abstract The chemical composition, content of minerals and the profiles of amino acids and fatty acids were analyzed in fish bones from eight different species of fish. Fish bones varied significantly in chemical composition. The main difference was lipid content ranging from 23 g/kg in cod (Gadus morhua) to 509 g/kg in mackerel (Scomber scombrus). In general fatty fish species showed higher lipid levels in the bones compared to lean fish species. Similarly, lower levels of protein and ash were observed in bones from fatty fish species. Protein levels differed from 363 g/kg lipid free dry matter (dm) to 568 g/kg lipid free dm with a concomitant inverse difference in ash content. Ash to protein ratio differed from 0.78 to 1.71 with the lowest level in fish that naturally have highest swimming and physical activity. Saithe (Pollachius virens) and salmon (Salmo salar) were found to be significantly different in the levels of lipid, protein and ash, and ash/protein ratio in the bones. Only small differences were observed in the level of amino acids although species specific differences were observed. The levels of Ca and P in lipid free fish bones were about the same in all species analyzed. Fatty acid profile differed in relation to total lipid levels in the fish bones, but some minor differences between fish species were observed. © 2006 Elsevier Inc. All rights reserved. Keywords: Amino acids; Chemical composition; Fatty acids; Fish bone; Lipids; Minerals
1. Introduction Bones are mainly regarded as rich in minerals such as calcium and phosphorus and collagen proteins, but some special carbohydrate and lipids are also found (Johns, 1977). The bone tissue is mainly built up of an organic extra cellular matrix covered with hydroxyapatite (Ca5(PO4)3OH2). Bone tissue is an important depot for storage of calcium and phosphates and is essential in the regulation of plasma concentrations of these minerals (Nordin, 1976). Due to the physiological importance of calcium and phosphorous in the soft tissues, calcium and phosphate present in the bones may be relocated to other tissues when the dietary supply do not meet the requirement. Bones constitute a significant part of the fish; approximately 10–15% of total fish biomass are bones from the head and vertebrae. Knowledge of the chemical composition of fish bones is limited, but may be interesting for several reasons. In a recent
⁎ Corresponding author. Tel.: +47 55 50 12 00; fax: +47 55 50 12 99. E-mail address:
[email protected] (J. Toppe). 1096-4959/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2006.11.020
study dried fish bones were used as feed ingredient in diets for cod, showing a positive effect on growth and feed efficiency compared to traditional diets (Toppe et al., 2006). Fish meal produced from fish by-products or whole fish contains about 10% minerals, especially high in calcium and phosphorus, and represent an important source of minerals when included in feed. The digestibility of minerals from fish meal shows great variation, but is generally low, and the presence of phytic acid from vegetable protein sources may further decrease the availability. The level of available phosphorus in feeds for aquaculture might be limited for optimal growth and fish health (Nordum et al., 1997). However, processing of fish bones may improve the availability of the present minerals (Aksnes, unpublished). Fish bones represent a significant part of the cut offs from the filleting industry and a better utilization of this raw material for various applications is a matter of great scientific interest. Bone minerals for human health products may be of interest as well as the protein from the collagen bone matrix (Liaset et al., 2003; Kim and Mendis, 2006). Information about the chemical composition of fish bones is therefore of interest for several reasons. It is important in
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understanding the physiological role of bone tissue, and for evaluating the potential of processing bone rich by-products from the fish processing industry. Bones from whole fish and cut offs could be used as a raw material for health products and as ingredient in feeds for aquaculture (Toppe et al., 2006). The aim of the present work is to obtain more information about the differences in chemical composition in bones from various fish species with special focus on fish species of interest for aquaculture, either as farmed fish or as a potential raw material for fish feed. 2. Materials and methods
into the boiling water. The temperature of the water then dropped to 80–85 °C. After simmering for 2.5 min, the bones and heads were taken out, and the remaining meat on the bones could easily be removed by using running cold water. For the largest fish samples (salmon, trout, saithe and cod) the heads were allowed to simmer for an additional minute, allowing all the meat to coagulate and subsequently easily be removed. The clean bones where then frozen at − 20 °C and lyophilized (final plate temperature 24 °C). The pressure in the freeze dryer was equalized to atmospheric pressure with nitrogen gas, to avoid oxidation of components in the samples. The dried samples were then ground in a Retsch mill (b 1 mm filter bags).
