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Comparison of IEF patterns of sarcoplasmic proteins of fish from North Atlantic and Aegean Sea
Food Control 20 (2009) 980–985
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Comparison of IEF patterns of sarcoplasmic proteins of fish from North Atlantic and Aegean Sea Can Altinelataman a, Rainer Kündiger b, Sukran Cakli a, Hartmut Rehbein b,* a b
Ege University, Fisheries Faculty, 35100 Bornova/Izmir, Turkey Max Rubner-Institute, Department for Safety and Quality of Milk and Fish Products, Palmaille 9, 22767 Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 27 June 2008 Received in revised form 13 November 2008 Accepted 21 November 2008
Keywords: Fish species identification IEF Mediterranean Sea
a b s t r a c t Identification of processed fish lacking morphological characteristics is of growing importance in the times of increasing global trade and illegal fishery. Protein electrophoresis techniques, like isoelectric focusing (IEF) of water-soluble proteins, are fast and inexpensive tools for identification of raw fishery products. Protein profiles of raw fish or fillet of the same species were found to be remarkably constant. However, it cannot be excluded that protein patterns of fish of the same species from distant stocks may show some variation. The protein profiles of commercially important fish from different fishing grounds were determined by collecting samples of 38 fish species from Aegean Sea and Turkish fish farms and of five species from North Atlantic waters. The raw fillet of the fish was analysed by IEF of water-soluble (sarcoplasmic) proteins. Species-specific protein profiles were obtained suited for identification of raw fish or fillet. When protein patterns and pI values were compared between Mediterranean and North Atlantic fish of the respective species, only minor differences were observed. These differences were related to the intensity of protein bands, but not to the position within the pH gradient of the IEF gel. The pI values (isoelectric points) of the prominent protein bands of the fish were determined to be used as a database. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction In recent years world-wide trade of fishery products has steadily increased. As a result, in many countries new species appeared on the market. In cases where external biological characteristics had been removed by processing, e.g. in case of fillets, objective analytical methods have to be used for control of product labelling to safeguard fair trade and to protect consumers against fraud (Berrini, Tepedino, Borromeo, & Secchi, 2006; Martinez, 2003). In West Europe fish from the Mediterranean Sea has gained increased popularity. This development prompted us to establish a catalogue of species-specific protein patterns for fish species from Aegean Sea. The patterns have been generated by isoelectric focusing (IEF) of water-soluble (sarcoplasmic) proteins, as this technique has been proven to give fast and reliable results for raw fish, respectively fillet (Berrini et al., 2006; Rehbein, 2003). Analysis by IEF of water-soluble proteins (WSP) offers several advantages against electrophoresis of myofibrillar proteins: (i) greater stability of WSP during storage of chilled or frozen fish or fillet, (ii) easy and
* Corresponding author. Tel.: +49 40 38905 167; fax: +49 40 38905 262. E-mail address: [email protected] (H. Rehbein). 0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.11.007
rapid extractability of WSP, and (iii) WSP are giving protein profiles with a large number of bands of different pI values (Mackie, 1996). Preliminary results obtained with the ready-to-use PAG-Plates (Amersham Biosciences, Freiburg, Germany) for IEF have been reported recently (Ataman, Celik, & Rehbein, 2006). 2. Materials and methods 2.1. Origin of samples In February 2001, 36 samples of Aegean fish species were bought as wet fish on the fish market in Izmir and two samples were obtained from local fish farmers (Table 1). The fish was identified by their biological characteristics, stored at 12 °C overnight and transported in coolers to Hamburg by air-cargo within 6 h. After arrival in the institute in Hamburg samples were frozen immediately at 25 °C and stored at this temperature until analysed. After thawing, samples were checked for freshness by determination of the volatile amines (di- and trimethylamine–nitrogen (DMA-N, TMA-N), ammonia–nitrogen (NH3–N), and total volatile basic nitrogen TVB-N)). Having guaranteed by these methods that the samples showed no signs of spoilage, they were analysed by IEF. Fish of five species from North Atlantic waters were collected at a cruise of the fishery research vessel ‘‘Solea” in April 2001, or were
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C. Altinelataman et al. / Food Control 20 (2009) 980–985 Table 1 Fish samples. Number and origin Aegean Sea 1 2 3 4
Common name
5
Chub mackerel Atlantic mackerel Horse mackerel Mediterranean horse mackerel Twaite shad
6 7 8 9 10
Sea bass Tub gurnard Red scorpionfish John dory Hake
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Large eye dentex Anchovy European sprat Sardine Solenette Flathead mullet Red mullet Leaping grey mullet Striped mullet Thin lipped grey mullet Angler fish Bogue Salema Picarel Painted comber Gav fish Shark Sole Gilthead seabream Stargazer Common pandorra Saddled sea bream White sea bream Striped sea bream
35
Two banded sea bream
Scientific name
36
Sharp snout sea bream
Scomber japonicus (Houttuyn, 1782) Scomber scombrus (Linnaeus, 1758) Trachurus trachurus (Linnaeus, 1758) Trachurus mediterraneus (Steindachner, 1868) Alosa fallax nilotica (Geoffroy SaintHilaire, 1808) Dicentrarchus labrax (Linnaeus, 1758) Trigla lucerna (Linnaeus, 1758) Scorpaena scrofa (Linnaeus, 1758) Zeus faber (Linnaeus, 1758) Merluccius merluccius (Linnaeus, 1758) Dentex macrophthalmus (Bloch, 1791) Engraulis encrasicolus (Linnaeus, 1758) Sprattus sprattus (Linnaeus, 1758) Sardina pilchardus (Walbaum, 1792) Buglossidium luteum (Risso, 1810) Mugil cephalus (Linnaeus, 1758) Mullus barbatus (Linnaeus, 1758) Lizal saliens (Risso, 1810) Mullus surmuletus (Linnaeus, 1758) Liza ramada (Risso, 1826) Lophius piscatorius (Linnaeus, 1758) Boops boops (Linnaeus, 1758) Sarpa salpa (Linnaeus, 1758) Spicara smaris (Linnaeus, 1758) Serranus scriba (Linnaeus, 1758) Belone belone (Linnaeus, 1761) Mustelus mustelus (Linnaeus, 1758) Solea solea (Linnaeus, 1758) Sparus aurata (Linnaeus, 1758) Uranoscopus scaber (Linnaeus, 1758) Pagellus erythrinus (Linnaeus, 1758) Oblada melanura (Linnaeus, 1758) Diplodus sargus (Linnaeus, 1758) Lithognathus mormyrus (Linnaeus, 1758) Diplodus vulgaris (E. Geoffroy SaintHilaire, 1817) Diplodus puntazzo (Gmelin, 1789)
Turkish fish farm 37 38
Gilthead seabream Sea bass
