High resolution two-dimensional electrophoresis as a tool to differentiate wild from farmed cod (Gadus morhua) and to assess the protein composition of klipfish

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Abstract

Tris and CHAPS–urea extracts from wild and farmed cod muscle and from rehydrated cod klipfish fillets were analyzed by one (1DE) and two-dimensional electrophoresis (2DE). 2DE maps of tris extracts from farmed cod differed from the wild in a series of spots of Mw 35 and 45 kDa. The CHAPS–urea extracts from farmed cod had a several spots of Mw between 100 and 45 kDa, which were hardly detectable in wild cod and very prominent in klipfish. Klipfish was clearly different from the other samples: the myosin heavy chain was hardly detectable in these samples, and the tris extracts contained fewer, and the CHAPS–urea more spots than the corresponding extracts from the raw muscles. Further identification of these potentially diagnostic spots will make it easier the differentiation of farmed from wild cod and the evaluation of klipfish processing on the protein content of the product.

Introduction

European citizens are entitled by law (CR-EC Nos. 2065/2001 and 104/2000) to information on the scientific name, method of production (farmed or wild), and the area in which wild fish was caught or farmed fish underwent the final developmental stage. Additional legal requirements for the implementation of traceability systems in the food and feed supply chains in Europe, are laid down in the General Food Law, Regulation 178/2002/EC, whose article number 18 referring to traceability has become effective since 1st January 2005. The EU Food Law defines traceability as “the ability to trace and follow a food, feed, food-producing animal or substance intended to be, or expected to be incorporated into a food or feed, through all stages of production, processing and distribution”.

Unfortunately, globalization and the consequent availability of unknown, novel species in certain markets, together with the consumer’s willingness to pay higher prices for certain production methods (i.e. organically produced or environmentally friendly) and the lack of analytical methods to verify some product claims, allows the intervention of opportunistic elements and the fraudulent falsification of product information. The consequences can be very serious, including allergies and death, in the case of spoiled or contaminated products.

There are several methods suitable for species identification in seafood, but not for the unequivocal determination of wild and farmed cod, or for the life-history of the product, which are data necessary to verify the traceability documentation of a product and to detect fraud (Martinez et al., 2003).

We are interested in developing analytical techniques to verify product composition and claims that may be of relevance for the customers. Our previous works have dealt with the analyses of proteins and DNA for species and breeding stock identification (Martinez et al., 2003, Martinez and Friis, 2004) and application of nuclear magnetic resonance techniques for the authentication of fish products (Aursand et al., 1994, Martinez et al., 2003) and of small water soluble bioactive molecules (Martinez et al., 2005). Complementing these works, we decided to examine the suitability of muscle protein analysis to differentiate farmed from wild cod due to the fact that proteins are the most abundant component in cod muscle and also because it has been shown that the feed composition, and in particular the content of vegetable protein components in the feed, induce alterations in the protein expression in rainbow trout liver (Martin et al., 2003). However, we are not aware of works examining the protein expression in muscle, which is the most common edible part and therefore available for analysis all along the production chain, from the farm to the dish.

The protein profile of klipfish was also examined in order to map changes due to the processing and preserving conditions because that information may be of relevance regarding the protein composition of these products. In our recent work we have documented that klipfish is poorer in bioactive components than fresh fish (Martinez et al., 2005). Proteins from marine organisms have been shown to exert positive effects on human health, for example on the blood lipid profiles (Wergedahl et al., 2004) and skeletal muscle insulin responsiveness (Tremblay, Lavigne, Jacques, & Marette, 2003). However, these works used isolated and heat-treated salmon protein hydrolysates and purified cod protein respectively, while human diets are made up, usually, of processed fish containing whole proteins and therefore the protein profile resulting from certain processing conditions deserves closer examination. The suitability of a proteomics approach to map the composition of seafoods and changes in fish muscle due to loss of freshness has already been examined by several authors (Kjærsgård and Jessen, 2003, Martinez and Friis, 2004, Piñeiro et al., 2003). However, we are aware of only one work analyzing the effect of the processing conditions on the protein profile of surimi (Martinez, Solberg, Lauritzsen, & Ofstad, 1992), and of none aiming at identifying the protein profile of klipfish, a popular product consumed by millions of persons in many European and American countries.

This work had two aims: the first was to examine the suitability of two-dimensional electrophoresis of muscle extracts to differentiate farmed from wild cod and the second to assess the effect of klipfish processing on the protein composition of cod muscle.

Section snippets

Fish samples

The cod (Gadus morhua) used were wild specimens captured in the Trondheimsfjord in April (n = 10), farmed cod from the Trondelag region slaughtered in February (n = 5) and rehydrated klipfish (n = 5). The wild and farmed fish arrived iced at our laboratory within a few hours (under 5 h) of having been captured or slaugthered. The cod were eviscerated and washed, and chops of about 3 cm in width were cut and immediately frozen and stored at −80 °C. For protein extraction, the chops were placed in a

Analyses of wild and farmed cod

Fig. 1 shows the 1D SDS–PAGE analyses of the Tris (Fig. 1a) and CHAPS–urea (Fig. 1b) extracts from farmed and wild cod. The bands corresponding to the myosin heavy chain, actin, tropomyosin and fast myosin light chain types 1, 2 and 3, which have been identified by electrophoretic analyses of purified native myosin and actomyosin in previous works (Martinez et al., 1990, Martinez et al., 1991, Martinez et al., 1992, Martinez et al., 1993) are indicated in the figure.

Some of the differences

Conclusions

The protein patterns of wild and farmed cod seemed to indicate that farmed cod muscle had a different protein expression and/or different post mortem degradation pattern than wild cod. This may be due to stress during cultivation, to differences in post mortem muscle conditions (for example pH, that is altered by feed intake at around the time of death), and/or to qualitative and quantitative differences in the expression or regulation of proteases with a role in post mortem muscle

Acknowledgement

The financial support of the Norwegian Research Council (Project 154 137/130) is gratefully acknowledged.

References (37)

  • G.B. Olsson et al.

    Post mortem structural characteristics and water-holding capacity in Atlantic halibut muscle

    LWT- Food Science and Technology

    (2003)
  • H. Wergedahl et al.

    Fish protein hydrolysate reduces plasma total cholesterol, increases the proportion of HDL cholesterol, and lowers acyl-CoA: cholesterol acyltransferase activity in liver of Zucker rats

    Journal of Nutrition

    (2004)
  • C.W. Anderson et al.

    Processing of adenovirus 2-induced proteins

    Journal of Virology

    (1973)
  • J.F. Ang et al.

    Denaturation of cod myosin during freezing after modification with formaldehyde

    Journal of Food Science

    (1989)
  • W. Ansorge

    Fast visualization of protein bands by impregnation in potassium permanganate and silver nitrate

  • M. Aursand et al.

    Quantitative high resolution 13C and 1H nuclear magnetic resonance of omega−3 fatty acids from white muscle of Atlantic salmon (Salmo salar)

    Journal of the Americal Oil Chemists Society

    (1994)
  • Bio-Rad., (2001). In D. Garfin & L. Heerdet (Eds.), 2-D Electrophoresis for proteomics: A methods and product manual....
  • E. Carpené et al.

    Biochemical differences in lateral muscle of wild and farmed gilthead sea bream (series Sparus aurata L.)

    Fish Physiology and Biochemistry

    (1998)
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