2.1. Biological material 2.3. Analysis Thirteen different samples of fish were collected and stored on ice or at − 20 °C before being processed to separate the bones from the remaining fish. The samples represented eight different species, including three samples of saithe, three samples of salmon and two samples of herring. The fish samples represent different locations, seasons or size (Table 1). All the samples were collected from coastal areas of western Norway and the Norwegian sea. The salmon, trout, mackerel, herring (small) and saithe were purchased from local seafood retailers. The cod was farmed at local facilities at Austevoll, Bergen. The remaining three samples were fish used for industrial processing; blue whiting, herring (big) and horse mackerel caught in the North Sea. Further details of the samples are shown in Table 1. 2.2. Processing Before processing, the frozen samples where thawed overnight at 15 °C. One sample of fish was processed at a time, consisting of minimum 5 fish, and a total mass of at least 4.5 kg. The fish was gutted, filleted and gills were removed. As much as possible of the remaining meat was removed by scraping the backbone with a small knife. A casserole of 20 L was filled with 15 L of water and the bones and heads were put
Crude protein (N × 6.25) was determined by the Kjeldahl method (ISO 5983-1997), moisture gravimetrically after drying for 4 h at 105 °C (ISO 6496-1999) and ash after combustion for 16 h at 550 °C (ISO 5984-2002). Phosphorus was determined by a spectrometric method (ISO 6491-1998) and calcium by atomic absorption spectrometry (ISO 6869-2000). The remaining mineral analyses were done by ICP-MS. Samples for analyses of total amino acids were hydrolyzed in 6 M HCl for 22 h at 110 °C and analyzed by HPLC using a fluorescence technique for detection (Cohen and Michaud, 1993). Total lipid was determined both by Soxhlet extraction (AOCS, Ba 3-38) and the Bligh and Dyer method (Bligh and Dyer, 1959). Fatty acid analyses were carried out by AOCS Official Method (Ce 1b-89). All analyses were carried out in duplicate samples of at least 5 fish each. Data are given as mean ± standard deviation. Where analyses of sample parallels differed significantly, analyses were repeated. All analyses, except ICP-MS analyses were run at Fiskeriforskning, Bergen. Eurofins Norway arranged for the ICP-MS analyses. Proximate analyses (except Bligh and Dyer method) and analyses on phosphorus are accredited analyses. Analytical data were subjected to a oneway analysis of variance (ANOVA) using SYSTAT 5.0 for
Table 1 Fish samples used for analyses of fish bones Species
Latin name
Origin in Norway
Date of catch
Number of fish
Average fish mass (g/fish)
Codab Saithe1bd Saithe2bd Saithe3 (big)bd Blue whitingc Salmon1abd Salmon2abd Salmon3abd Troutabd Herring1 (small)b Herring2 (big) c Mackerelb Horse mackerelc
Gadus morhua Pollachius virens Pollachius virens Pollachius virens Micromesistius poutassou Salmo salar Salmo salar Salmo salar Salmo trutta Clupea harengus Clupea harengus Scomber scombrus Trachurus trachurus
Austevoll Sotra Nordhordland Nordhordland North Sea Frøyfjord Sekkingstad Brandasund Sognefjord Hardangerfjord North Sea Hardangerfjord North Sea
20 July 2004 10 September 2004 18 October 2006 11 October 2006 28 June 2003 5 July 2004 11 October 2006 18 October 2006 8 July 2004 11 July 2004 October 2004 18 July 2004 November 2004
5 6 6 6 65 6 6 6 6 75 14 15 12
1566 1230 1270 2714 69 2495 2709 2714 2567 63 369 326 429
a
Farmed. Stored on ice before processing. c Stored at −20 °C before processing. d Gutted. b