Sparus aurata (Linnaeus, 1758) Dicentrarchus labrax (Linnaeus, 1758)
North Atlantic waters 39 40 41 42 43
North Atlantic hake Horse mackerel Atlantic mackerel Herring European sprat
Merluccius merluccius (Linnaeus, 1758) Trachurus trachurus (Linnaeus, 1758) Scomber scombrus (Linnaeus, 1758) Clupea harengus (Linnaeus, 1758) Sprattus sprattus (Linnaeus, 1758)
purchased on the fish market in Hamburg–Altona (Table 1). The fish species were identified by their biological characteristics and samples were stored at 25 °C until analysed by IEF. 2.2. Analytical methods 2.2.1. Determination of volatile amines Amines were extracted from fish muscle by homogenising 10 g of muscle with 90 ml of pre-cooled 6% (w/v) perchloric acid using the Ultra Turrax. The homogenate was clarified by filtration and kept frozen until analysis. DMA-N and TMA-N were determined in the perchloric acid extract using a gas chromatograph equipped with a nitrogen–phosphorus detector (Oetjen & Karl, 1999). For measurement of TVB-N values the official German method (Anonymous, 1988) was used. The ammonia content of perchloric
Table 2 Volatile amines (mg N/100 g wet weight) of fish from Aegean Sea and Turkish fish farms. Common name
Scientific name
TMA-N/ DMA-N
NH3– N
TVBN
Chub mackerel Horse mackerel Twaite shad Sea bass Tub gurnard Red scorpionfish John dory Hake
Scomber japonicus Trachurus trachurus Alosa fallax nilotica Dicentrarchus labrax Trigia lucerna Scorpaena scrofa Zeus faber Merluccius merluccius Dentex macrophthalmus Engraulis engrasicolus Mugil cephalus Lophius piscatorius Boops boops Sarpa salpa Trachurus mediterraneus Spicara smaris Serranus scriba Sardina pilchardus Belone belone Mustelus mustelus Mullus barbatus Sparus aurata Oblada melanura Diplodus sargus Solea solea Liza saliens Lithognatus mormyrus Uranoscopus scaber Diplodus vulgaris Diplodus puntazzo Mullus surmuletus Scomber scombrus Pagellus erythrinus Liza ramada Sprattus sprattus Buglossidium luteum Sparus aurata Dicentrarchus labrax
0.8/<0.4 0.7/<0.4 0.7/0.6 0.6/<0.3 0.1/<0.1 1.3/0.7 0.5/1.4 <0.3/0.6
18.18 13.23 15.68 14.15 12.72 9.58 8.07 11.63
19.35 15.35 17.72 15.88 14.13 12.47 26.15 14.78
<0.1/<0.1
14.60
17.49
<0.1/<0.3
23.96
25.60
<0.3/<0.4 2.2/<0.3 0.8/<0.3 1.0/<0.3 1.9/n.d
17.19 10.32 12.45 15.57 12.68
19.42 11.92 15.68 18.63 15.46
0.9/<0.3 <0.2/<0.1 0.5/<0.3 0.7/0.6 2.2/1.0 3.9/0.9 <0.4/<0.3 <0.1/<0.4 0.8/0.5 0.8/<0.4 <0.1/<0.1 <0.1/<0.1
15.65 13.88 14.01 17.57 13.93 11.94 12.27 16.26 14.70 7.14 11.39 7.62
18.34 21.22 14.91 19.74 18.48 17.64 13.72 18.38 16.87 9.66 13.72 9.24
<0.1/<0.2 2.2/<0.1 0.8/<0.1 2.2/<0.1 <0.3/0.5 0.5/<0.4 0.6/0.5 1.1/<0.1 0.5/<0.4 0.5/<0.3 0.8/0.6
9.95 13.49 14.38 15.99 12.84 16.49 12.44 14.02 9.09 11.33 10.84
12.33 16.10 17.03 19.74 16.11 19.52 14.83 16.76 12.65 13.44 12.04
Large eye dentex Anchovy Flathead mullet Angler fish Bogue Salema Mediterranean horse mackerel Picarel Painted omber Sardine Gav fish Shark Red mullet Gilthead seabream Saddled seabream White bream Sole Leaping grey mullet Striped bream Stargazer Two banded bream Sharpsnout seabream Striped mullet Atlantic mackerel Red pandorra Thin lipped grey mullat Sprat Solenette Gilthead seabream Sea bass
acid extracts was determined enzymatically (Rehbein & Oehlenschläger, 1982) according to Bergmeyer and Beutler (1985). 2.2.2. Extraction of water-soluble proteins 2 g of thawed light muscle of fish were homogenised with 8 ml of pre-cooled (8 °C) distilled water in a beaker placed in an ice bath using the Ultra Turrax (IKA Labortechnik, Staufen, Germany) for 30 s. Insoluble material was precipitated by centrifugation for 20 min at 14,000 rpm (2630g) at 5 °C (centrifuge Universal 16 R, rotor E 806, Hettich, Tuttlingen, Germany). The supernatant was stored at 4 °C until being analysed by isoelectric focusing (IEF). 2.2.3. Isoelectric focusing IEF was performed according to Rehbein and Kündiger (1984) using Servalyt Precote 3–10 of 0.3 mm gel thickness (SERVA, Heidelberg, Germany); sample volume was 7.5 ll. Proteins were stained with SERVA Violet 17. 2.2.4. Calculation of pI values The pI values of characteristic bands of sarcoplasmic proteins were determined by comparing the position of marker proteins (Broad pI Kit, pH 3.5–9.3; Amersham Biosciences) and sample bands using the Bio-Rad Gel Doc 2000 System and the software
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pI 8.15
7.35
6.55
5.20
4.65
Horse mackerel Horse mackerel (N) Medit. Horse mackerel Mackerel Mackerel (N)
Mackerel (N) Chub mackerel Gav fish Fig. 1. Patterns obtained by IEF of sarcoplasmic proteins of mackerel and horse mackerel. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
pI 8.15
7.35
6.55
5.85
4.55
3.50
White sea bream Saddled sea bream Gilthead sea bream Large eye dentex Common pandorra Two banded sea bream Striped sea bream Fig. 2. Patterns obtained by IEF of sarcoplasmic proteins of sea breams. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3.
Quantity One Version 4.1. In the first step an image of the gel was captured with Gel Doc System, followed by analysis with lanebased functions. Selection of lanes and bands of markers (of known pIs) and samples was performed manually, calculation of sample pIs was done by the software.
pI 8.15
6.00
4.50
3.50
Hake (N) Hake
3. Results and discussion 3.1. Indication of quality of fish samples To ensure that the quality of the samples had not suffered from transport, absence of signs of spoilage was proved by determina-
Fig. 3. Patterns obtained by IEF of sarcoplasmic proteins of North Atlantic hake. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
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pI 8.15 7.35
6.55
5.20
3.50
Sardine Twaite shad Herring (N) European sprat (N) Cultered sea bass Painted comber Fig. 4. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
tion of the content of volatile amines. The content of TMA-N was below 1 mg/100 g wet weight (ww) for most of the samples, the DMA-N content was less than 1.5 mg/100 g ww, and the values for TVB-N and NH3–N did not exceed 20 mg/100 g ww in most cases, indicating the high quality of the samples (Table 2) (Oehlenschläger, 1992). In case of three samples, John Dory, painted comber and anchovy, higher TVB-N values were found (Table 2). On the other hand, content of DMA-N, TMA-N and NH3–N was low for the first two
pI
8.15
7.35
6.55
5.85
samples. As TVB-N is composed of DMA-N, TMA-N and NH3–N, the difference between TVB-N and the sum of DMA-N, TMA-N, NH3–N in case of John Dory and painted comber is indicating an experimental error in TVB-N measurement. It was concluded that in general the fish samples obtained from Turkey were well suited for analysis by IEF, as the content of TVB-N was well below the cut off level for flatfish (30 mg TVB-N/100 g ww) and gadoids or hakes (35 mg TVB-N/100 g ww) (European Commission, 2005).