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Windows (Wilkinson et al., 1992), and differences between means were tested using t-test. Effects with a probability p b 0.05 were considered significant. Data are given as mean ± SD. 3. Results and discussion The fish samples used in this work were all collected along the Norwegian coast. Fish used for human consumption were taken either in the fjords (salmon, trout, small herring and mackerel) with some reduced sea water salinity or close to the coast (cod and saithe). The fish species normally used for industrial processing (blue whiting, horse mackerel and large herring) were all caught in the North Sea (Table 1). The various fish species varies naturally in size. The sampling of at least 4.5 kg and thus 5–75 fish per sample is supposed to give a reasonable average. The chemical composition of fish bones may vary with season, size and fish age. The samples of saithe and herring contain different fish sizes. This may give an indication of the effect of fish size on the chemical composition of the respective fish bones. 3.1. Chemical composition The chemical composition of fish bone differs significantly in the level of proteins, ash and lipids between species. The most pronounced is the difference in level of lipid in the fish bones (Table 2). The fatty fish; salmon, trout, herring and mackerel, that store lipid in the muscle, have a much higher content of lipids in bones (318 ± 101 g/kg, n = 8) compared to the lean fish; cod, saithe and blue whiting (20.6 ± 16.0 g/kg, n = 5) that store lipid in the liver (p b 0.001). Significant difference in lipid level was also observed between saithe and salmon (p b 0.05). As a natural consequence, lean fish showed the highest levels of ash and protein. Lipids adsorbed to the bone surface could partly explain the difference; however Lee et al. (1975) reported even higher levels of lipid in fish bones
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from three different marine fish species. Lipid levels in animal bones are shown to range from 1–27% (Johns, 1977). The difference in extraction yield using apolar Soxhlet extraction compared to the more polar Bligh and Dyer extraction shows some variation (1.6–37.7 g/kg), indicating that the level of more polar lipids (e.g. phospholipids) slightly differ (Table 2). Saithe and salmon also showed significant differences in the levels of protein and ash (p b 0.05). Due to the difference in lipid content in bones from different species, the comparison of protein and ash levels was done on data adjusted for lipid level (soxhlet extraction), giving theoretical lipid free samples. Protein and ash levels were significantly different (p b 0.05) when comparing lean and, fatty fish. Protein and ash levels in lipid free samples were respectively 480 ± 60 and 482 ± 62 g/kg in fatty fish species, and 404 ± 40 and 570 ± 40 g/kg in the corresponding lean fish. A significant inverse correlation was observed between lipid free ash and protein levels (r2 = 0.98, p b 0.01). Protein in lipid free bones showed the highest value for mackerel, salmon and trout (average 513 ± 30 g/kg, n = 5). The ash/protein ratio was significantly highest (average 1.42 ± 0.22) for the lean fish (p b 0.05), although big herring (1.34) and horse mackerel (1.71) caught in the North Sea had a high ash/protein ratio. Salmon, trout and mackerel had an average ratio of 0.92 ± 0.09. However, salmon1 (caught in 2004) had an ash/protein ratio of 0.78 compared to 0.98 for salmon2 and salmon3 (caught in 2006). Improved formulation of commercial diets for salmon could explain an increased ash/protein ratio in the bones, indicating an improved bone mineralization. Bone mineralization is associated with hardness of the bones. The higher the Ca and P levels the harder is the bone. Salmon, trout and mackerel are species with a high swimming activity and the low ash/ protein level may indicate a need for better elasticity of the bones to support the high physical activity. Few data are given for the chemical composition of fish bones. In fish bones fractionated from fish meal of blue whiting, Shearer et al.
Table 2 Proximate composition of fish bones from fish caught in Norway given as g/kg sample or g/kg lipid free dry matter
g/kg sample Water Protein (N⁎6.25) a Ash b Lipid (Soxhlet) Lipid (Bligh and Dyer) g/kg lipid free dm c Protein (N⁎6.25) Ash Ash/raw protein
Cod
Saithe1, 2 (small)
Saithe3 (large)
Blue whiting
Salmon1,2,3
Trout
Herring1 (small)
Herring2 (large)
Mackerel
Horse mackerel
n=2
n=4
n=2
n=2
n=6
n=2
n=2
n=2
n=2
n=2
77.7 ± 0.4 357.8 ± 1.6 526.4 ± 0.4 11.4 ± 0.3 23.1 ± 0.4
61.9 ± 2.4 369.7 ± 2.2 538.2 ± 7.4 14.1 ± 2.0 24.8 ± 8.0
52.1 ± 0.1 335.9 ± 1.0 565 ± 1.1 14.9 ± 0.2 23.0 ± 0.6
64.1 ± 0.1 418.0 ± 0.1 445.8 ± 0.3 49.1 ± 0.7 73.1 ± 1.0
49.6 ± 3.3 292.0 ± 31.6 263.7 ± 20.4 381.2 ± 32.4 382.8 ± 37.9
53.3 ± 0.1 314.0 ± 4.1 265.5 ± 3.0 343.7 ± 3.6 360.1 ± 2.5
71.5 ± 0.5 373.1 ± 6.2 368.7 ± 1.9 152.5 ± 0.5 175.6 ± 1.1
40.7 ± 0.0 301.2 ± 0.7 357.1 ± 2.3 266.7 ± 3.3 265.0 ± 4.1
44.2 ± 0.4 261.3 ± 1.1 212.4 ± 0.8 471.8 ± 0.1 509.5 ± 5.3
26.2 ± 0.2 270.2 ± 1.4 463.0 ± 0.2 226.1 ± 1.9 231.5 ± 7.5
393 577 1.47
399 576 1.44
399 590 1.48
Values are given as mean ± SD. a Maximum acceptable deviation between replicates 3 g/kg. b Maximum acceptable deviation between replicates 10 g/kg. c Calculated by using lipid values based on Soxhlet extraction.
471 503 1.07
543 424 0.78
521 441 0.84
480 475 0.99
435 516 1.34
539 438 0.92
361 619 1.71
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Table 3 Mineral composition of fish bones given as g/kg or mg/kg lipid free dry matter
Calcium (Ca) Phosphorous (P) Magnesium (Mg) Iron (Fe) Zinc (Zn) Copper (Cu) Chromium (Cr) Sodium (Na) Potassium (K) Selenium (Se) Iodine (I) Chlorine (Cl) Fluorine (F) Arsenic (As) Cadmium (Cd) Mercury (Hg) Lead (Pb)
g/kg g/kg g/kg mg/kg mg/kg mg/kg mg/kg g/kg mg/kg mg/kg mg/kg g/kg g/kg mg/kg mg/kg mg/kg mg/kg
Cod
Saithe1
Blue whiting
Salmon1
Trout
Herring1 (small)
Herring2 (large)
Mackerel
Horse mackerel
190 113 3.0 49 98 1.0 10.8 7.7 5.2 n.d. 3.7 4.8 0.19 n.d. 0.01 0.01 n.d.
199 108 3.0 44 70 1.2 9.8 7.1 4.9 n.d. 2.6 4.1 0.17 0.3 0.02 0.01 0.07
170 87 3.2 135 72 3.0 16.9 4.6 2.6 n.d. 1.4 1.9 0.07 0.6 0.13 0.04 0.12
135 81 2.2 32 233 0.9 5.5 5.7 8.2 n.d. 2.7 4.4 0.10 1.0 n.d. 0.02 n.d.
147 87 2.4 32 126 0.9 6.7 5.8 7.7 n.d. 2.5 4.2 0.09 1.2 0.02 0.01 n.d.