5.20 4.55
3.50
Shark Sole Cuttlefish White sea bream Red scorpionfish Fig. 5. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
Marker Thin lipped grey mullet Leaping grey mullet Flathead mullet Striped mullet Red mullet Fig. 6. Patterns obtained by IEF of sarcoplasmic proteins of different mullet species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins which are shown in the top lane. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
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8.15
7.35 6.90 6.55
5.85
5.20 4.55
3.50
Marker Salema Stargazer Sharpsnout sea bream Saurey pike Painted comber Red scorpionfish Bogue Fig. 7. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins which are shown in the top lane. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
3.2. Protein patterns of fish from Turkish and North Atlantic waters In total the sarcoplasmic proteins of 38 fish species from Aegean Sea or Turkish fish farms and five species from North Atlantic waters were analysed by IEF, as shown in Figs. 1–8. As in some cases only one specimen of a given species has been analysed the results have to be considered as being somewhat preliminary. On the other hand the intra-species variability of IEF patterns has been reported to be very low for most of the fish species studied (Rehbein, 1990). The protein profiles of horse mackerel (Trachurus trachurus) and Mediterranean horse mackerel ( Trachurus mediterraneus) were clearly different, whereas the protein patterns for mackerel ( Scomber scombrus) and chub mackerel (Scomber japonicus) resembled each other by showing only protein bands in the cathodic part of the gel (Fig. 1). No differences in protein profiles were observed between fish from North Atlantic waters and Aegean Sea in case of horse mackerel or mackerel, apart from varying intensity of some bands (Fig. 1). The protein pattern of gav fish (Belone belone) has not been published up to now to our knowledge.
pI 9.30 8.15
6.85
5.85
5.20
4.55 3.5
John dory Angler fish
The protein patterns of five sea bream species (Diplodus sargus, Diplodus vulgaris, Lithognathus mormyrus, Sparus aurata, Oblada melanura), as well as large eye dentex (Dentex macrophthalmus), common pandorra ( Pagellus erythrinus) and bogue ( Boops boops) are compiled in Fig. 7. In case of white sea bream and two banded sea bream protein patterns were very similar, and more specimens of these two species have to be analysed until a conclusion can be drawn on their differentiation by IEF. With this exception, each of the species of Fig. 2 expressed a characteristic pattern of bands ranging from the cathode to the anode. The pattern of another sea bream species, sharp snout sea bream (Diplodus puntazzo), is given in Fig. 7. Specimens of North Atlantic hake (Merluccius merluccius) from Aegean Sea and North Atlantic waters could not be distinguished by IEF (Fig. 3), although they are belonging to different populations (Roldan, Garcia-Marin, Utter, & Pla, 1998). Fig. 4 shows the patterns for sardine, herring, sprat, twaite shad, cultured sea bass and painted comber, and in Fig. 5 the patterns of shark, sole, cuttle fish, white sea bream and red scorpion fish are presented. All of these patterns were found to be species specific. Five mullet species could be differentiated by means of their protein patterns, as shown in Fig. 6. Like many other fish species, each of the mullet species possessed several strong protein bands in the anodic part of the gel, which had been identified as parvalbumins (Rehbein, Kündiger, Pineiro, & Perez-Martin, 2000). A collection of various Mediterranean fish species is compiled in Figs. 7 and 8, most of them have not been analysed by IEF up to now by our knowledge.
Salema Picarel Anchovy Tub gurnard Solenette Fig. 8. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3.
3.3. Compilation of pI values of fish from Aegean Sea and Turkish fish farms The pI values of prominent protein bands of each of the 38 species are listed in Table 3. The accompanying bands can be identified in the figures by the arrowheads. The compilation of the pI values may be useful in the identification of unknown samples (Ukishima, Narita, Masui, Matsubara, & Okada, 1986), as the protein profiles are depending on the type and pH gradient of the applied IEF gel (FDA., 2001). Comparison of patterns and pI values for several Aegean Sea fish species determined using either PAG plates (Ataman et al., 2006) or Servalyte Precotes (this study) showed that protein profiles were similar overall, but that differences in number and pI
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C. Altinelataman et al. / Food Control 20 (2009) 980–985 Table 3 pI values of sarcoplasmic proteins of fish species from Aegean Sea and Turkish fish farms. The pI values refer to the bands indicated in Figs. 1–8 by arrow heads. Nr
Common name
Scientific name
pI values of bands from anode to cathode
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Chub mackerel Atlantic mackerel Horse mackerel Mediterranean horse mackerel Twaite shad Sea bass Tub gurnard Red scorpionfish John dory North Atlantic hake Large eye dentex Anchovy European sprat Sardine Solenette Flathead mullet Red mullet Leaping grey mullet Striped mullet Thin lipped grey mullet Angler fish Bogue Salema Picarel Painted comber Gav fish Shark Sole Gilthead seabream Stargazer Common pandorra Saddled sea bream White sea bream Striped sea bream Two banded sea bream Sharp snout sea bream Gilthead sea bream, farmed Sea bass, farmed