161 94 2.6 61 191 2.6 3.6 3.3 5.0 n.d. 1.2 1.4 0.03 1.0 0.03 0.03 0.26
197 95 2.9 72 124 0.8 2.4 7.8 7.7 0.3 3.6 4.5 n.d. 3.8 0.06 0.02 0.09
143 86 2.6 73 125 2.2 3.9 6.5 6.7 n.d. 2.2 4.1 0.26 2.4 0.06 0.03 0.24
233 111 3.6 56 70 0.5 5.5 7.1 4.4 n.d. 2.1 4.0 n.d. 2.7 0.03 0.02 0.11
(1992) and Toppe et al. (2006) refer to similar ash levels and some higher lipid and protein levels compared to the present data given in Table 2. The higher levels of proteins and lipids are probably due to particles adsorbed to the bones during fractionation. There is therefore good correspondence between present data and the results presented by Shearer et al. (1992). Available data for salmon bones differ significantly. Liaset et al. (2003) presents data for salmon bones isolated after enzymatic treatment of salmon frames that show significant lower lipid and protein content than the present data. Consequently the ash content was high; 514 g/kg. This difference may be due to the enzymatic treatment with proteolytic enzymes and the following heat treatment to inactivate enzymes (98 °C for 105 min) dissolving protein and lipid and increasing the ash content.
Helland et al. (2005) reported however, ash levels in salmon bones that were similar to the present data; 250 g/kg dm. The bone samples from saithe and herring constitute different fish sizes. Comparing the effect of fish size for these samples, indicates that larger (older) fish have higher levels of lipid and ash, and a higher ash/protein ratio. Larger fish seems to have a lower protein level than smaller fish. 3.2. Minerals In general the levels of macro minerals correlated to the level of ash in the bones (Table 3). The levels of the macro minerals calcium (Ca), phosphorous (P) and magnesium (Mg) were lowest in bones from the salmonid species (salmon and trout),
Table 4 Amino acids in fish bones in g/kg raw protein
Aspartic acid a Glutamic acid b Hydroxyproline Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Cysteine/cystine Tryptophan Total amino acids
Cod
Saithe1
Blue whiting
Salmon1
Trout
Herring1 (small)
Herring2 (large)
Mackerel
Horse mackerel
77 112 52 58 172 18 82 33 73 80 20 31 24 22 43 25 42 20 4 988
74 110 54 55 175 18 82 36 74 86 22 31 23 22 40 26 41 17 4 990
88 126 29 49 115 19 77 39 68 63 31 41 30 33 59 35 61 12 8 983
78 112 56 48 173 22 78 32 73 86 19 30 26 22 41 26 44 14 4 984
78 112 57 49 173 22 78 32 71 82 18 29 26 22 39 26 43 12 4 973
82 115 40 48 134 19 77 36 69 72 27 40 29 27 52 34 50 12 7 970
70 103 56 50 177 16 77 35 79 91 17 34 25 20 44 29 44 55 6 1028
78 110 45 50 138 24 81 35 70 73 23 36 24 27 51 31 52 13 6 967
66 103 67 46 191 16 81 34 87 103 15 29 22 20 40 26 43 72 5 1066
All data are based on replicates (n = 2), general analytical deviation from mean b3.5%. a Represents the sum of aspartic acid and asparagine. b Represents the sum of glutamic acid and glutamine.
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Table 5 Distribution of different lipid classes in fish bones, in g/kg extracted lipid
Total saturated fatty acids Total monoene fatty acids Total PUFA (n-6) f.a. Total PUFA (n-3) f.a. Total PUFA Total fatty acids
Cod
Saithe1
Blue whiting
Salmon1
Trout
Herring1 (small)
Herring2 (large)
Mackerel
Horse mackerel
124 167 19 126 148 439
114 215 12 113 127 457
101 213 07 91 101 415
187 386 40 233 284 857
205 408 31 213 255 868
164 340 11 95 117 621
204 492 12 143 162 858
204 360 18 188 215 778
250 351 16 195 221 821
All data are based on replicates (n = 2), general analytical deviation from mean b5%.