Scomber japonicus (Houttuyn, 1782) Scomber scombrus (Linnaeus, 1758) Trachurus trachurus (Linnaeus, 1758) Trachurus mediterraneus (Steindachner, 1868) Alosa fallax nilotica (Geoffroy Saint-Hilaire, 1808) Dicentrarchus labrax (Linnaeus, 1758) Trigla lucerna (Linnaeus, 1758) Scorpaena scrofa (Linnaeus, 1758) Zeus faber (Linnaeus, 1758) Merluccius merluccius (Linnaeus, 1758) Dentex macrophthalmus (Bloch, 1791) Engraulis encrasicolus (Linnaeus, 1758) Sprattus sprattus (Linnaeus, 1758) Sardina pilchardus (Walbaum, 1792) Buglossidium luteum (Risso, 1810) Mugil cephalus (Linnaeus, 1758) Mullus barbatus (Linnaeus, 1758) Lizal saliens (Risso, 1810) Mullus surmuletus (Linnaeus, 1758) Liza ramada (Risso, 1826) Lophius piscatorius (Linnaeus, 1758) Boops boops (Linnaeus, 1758) Sarpa salpa (Linnaeus, 1758) Spicara smaris (Linnaeus, 1758) Serranus scriba (Linnaeus, 1758) Belone belone (Linnaeus, 1761) Mustelus mustelus (Linnaeus, 1758) Solea solea (Linnaeus, 1758) Sparus aurata (Linnaeus, 1758) Uranoscopus scaber (Linnaeus, 1758) Pagellus erythrinus (Linnaeus, 1758) Oblada melanura (Linnaeus, 1758) Diplodus sargus (Linnaeus, 1758) Lithognathus mormyrus (Linnaeus, 1758) Diplodus vulgaris (E. Geoffroy Saint-Hilaire, 1817) Diplodus puntazzo (Gmelin, 1789) Sparus aurata (Linnaeus, 1758) Dicentrarchus labrax (Linnaeus, 1758)
6.68 6.71 4.20 4.21 5.36 3.76 3.99 3.95 4.18 4.02 4.09 4.59 5.17 4.65 4.04 4.19 3.91 3.89 3.99 3.96 3.99 4.06 4.54 4.57 3.78 4.44 6.58 4.20 3.84 4.26 3.89 3.64 3.60 3.77 3.71 4.02 4.42 3.76
value of bands were observed being caused by the different type of ampholytes present in both gels. References Anonymous (1988). Bestimmung des Gehaltes von flüchtigen stickstoffhaltigen Basen (TVB-N) in Fischen und Fischerzeugnissen. Amtliche Sammlung von Untersuchungsverfahren nach §35 LMBG, Methode L 10.00/3. Beuth-Verlag, Berlin. Ataman, C., Celik, U., & Rehbein, H. (2006). Identification of some Aegean fish species by native isoelectric focusing. European Food Research and Technology, 222, 99–104. Bergmeyer, H. U., & Beutler, H.-O. (1985). Ammonia (3rd ed.. In H. U. Bergmeyer, J. Bergmeyer, & M. Grabl (Eds.). Method of enzymatic analysis (Vol. VIII, pp. 454–461). Weinheim: VCH. Berrini, A., Tepedino, V., Borromeo, V., & Secchi, C. (2006). Identification of fresh water fish commercially labelled ‘‘perch” by isoelectric focusing and twodimensional electrophoresis. Food Chemistry, 96, 163–168. European Commission Regulation (EC) No. 2074/2005 (2005). Official Journal of the European Union, L338/27–L338/59. FDA (2001). Regulatory fish encyclopedia (). Mackie, I. M. (1996). Authenticity of fish. In P. R. Ashurst & M. J. Dennis (Eds.), Food authentication (pp. 140–170). London: Blackie Academic and Professional. Martinez, I. (2003). Application of protein electrophoretic analysis and DNA fingerprinting to species identification. In R. Perez-Martin & C. G. Sotelo (Eds.), Authenticity of species in meat and seafood products. Association International Congress on Authenticity of Species in Meat and Seafood Products (pp. 177–188). Vigo.
6.77 6.73 4.50 4.28 6.65 5.06 4.24 4.25 5.94 4.13 4.69 6.79 6.57 6.51 4.28 4.56 4.99 5.11 4.41 4.96 6.06 5.89 5.90 4.97 4.33 6.37 6.65 5.28 4.42 4.69 4.51 3.95 3.81 4.58 3.89 4.33 4.96 5.06
7.57 7.70 (Fig. 1) 6.76 7.24 (Fig. 1) 5.95 6.57 7.30 (Fig. 1) 6.06 6.17 7.31 (Fig. 1) 6.80 7.24 (Fig. 4) 5.75 5.84 6.02 6.80 (Fig. 4) 4.82 6.04 6.59 6.65 6.70 7.07 8.39 (Fig. 8) 5.50 5.61 6.36 6.96 (Fig. 5) 6.80 (Fig. 8) 4.52 5.93 6.11 6.30 6.70 7.65 (Fig. 3) 5.12 5.39 5.75 5.93 6.76 7.15 (Fig. 2) 6.84 6.92 7.07 8.11 8.31 (Fig. 8) 6.63 7.30 (Fig. 4) 6.62 6.67 6.72 6.78 7.48 (Fig. 4) 5.16 5.37 6.36 6.59 6.84 (Fig. 8) 5.43 6.75 (Fig. 6) 5.36 6.20 6.49 (Fig. 6) 6.82 (Fig. 6) 4.99 5.41 6.20 6.28 6.43 (Fig. 6) 6.82 (Fig. 6) (Fig. 8) 5.96 6.31 6.70 (Fig. 7) 7.11 (Fig. 7) 5.92 6.04 6.18 6.81 7.36 (Fig. 8) 5.16 5.38 (Fig. 7) 6.53 7.59 (Fig. 1) 6.77 6.89 (Fig. 5) 5.72 6.65 8.67 (Fig. 5) 4.96 5.38 5.68 5.81 5.93 6.78 7.20 (Fig. 2) 5.37 6.50 6.71 (Fig. 7) 5.18 5.43 5.92 6.08 6.24 6.46 7.17 7.73 (Fig. 2) 4.19 5.39 5.59 5.68 5.81 6.68 7.16 7.79 (Fig. 2) 4.49 5.37 5.57 5.66 6.85 (Figs. 2 and 5) 5.15 5.40 5.86 5.99 6.80 7.12 (Fig. 2) 4.63 5.60 6.81 (Fig. 2) 5.17 5.64 7.09 8.61 (Fig. 2) 5.38 5.68 5.81 5.93 6.78 7.20 (Fig. 2) 5.75 5.84 6.02 6.80 (Fig. 4)
Oehlenschläger, J. (1992). Evaluation of some well established and some underrated indices for the determination of freshness and/or spoilage of ice stored wet fish. In H. H. Huss, M. Jakobsen, & J. Liston (Eds.), Quality assurance in the fish industry (pp. 339–350). Amsterdam: Elsevier. Oetjen, K., & Karl, H. (1999). Improvement of gas chromatographic determination methods of volatile amines in fish and fishery products. Deutsche LebensmittelRundschau, 95, 403–407. Rehbein, H. (1990). Electrophoretic techniques for species identification of fishery products. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung, 191, 1–10. Rehbein, H. (2003). Identification of fish species by protein- and DNA-analysis. In R. Perez-Martin, C. G. Sotelo (Eds.), Authenticity of species in meat and seafood products. Association International Congress on Authenticity of Species in Meat and Seafood Products (pp. 83–101). Vigo. Rehbein, H., & Kündiger, R. (1984). Comparison of the isoelectric focusing patterns of sarcoplasmic proteins from red and white muscle of various fish species. Archiv Fur Fischerei Wissenschaft, 35, 7–16. Rehbein, H., Kündiger, R., Pineiro, C., & Perez-Martin, R. (2000). Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing. Electrophoresis, 21, 1458–1463. Rehbein, H., & Oehlenschläger, J. (1982). Zur Zusammensetzung der TVB-N-Fraktion (flüchtige Basen) in sauren Extrakten und alkalischen Destillaten von Seefischfilets. Archiv Fur Lebensmittelhygiene, 33, 44–48. Roldan, M. I., Garcia-Marin, J. L., Utter, F. M., & Pla, C. (1998). Population genetic structure of European hake, Merluccius merluccius. Heredity, 81, 327–334. Ukishima, Y., Narita, H., Masui, T., Matsubara, S., & Okada, S. (1986). Studies of the application of thin layer isoelectric focusing to food analysis II. Identification of fish species by numerical value of isoelectric focusing pattern. Journal of the Hygienic Chemistry, 32, 176–184.
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Comparison of IEF patterns of sarcoplasmic proteins of fish from North Atlantic and Aegean Sea Can Altinelataman a, Rainer Kündiger b, Sukran Cakli a, Hartmut Rehbein b,* a b
Ege University, Fisheries Faculty, 35100 Bornova/Izmir, Turkey Max Rubner-Institute, Department for Safety and Quality of Milk and Fish Products, Palmaille 9, 22767 Hamburg, Germany
a r t i c l e
i n f o
Article history: Received 27 June 2008 Received in revised form 13 November 2008 Accepted 21 November 2008
Keywords: Fish species identification IEF Mediterranean Sea
a b s t r a c t Identification of processed fish lacking morphological characteristics is of growing importance in the times of increasing global trade and illegal fishery. Protein electrophoresis techniques, like isoelectric focusing (IEF) of water-soluble proteins, are fast and inexpensive tools for identification of raw fishery products. Protein profiles of raw fish or fillet of the same species were found to be remarkably constant. However, it cannot be excluded that protein patterns of fish of the same species from distant stocks may show some variation. The protein profiles of commercially important fish from different fishing grounds were determined by collecting samples of 38 fish species from Aegean Sea and Turkish fish farms and of five species from North Atlantic waters. The raw fillet of the fish was analysed by IEF of water-soluble (sarcoplasmic) proteins. Species-specific protein profiles were obtained suited for identification of raw fish or fillet. When protein patterns and pI values were compared between Mediterranean and North Atlantic fish of the respective species, only minor differences were observed. These differences were related to the intensity of protein bands, but not to the position within the pH gradient of the IEF gel. The pI values (isoelectric points) of the prominent protein bands of the fish were determined to be used as a database. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction In recent years world-wide trade of fishery products has steadily increased. As a result, in many countries new species appeared on the market. In cases where external biological characteristics had been removed by processing, e.g. in case of fillets, objective analytical methods have to be used for control of product labelling to safeguard fair trade and to protect consumers against fraud (Berrini, Tepedino, Borromeo, & Secchi, 2006; Martinez, 2003). In West Europe fish from the Mediterranean Sea has gained increased popularity. This development prompted us to establish a catalogue of species-specific protein patterns for fish species from Aegean Sea. The patterns have been generated by isoelectric focusing (IEF) of water-soluble (sarcoplasmic) proteins, as this technique has been proven to give fast and reliable results for raw fish, respectively fillet (Berrini et al., 2006; Rehbein, 2003). Analysis by IEF of water-soluble proteins (WSP) offers several advantages against electrophoresis of myofibrillar proteins: (i) greater stability of WSP during storage of chilled or frozen fish or fillet, (ii) easy and
* Corresponding author. Tel.: +49 40 38905 167; fax: +49 40 38905 262. E-mail address: [email protected] (H. Rehbein). 0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.11.007
rapid extractability of WSP, and (iii) WSP are giving protein profiles with a large number of bands of different pI values (Mackie, 1996). Preliminary results obtained with the ready-to-use PAG-Plates (Amersham Biosciences, Freiburg, Germany) for IEF have been reported recently (Ataman, Celik, & Rehbein, 2006). 2. Materials and methods 2.1. Origin of samples In February 2001, 36 samples of Aegean fish species were bought as wet fish on the fish market in Izmir and two samples were obtained from local fish farmers (Table 1). The fish was identified by their biological characteristics, stored at 12 °C overnight and transported in coolers to Hamburg by air-cargo within 6 h. After arrival in the institute in Hamburg samples were frozen immediately at 25 °C and stored at this temperature until analysed. After thawing, samples were checked for freshness by determination of the volatile amines (di- and trimethylamine–nitrogen (DMA-N, TMA-N), ammonia–nitrogen (NH3–N), and total volatile basic nitrogen TVB-N)). Having guaranteed by these methods that the samples showed no signs of spoilage, they were analysed by IEF. Fish of five species from North Atlantic waters were collected at a cruise of the fishery research vessel ‘‘Solea” in April 2001, or were
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C. Altinelataman et al. / Food Control 20 (2009) 980–985 Table 1 Fish samples. Number and origin Aegean Sea 1 2 3 4
Common name
5
Chub mackerel Atlantic mackerel Horse mackerel Mediterranean horse mackerel Twaite shad
6 7 8 9 10
Sea bass Tub gurnard Red scorpionfish John dory Hake
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Large eye dentex Anchovy European sprat Sardine Solenette Flathead mullet Red mullet Leaping grey mullet Striped mullet Thin lipped grey mullet Angler fish Bogue Salema Picarel Painted comber Gav fish Shark Sole Gilthead seabream Stargazer Common pandorra Saddled sea bream White sea bream Striped sea bream
35
Two banded sea bream
Scientific name
36
Sharp snout sea bream
Scomber japonicus (Houttuyn, 1782) Scomber scombrus (Linnaeus, 1758) Trachurus trachurus (Linnaeus, 1758) Trachurus mediterraneus (Steindachner, 1868) Alosa fallax nilotica (Geoffroy SaintHilaire, 1808) Dicentrarchus labrax (Linnaeus, 1758) Trigla lucerna (Linnaeus, 1758) Scorpaena scrofa (Linnaeus, 1758) Zeus faber (Linnaeus, 1758) Merluccius merluccius (Linnaeus, 1758) Dentex macrophthalmus (Bloch, 1791) Engraulis encrasicolus (Linnaeus, 1758) Sprattus sprattus (Linnaeus, 1758) Sardina pilchardus (Walbaum, 1792) Buglossidium luteum (Risso, 1810) Mugil cephalus (Linnaeus, 1758) Mullus barbatus (Linnaeus, 1758) Lizal saliens (Risso, 1810) Mullus surmuletus (Linnaeus, 1758) Liza ramada (Risso, 1826) Lophius piscatorius (Linnaeus, 1758) Boops boops (Linnaeus, 1758) Sarpa salpa (Linnaeus, 1758) Spicara smaris (Linnaeus, 1758) Serranus scriba (Linnaeus, 1758) Belone belone (Linnaeus, 1761) Mustelus mustelus (Linnaeus, 1758) Solea solea (Linnaeus, 1758) Sparus aurata (Linnaeus, 1758) Uranoscopus scaber (Linnaeus, 1758) Pagellus erythrinus (Linnaeus, 1758) Oblada melanura (Linnaeus, 1758) Diplodus sargus (Linnaeus, 1758) Lithognathus mormyrus (Linnaeus, 1758) Diplodus vulgaris (E. Geoffroy SaintHilaire, 1817) Diplodus puntazzo (Gmelin, 1789)
Turkish fish farm 37 38
Gilthead seabream Sea bass
Sparus aurata (Linnaeus, 1758) Dicentrarchus labrax (Linnaeus, 1758)
North Atlantic waters 39 40 41 42 43