showing ranges of 135–147 g/kg for Ca, 81–87 g/kg for P and 2.2–2.4 g/kg for Mg in lipid free dry matter. The corresponding levels in lean fish species were; Ca: 186 ± 15 g/kg, P: 102 ± 14 g/ kg, Mg:3.1 ± 0.1 g/kg (n = 3) However, potassium (K) levels were highest in salmon and trout with a range of 7.7–8.2 g/kg. Horse mackerel showed the highest levels of Ca (233 g/kg) and Mg (3.6 g/kg). Blue whiting had the lowest level of K (2.6 g/ kg). The gadoid species cod and saithe showed Ca, P, Mg and K ranges of 190–199, 108–113, 3.0 and 4.9–5.2 g/kg, respectively. The level of Ca in ash is about similar for all species ranging from 304 g/kg ash in salmon to 325 g/kg ash in saithe, except for large herring and horse mackerel showing Ca values of 367 g/kg ash. The value found in salmon is in good correspondence with values reported by Liaset et al. (2003) 325 g/kg ash and Helland et al. (2005) 272 g/kg ash. The Ca level in blue whiting of 316 g/kg ash is also in good correspondence with the value reported by Shearer et al. (1992) (294 g/kg ash) and by Toppe et al. (2006) (297 g/kg ash).
The level of P is also about equal and independent of fish species although blue whiting showed some lower level (161 g/ kg ash). This is however still higher than the P level reported by Shearer et al. (1992) (121 g/kg ash). Generally, the mineral bone matrix contains about similar levels of the main structural minerals, Ca and P, giving a similar Ca/P ratio in all samples. This is in accordance with the results presented by Komárková and Bilvyk (1973). Sodium and chlorine levels followed each other in the study and about similar values were found for all fish species, although blue whiting and small herring showed some lower levels. By grouping into lean gadoid fish (cod, saithe and blue whiting) and more fatty fish (salmon, trout, herring, mackerel and horse mackerel), a few differences were found with regard to micro minerals. With the exception of horse mackerel, the level of zinc (Zn), on lipid free dry matter, was significantly higher in the more fatty fish (145 ± 58 mg/kg), compared to (80 ± 15 mg/kg
Table 6 Fatty acids in fish bones in g/kg extracted lipid
14:0 16:0 18:0 20:0 22:0 16:1 n-7 18:1 (n-9) + (n-7) + (n-5) 20:1 (n-9) + (n-7) 22:1 (n-11) + (n-9) + (n-7) 24:1 n-9 16:2 n-4 16:3 n-4 16:4 n-1 18:2 n-6 18:3 n-6 20:2 n-6 20:3 n-6 20:4 n-6 22:4 n-6 18:3 n-3 18:4 n-3 20:3 n-3 20:4 n-3 20:5 n-3 21:5 n-3 22:5 n-3 22:6 n-3
Cod
Saithe1
Blue whiting
Salmon1
Trout
Herring1 (small)
Herring2 (large)
Mackerel
Horse mackerel
9 87 28 b1 b1 12 112 16 11 16 2 b1 1 10 b1 1 b1 7 b1 1 2 b1 1 46 b1 6 69
16 73 24 1 b1 14 111 37 34 19 2 b1 1 4 b1 1 b1 7 b1 2 4 b1 2 37 b1 4 63
15 65 21 1 b1 11 92 46 47 17 2 b1 1 3 b1 1 b1 3 b1 2 3 b1 2 32 b1 4 49
44 119 23 1 b1 54 150 90 86 6 6 2 3 29 b1 3 1 7 b1 7 15 5 12 65 3 25 101
51 130 23 1 b1 56 164 94 88 6 6 1 3 25 b1 2 1 4 b1 7 14 1 11 58 3 17 102
54 98 10 2 1 53 65 83 134 6 5 2 4 9 b1 1 b1 1 b1 4 10 b1 2 41 b1 03 34
78 113 11 2 1 35 123 122 204 8 5 1 2 10 b1 1 b1 1 b1 7 16 b1 3 46 1 5 65
54 125 22 2 1 32 113 75 130 10 6 1 2 12 b1 2 b1 4 b1 11 28 b1 7 49 2 10 81
63 157 28 1 b1 42 172 51 77 8 6 1 2 10 1 2 b1 4 b1 7 16 1 6 52 2 12 99
All data are based on replicates (n = 2), general analytical deviation from mean b5%.