North Atlantic hake Horse mackerel Atlantic mackerel Herring European sprat
Merluccius merluccius (Linnaeus, 1758) Trachurus trachurus (Linnaeus, 1758) Scomber scombrus (Linnaeus, 1758) Clupea harengus (Linnaeus, 1758) Sprattus sprattus (Linnaeus, 1758)
purchased on the fish market in Hamburg–Altona (Table 1). The fish species were identified by their biological characteristics and samples were stored at 25 °C until analysed by IEF. 2.2. Analytical methods 2.2.1. Determination of volatile amines Amines were extracted from fish muscle by homogenising 10 g of muscle with 90 ml of pre-cooled 6% (w/v) perchloric acid using the Ultra Turrax. The homogenate was clarified by filtration and kept frozen until analysis. DMA-N and TMA-N were determined in the perchloric acid extract using a gas chromatograph equipped with a nitrogen–phosphorus detector (Oetjen & Karl, 1999). For measurement of TVB-N values the official German method (Anonymous, 1988) was used. The ammonia content of perchloric
Table 2 Volatile amines (mg N/100 g wet weight) of fish from Aegean Sea and Turkish fish farms. Common name
Scientific name
TMA-N/ DMA-N
NH3– N
TVBN
Chub mackerel Horse mackerel Twaite shad Sea bass Tub gurnard Red scorpionfish John dory Hake
Scomber japonicus Trachurus trachurus Alosa fallax nilotica Dicentrarchus labrax Trigia lucerna Scorpaena scrofa Zeus faber Merluccius merluccius Dentex macrophthalmus Engraulis engrasicolus Mugil cephalus Lophius piscatorius Boops boops Sarpa salpa Trachurus mediterraneus Spicara smaris Serranus scriba Sardina pilchardus Belone belone Mustelus mustelus Mullus barbatus Sparus aurata Oblada melanura Diplodus sargus Solea solea Liza saliens Lithognatus mormyrus Uranoscopus scaber Diplodus vulgaris Diplodus puntazzo Mullus surmuletus Scomber scombrus Pagellus erythrinus Liza ramada Sprattus sprattus Buglossidium luteum Sparus aurata Dicentrarchus labrax
0.8/<0.4 0.7/<0.4 0.7/0.6 0.6/<0.3 0.1/<0.1 1.3/0.7 0.5/1.4 <0.3/0.6
18.18 13.23 15.68 14.15 12.72 9.58 8.07 11.63
19.35 15.35 17.72 15.88 14.13 12.47 26.15 14.78
<0.1/<0.1
14.60
17.49
<0.1/<0.3
23.96
25.60
<0.3/<0.4 2.2/<0.3 0.8/<0.3 1.0/<0.3 1.9/n.d
17.19 10.32 12.45 15.57 12.68
19.42 11.92 15.68 18.63 15.46
0.9/<0.3 <0.2/<0.1 0.5/<0.3 0.7/0.6 2.2/1.0 3.9/0.9 <0.4/<0.3 <0.1/<0.4 0.8/0.5 0.8/<0.4 <0.1/<0.1 <0.1/<0.1
15.65 13.88 14.01 17.57 13.93 11.94 12.27 16.26 14.70 7.14 11.39 7.62
18.34 21.22 14.91 19.74 18.48 17.64 13.72 18.38 16.87 9.66 13.72 9.24
<0.1/<0.2 2.2/<0.1 0.8/<0.1 2.2/<0.1 <0.3/0.5 0.5/<0.4 0.6/0.5 1.1/<0.1 0.5/<0.4 0.5/<0.3 0.8/0.6
9.95 13.49 14.38 15.99 12.84 16.49 12.44 14.02 9.09 11.33 10.84
12.33 16.10 17.03 19.74 16.11 19.52 14.83 16.76 12.65 13.44 12.04
Large eye dentex Anchovy Flathead mullet Angler fish Bogue Salema Mediterranean horse mackerel Picarel Painted omber Sardine Gav fish Shark Red mullet Gilthead seabream Saddled seabream White bream Sole Leaping grey mullet Striped bream Stargazer Two banded bream Sharpsnout seabream Striped mullet Atlantic mackerel Red pandorra Thin lipped grey mullat Sprat Solenette Gilthead seabream Sea bass
acid extracts was determined enzymatically (Rehbein & Oehlenschläger, 1982) according to Bergmeyer and Beutler (1985). 2.2.2. Extraction of water-soluble proteins 2 g of thawed light muscle of fish were homogenised with 8 ml of pre-cooled (8 °C) distilled water in a beaker placed in an ice bath using the Ultra Turrax (IKA Labortechnik, Staufen, Germany) for 30 s. Insoluble material was precipitated by centrifugation for 20 min at 14,000 rpm (2630g) at 5 °C (centrifuge Universal 16 R, rotor E 806, Hettich, Tuttlingen, Germany). The supernatant was stored at 4 °C until being analysed by isoelectric focusing (IEF). 2.2.3. Isoelectric focusing IEF was performed according to Rehbein and Kündiger (1984) using Servalyt Precote 3–10 of 0.3 mm gel thickness (SERVA, Heidelberg, Germany); sample volume was 7.5 ll. Proteins were stained with SERVA Violet 17. 2.2.4. Calculation of pI values The pI values of characteristic bands of sarcoplasmic proteins were determined by comparing the position of marker proteins (Broad pI Kit, pH 3.5–9.3; Amersham Biosciences) and sample bands using the Bio-Rad Gel Doc 2000 System and the software
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pI 8.15
7.35
6.55
5.20
4.65
Horse mackerel Horse mackerel (N) Medit. Horse mackerel Mackerel Mackerel (N)
Mackerel (N) Chub mackerel Gav fish Fig. 1. Patterns obtained by IEF of sarcoplasmic proteins of mackerel and horse mackerel. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
pI 8.15
7.35
6.55
5.85
4.55
3.50
White sea bream Saddled sea bream Gilthead sea bream Large eye dentex Common pandorra Two banded sea bream Striped sea bream Fig. 2. Patterns obtained by IEF of sarcoplasmic proteins of sea breams. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3.
Quantity One Version 4.1. In the first step an image of the gel was captured with Gel Doc System, followed by analysis with lanebased functions. Selection of lanes and bands of markers (of known pIs) and samples was performed manually, calculation of sample pIs was done by the software.
pI 8.15
6.00
4.50
3.50
Hake (N) Hake
3. Results and discussion 3.1. Indication of quality of fish samples To ensure that the quality of the samples had not suffered from transport, absence of signs of spoilage was proved by determina-
Fig. 3. Patterns obtained by IEF of sarcoplasmic proteins of North Atlantic hake. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
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pI 8.15 7.35
6.55
5.20
3.50
Sardine Twaite shad Herring (N) European sprat (N) Cultered sea bass Painted comber Fig. 4. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
tion of the content of volatile amines. The content of TMA-N was below 1 mg/100 g wet weight (ww) for most of the samples, the DMA-N content was less than 1.5 mg/100 g ww, and the values for TVB-N and NH3–N did not exceed 20 mg/100 g ww in most cases, indicating the high quality of the samples (Table 2) (Oehlenschläger, 1992). In case of three samples, John Dory, painted comber and anchovy, higher TVB-N values were found (Table 2). On the other hand, content of DMA-N, TMA-N and NH3–N was low for the first two
pI
8.15
7.35
6.55
5.85
samples. As TVB-N is composed of DMA-N, TMA-N and NH3–N, the difference between TVB-N and the sum of DMA-N, TMA-N, NH3–N in case of John Dory and painted comber is indicating an experimental error in TVB-N measurement. It was concluded that in general the fish samples obtained from Turkey were well suited for analysis by IEF, as the content of TVB-N was well below the cut off level for flatfish (30 mg TVB-N/100 g ww) and gadoids or hakes (35 mg TVB-N/100 g ww) (European Commission, 2005).