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in the lean fish (p b 0.05). Arsenic (As) levels were also significantly highest in fatty fish, with average value of 2.0 ± 1.1 mg/kg, with the highest level in big herring (3.8 mg/kg). Lean fish showed an average As level of 0.3 ± 0.3 mg/kg. Levels of chromium (Cr) were highest in lean fish (9.8–16.9 mg/kg), compared to 2.4–6.7 mg/kg in the fatty fish. Highest levels of fluorine (F) were found in mackerel (0.25 g/kg), followed by cod and saithe with 0.19 and 0.17 g/kg respectively. Blue whiting showed the most extreme levels of several minerals studied. It showed the lowest level of K, but the highest levels of heavy metals such as, cadmium (Cd), 0.13 mg/ kg and mercury (Hg), 0.04 mg/kg. 3.3. Amino acids The differences in the amino acid profiles are in general small (Table 4). Some minor differences should however be commented on. Bones from farmed cod, salmon, trout, saithe and large herring have the same amino acid profiles. Blue whiting, small herring and mackerel show lower bone levels of the collagen associated amino acids glycine, proline and hydroxyprolin, and higher levels of tyrosine, valine, isoleucine and tryptophan. The amino acid profile of horse mackerel is different from the other species analysed, by having higher levels of glycine, alanine, proline, hydroxyproline and cysteine/cystine, and lower levels of histidine, tyrosine and isoleucine. The differences mentioned above are not associated to differences in protein, ash or lipid composition although some positive correlation to Ca level is indicated. Comparing small and large fish (saithe and herring), the large fish (of the same species) contain lower levels of protein and a higher level of ash. Further analyses on the herring samples showed higher Ca and higher levels of amino acids associated with gelatine structure. This corresponds to other results showing that mineralization increases by age (Johns, 1977). The total sum of amino acids ranges from 970 to 1066 g/kg raw protein, indicating that analysed protein is mainly explained by the amino acids identified. The high sum for herring (big) and horse mackerel can partly be explained by the relative high levels of small sized amino acids such as glycine, proline, hydroxyproline and alanine. The amino acid composition is in line with the results described for bone meal from an unspecified fish species by Hendriks et al. (2006) showing about similar amino acid composition as for mackerel in Table 4. The values for salmon bone in Table 4 are also in good correspondence with the results given by Liaset et al. (2003) for salmon, except for a general lower level in the latter (sum amino acids 93%) and a higher proline level. 3.4. Lipid composition The large difference in total lipid in fish bones between species (Table 2), is also positively correlated (p b 0.05) to the lipid composition (Table 5). For all species except small herring, the relative difference in total lipid content is reflected by differences in amount of total saturated fatty acids, total
monoene fatty acids and polyunsaturated fatty acids (PUFA). For small herring there is a higher relative content of monoene fatty acids and a lower level of PUFA. The main reason for this is due to higher 22:1 fatty acids and lower 20:5 and 22:6 fatty acids. Blue whiting is special among the lean fishes; the fish bone lipids contain the lowest level of 20:5 and 22:6. Very little is known about the lipid composition of fish bones. Lee et al. (1975) reported that 64–97% of total bone oil was triglycerides consisting mainly of the fatty acids 18:1, 16:0 and 16:1 in addition to the PUFA 20:5 and 22:6. These data are in good correspondence with the present results (Table 6). However, the monoenes 20:1 and 22:1 were also present in significant amounts, especially in the fatty fish. The low sum of total fatty acids given in Table 5 for the lean fish indicates that phospholipids constitute a significant fraction of the lipid extracted from bones of this fish. Similarly, the fraction of phospholipids in the fatty fish is indicated to be much lower. Comparing the lipid profile data with that of fish oil extracted from whole fish, Young (1986) gives data for blue whiting, herring and mackerel caught in the same area and the same season. The comparison shows that the lipid profile of fat extracted from bones of fatty fishes is closer to the oil extracted from whole fish, compared to the results for lean fish. The higher lipid levels are therefore likely storage fat and not structural fat. A higher fraction of phospholipids in lean fish compared to fat fish is in accordance with the results presented by Lee et al. (1975) for other fish species. Acknowledgements Thanks to Jarle Wang-Andersen for skillful and accurate help with the analyses and Dr. Jan Pettersen for useful evaluation of the lipid data. References Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Cohen, S.A., Michaud, K.E., 1993. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal. Biochem. 211, 279–287. Helland, S., Refstie, S., Espmark, Å., Hjelde, K., Baeverfjord, G., 2005. Mineral balance and bone formation in fast-growing Atlantic salmon parr (Salmo salar) in response to dissolved metabolic carbon dioxide and restricted dietary phosphorus supply. Aquaculture 250, 364–376. Hendriks, W.H., Cottam, Y.H., Thomas, D.V., 2006. The effect of storage on the nutritional quality of meat and bone meal. Anim. Feed Sci. Technol. 127, 151–160. Johns, P., 1977. The structure and components of collagen containing tissues. In: Ward, A.G., Cours, A. (Eds.), The Science and Technology of Gelatin. Academic Press, London, pp. 31–72. Kim, S.-K., Mendis, E., 2006. Bioactive compounds from marine processing byproducts — a review. Food Res. Int. 39, 383–393. Komárková, A., Bilvyk, I., 1973. Organic acids and minerals in the bones of lower vertebrates. Comp. Biochem. Physiol. B 46, 37–41. Lee, R.F., Phleger, C.F., Horn, M.H., 1975. Composition of oil in fish bones: possible function in the neutral buoyancy. Comp. Biochem. Physiol. B 50, 13–16. Liaset, B., Julshamn, K., Espe, M., 2003. Chemical composition and theoretical nutritional evaluation of the produced fractions from enzyme hydrolysis of salmon frames with Protamex. Proc. Biochem. 38, 1747–1759.
J. Toppe et al. / Comparative Biochemistry and Physiology, Part B 146 (2007) 395–401 Nordin, B.E.C., 1976. Plasma calcium and plasma magnesium homeostasis. In: Nordin, B.E.C. (Ed.), Calcium, phosphate and magnesium metabolism. Churchill Livingstone, Edinburgh, pp. 186–216. Nordum, S., Åsgård, T., Shearer, K.D., Arnessen, P., 1997. Availability of phosphorus in fish bone meal and inorganic salts to Atlantic salmon (Salmo salar) as determined by retention. Aquaculture 157, 51–61. Shearer, K.D., Maage, A., Opstvedt, J., Mundheim, H., 1992. Effects of highash diets on growth, feed efficiency, and zinc status of juvenile Atlantic salmon (Salmo salar). Aquaculture 106, 345–355.
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Toppe, J., Aksnes, A., Hope, B., Albrektsen, S., 2006. Inclusion of fish bone and crab by-products in diets for Atlantic cod, Gadus morhua. Aquaculture 253, 636–645. Wilkinson, L., Hill, M.-A., Welna, J.P., Birkenbeuel, G.K., 1992. Statistics. SYSTAT Inc., Evanston, Illinois. 750 pp. Young, F.V.K., 1986. The chemical and physical properties of crude fish oils for refiners and hydrogenators. IAFMM. Fish Oil Bull. 18.