5.20 4.55
3.50
Shark Sole Cuttlefish White sea bream Red scorpionfish Fig. 5. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
Marker Thin lipped grey mullet Leaping grey mullet Flathead mullet Striped mullet Red mullet Fig. 6. Patterns obtained by IEF of sarcoplasmic proteins of different mullet species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins which are shown in the top lane. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
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8.15
7.35 6.90 6.55
5.85
5.20 4.55
3.50
Marker Salema Stargazer Sharpsnout sea bream Saurey pike Painted comber Red scorpionfish Bogue Fig. 7. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins which are shown in the top lane. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3. ‘‘N” is marking samples from North Atlantic waters.
3.2. Protein patterns of fish from Turkish and North Atlantic waters In total the sarcoplasmic proteins of 38 fish species from Aegean Sea or Turkish fish farms and five species from North Atlantic waters were analysed by IEF, as shown in Figs. 1–8. As in some cases only one specimen of a given species has been analysed the results have to be considered as being somewhat preliminary. On the other hand the intra-species variability of IEF patterns has been reported to be very low for most of the fish species studied (Rehbein, 1990). The protein profiles of horse mackerel (Trachurus trachurus) and Mediterranean horse mackerel ( Trachurus mediterraneus) were clearly different, whereas the protein patterns for mackerel ( Scomber scombrus) and chub mackerel (Scomber japonicus) resembled each other by showing only protein bands in the cathodic part of the gel (Fig. 1). No differences in protein profiles were observed between fish from North Atlantic waters and Aegean Sea in case of horse mackerel or mackerel, apart from varying intensity of some bands (Fig. 1). The protein pattern of gav fish (Belone belone) has not been published up to now to our knowledge.
pI 9.30 8.15
6.85
5.85
5.20
4.55 3.5
John dory Angler fish
The protein patterns of five sea bream species (Diplodus sargus, Diplodus vulgaris, Lithognathus mormyrus, Sparus aurata, Oblada melanura), as well as large eye dentex (Dentex macrophthalmus), common pandorra ( Pagellus erythrinus) and bogue ( Boops boops) are compiled in Fig. 7. In case of white sea bream and two banded sea bream protein patterns were very similar, and more specimens of these two species have to be analysed until a conclusion can be drawn on their differentiation by IEF. With this exception, each of the species of Fig. 2 expressed a characteristic pattern of bands ranging from the cathode to the anode. The pattern of another sea bream species, sharp snout sea bream (Diplodus puntazzo), is given in Fig. 7. Specimens of North Atlantic hake (Merluccius merluccius) from Aegean Sea and North Atlantic waters could not be distinguished by IEF (Fig. 3), although they are belonging to different populations (Roldan, Garcia-Marin, Utter, & Pla, 1998). Fig. 4 shows the patterns for sardine, herring, sprat, twaite shad, cultured sea bass and painted comber, and in Fig. 5 the patterns of shark, sole, cuttle fish, white sea bream and red scorpion fish are presented. All of these patterns were found to be species specific. Five mullet species could be differentiated by means of their protein patterns, as shown in Fig. 6. Like many other fish species, each of the mullet species possessed several strong protein bands in the anodic part of the gel, which had been identified as parvalbumins (Rehbein, Kündiger, Pineiro, & Perez-Martin, 2000). A collection of various Mediterranean fish species is compiled in Figs. 7 and 8, most of them have not been analysed by IEF up to now by our knowledge.
Salema Picarel Anchovy Tub gurnard Solenette Fig. 8. Patterns obtained by IEF of sarcoplasmic proteins of different fish species. The pH gradient in the gel is indicated at the top of the figure by pI values of marker proteins. Strong characteristic bands are indicated by arrowheads. The accompanying pI values are given in Table 3.
3.3. Compilation of pI values of fish from Aegean Sea and Turkish fish farms The pI values of prominent protein bands of each of the 38 species are listed in Table 3. The accompanying bands can be identified in the figures by the arrowheads. The compilation of the pI values may be useful in the identification of unknown samples (Ukishima, Narita, Masui, Matsubara, & Okada, 1986), as the protein profiles are depending on the type and pH gradient of the applied IEF gel (FDA., 2001). Comparison of patterns and pI values for several Aegean Sea fish species determined using either PAG plates (Ataman et al., 2006) or Servalyte Precotes (this study) showed that protein profiles were similar overall, but that differences in number and pI
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C. Altinelataman et al. / Food Control 20 (2009) 980–985 Table 3 pI values of sarcoplasmic proteins of fish species from Aegean Sea and Turkish fish farms. The pI values refer to the bands indicated in Figs. 1–8 by arrow heads. Nr
Common name
Scientific name
pI values of bands from anode to cathode
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Chub mackerel Atlantic mackerel Horse mackerel Mediterranean horse mackerel Twaite shad Sea bass Tub gurnard Red scorpionfish John dory North Atlantic hake Large eye dentex Anchovy European sprat Sardine Solenette Flathead mullet Red mullet Leaping grey mullet Striped mullet Thin lipped grey mullet Angler fish Bogue Salema Picarel Painted comber Gav fish Shark Sole Gilthead seabream Stargazer Common pandorra Saddled sea bream White sea bream Striped sea bream Two banded sea bream Sharp snout sea bream Gilthead sea bream, farmed Sea bass, farmed
Scomber japonicus (Houttuyn, 1782) Scomber scombrus (Linnaeus, 1758) Trachurus trachurus (Linnaeus, 1758) Trachurus mediterraneus (Steindachner, 1868) Alosa fallax nilotica (Geoffroy Saint-Hilaire, 1808) Dicentrarchus labrax (Linnaeus, 1758) Trigla lucerna (Linnaeus, 1758) Scorpaena scrofa (Linnaeus, 1758) Zeus faber (Linnaeus, 1758) Merluccius merluccius (Linnaeus, 1758) Dentex macrophthalmus (Bloch, 1791) Engraulis encrasicolus (Linnaeus, 1758) Sprattus sprattus (Linnaeus, 1758) Sardina pilchardus (Walbaum, 1792) Buglossidium luteum (Risso, 1810) Mugil cephalus (Linnaeus, 1758) Mullus barbatus (Linnaeus, 1758) Lizal saliens (Risso, 1810) Mullus surmuletus (Linnaeus, 1758) Liza ramada (Risso, 1826) Lophius piscatorius (Linnaeus, 1758) Boops boops (Linnaeus, 1758) Sarpa salpa (Linnaeus, 1758) Spicara smaris (Linnaeus, 1758) Serranus scriba (Linnaeus, 1758) Belone belone (Linnaeus, 1761) Mustelus mustelus (Linnaeus, 1758) Solea solea (Linnaeus, 1758) Sparus aurata (Linnaeus, 1758) Uranoscopus scaber (Linnaeus, 1758) Pagellus erythrinus (Linnaeus, 1758) Oblada melanura (Linnaeus, 1758) Diplodus sargus (Linnaeus, 1758) Lithognathus mormyrus (Linnaeus, 1758) Diplodus vulgaris (E. Geoffroy Saint-Hilaire, 1817) Diplodus puntazzo (Gmelin, 1789) Sparus aurata (Linnaeus, 1758) Dicentrarchus labrax (Linnaeus, 1758)
6.68 6.71 4.20 4.21 5.36 3.76 3.99 3.95 4.18 4.02 4.09 4.59 5.17 4.65 4.04 4.19 3.91 3.89 3.99 3.96 3.99 4.06 4.54 4.57 3.78 4.44 6.58 4.20 3.84 4.26 3.89 3.64 3.60 3.77 3.71 4.02 4.42 3.76
value of bands were observed being caused by the different type of ampholytes present in both gels. References Anonymous (1988). Bestimmung des Gehaltes von flüchtigen stickstoffhaltigen Basen (TVB-N) in Fischen und Fischerzeugnissen. Amtliche Sammlung von Untersuchungsverfahren nach §35 LMBG, Methode L 10.00/3. Beuth-Verlag, Berlin. Ataman, C., Celik, U., & Rehbein, H. (2006). Identification of some Aegean fish species by native isoelectric focusing. European Food Research and Technology, 222, 99–104. Bergmeyer, H. U., & Beutler, H.-O. (1985). Ammonia (3rd ed.. In H. U. Bergmeyer, J. Bergmeyer, & M. Grabl (Eds.). Method of enzymatic analysis (Vol. VIII, pp. 454–461). Weinheim: VCH. Berrini, A., Tepedino, V., Borromeo, V., & Secchi, C. (2006). Identification of fresh water fish commercially labelled ‘‘perch” by isoelectric focusing and twodimensional electrophoresis. Food Chemistry, 96, 163–168. European Commission Regulation (EC) No. 2074/2005 (2005). Official Journal of the European Union, L338/27–L338/59. FDA (2001). Regulatory fish encyclopedia (
6.77 6.73 4.50 4.28 6.65 5.06 4.24 4.25 5.94 4.13 4.69 6.79 6.57 6.51 4.28 4.56 4.99 5.11 4.41 4.96 6.06 5.89 5.90 4.97 4.33 6.37 6.65 5.28 4.42 4.69 4.51 3.95 3.81 4.58 3.89 4.33 4.96 5.06
7.57 7.70 (Fig. 1) 6.76 7.24 (Fig. 1) 5.95 6.57 7.30 (Fig. 1) 6.06 6.17 7.31 (Fig. 1) 6.80 7.24 (Fig. 4) 5.75 5.84 6.02 6.80 (Fig. 4) 4.82 6.04 6.59 6.65 6.70 7.07 8.39 (Fig. 8) 5.50 5.61 6.36 6.96 (Fig. 5) 6.80 (Fig. 8) 4.52 5.93 6.11 6.30 6.70 7.65 (Fig. 3) 5.12 5.39 5.75 5.93 6.76 7.15 (Fig. 2) 6.84 6.92 7.07 8.11 8.31 (Fig. 8) 6.63 7.30 (Fig. 4) 6.62 6.67 6.72 6.78 7.48 (Fig. 4) 5.16 5.37 6.36 6.59 6.84 (Fig. 8) 5.43 6.75 (Fig. 6) 5.36 6.20 6.49 (Fig. 6) 6.82 (Fig. 6) 4.99 5.41 6.20 6.28 6.43 (Fig. 6) 6.82 (Fig. 6) (Fig. 8) 5.96 6.31 6.70 (Fig. 7) 7.11 (Fig. 7) 5.92 6.04 6.18 6.81 7.36 (Fig. 8) 5.16 5.38 (Fig. 7) 6.53 7.59 (Fig. 1) 6.77 6.89 (Fig. 5) 5.72 6.65 8.67 (Fig. 5) 4.96 5.38 5.68 5.81 5.93 6.78 7.20 (Fig. 2) 5.37 6.50 6.71 (Fig. 7) 5.18 5.43 5.92 6.08 6.24 6.46 7.17 7.73 (Fig. 2) 4.19 5.39 5.59 5.68 5.81 6.68 7.16 7.79 (Fig. 2) 4.49 5.37 5.57 5.66 6.85 (Figs. 2 and 5) 5.15 5.40 5.86 5.99 6.80 7.12 (Fig. 2) 4.63 5.60 6.81 (Fig. 2) 5.17 5.64 7.09 8.61 (Fig. 2) 5.38 5.68 5.81 5.93 6.78 7.20 (Fig. 2) 5.75 5.84 6.02 6.80 (Fig. 4)
Oehlenschläger, J. (1992). Evaluation of some well established and some underrated indices for the determination of freshness and/or spoilage of ice stored wet fish. In H. H. Huss, M. Jakobsen, & J. Liston (Eds.), Quality assurance in the fish industry (pp. 339–350). Amsterdam: Elsevier. Oetjen, K., & Karl, H. (1999). Improvement of gas chromatographic determination methods of volatile amines in fish and fishery products. Deutsche LebensmittelRundschau, 95, 403–407. Rehbein, H. (1990). Electrophoretic techniques for species identification of fishery products. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung, 191, 1–10. Rehbein, H. (2003). Identification of fish species by protein- and DNA-analysis. In R. Perez-Martin, C. G. Sotelo (Eds.), Authenticity of species in meat and seafood products. Association International Congress on Authenticity of Species in Meat and Seafood Products (pp. 83–101). Vigo. Rehbein, H., & Kündiger, R. (1984). Comparison of the isoelectric focusing patterns of sarcoplasmic proteins from red and white muscle of various fish species. Archiv Fur Fischerei Wissenschaft, 35, 7–16. Rehbein, H., Kündiger, R., Pineiro, C., & Perez-Martin, R. (2000). Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing. Electrophoresis, 21, 1458–1463. Rehbein, H., & Oehlenschläger, J. (1982). Zur Zusammensetzung der TVB-N-Fraktion (flüchtige Basen) in sauren Extrakten und alkalischen Destillaten von Seefischfilets. Archiv Fur Lebensmittelhygiene, 33, 44–48. Roldan, M. I., Garcia-Marin, J. L., Utter, F. M., & Pla, C. (1998). Population genetic structure of European hake, Merluccius merluccius. Heredity, 81, 327–334. Ukishima, Y., Narita, H., Masui, T., Matsubara, S., & Okada, S. (1986). Studies of the application of thin layer isoelectric focusing to food analysis II. Identification of fish species by numerical value of isoelectric focusing pattern. Journal of the Hygienic Chemistry, 32, 176–184.