Scientific Alert Note

New understanding of how enzymes affect texture of rainbow trout

Picture 1 - Soft trout

Enzymatic processes in fish muscles are responsible for softening the flesh. Understanding the reactions involved in this process is crucial for producing seafood with high textural quality. A number of different enzymes (proteases) are suspected to be involved in this softening process. In the project LIPIDTEXT we have discovered how these proteases account for the deterioration of rainbow trout muscle texture. This work has been conducted by Helene Godiksen, Grethe Hyldig, Martine Morzel and Flemming Jessen.

 

 

 

  

 

 Author: Flemming Jessen, DTU Aqua, Denmark

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Texture is one of the most important quality attributes of fish and especially the softening of fish muscle is a huge problem. It is well known that a number of different parameters influence the softening. This includes both parameters in the production process such as time and temperature but also the state of the fish’s life before slaughtering has a great impact. In aquaculture we do have the opportunity to control many of the factors (e.g. feed composition, handling, stress and slaughtering method) that individually or combined can lead to a softer texture of the produced fish fillet. The mechanisms of this soft texture development are not fully understood, but many studies have shown that proteins in the fish muscle, both from the muscle cells and from the connective tissue are degraded by proteases during the softening. The proteases that are actually responsible for softening have not been identified yet but several studies have shown that different muscle proteases (particularly from the cathepsin family) might have a role in this alteration process.

 

Two types of investigations have been conducted in this respect, using either model systems or intact muscle. In the model systems purified probes of suspected enzymes are added to muscle extracts and it is then followed how they can degrade the muscle proteins. In this way proteins which are important for the muscle structure (e.g. myosin, troponin, alfa-actinin, tropomyosin, desmin, titin, nebulin, dystrophin and actin) and are therefore also regarded as important to texture are found to be degraded by different proteolytic enzymes. This type of approach demonstrates that a certain protease is able to digest a given protein but it does not prove in any way if this process will actually be relevant in the post mortem muscle. Presence of endogenous inhibitors in the muscle or compartmentalisation (enzyme and substrate are not in contact with each other) are possible reasons for this. In the studies using intact muscle no attempts have been done so far to identify the responsible proteases.

 

Picture 2 - Protein patterns of rainbow trout muscle when analysed by electrophoresis (SDS-PAGE)

In LIPIDTEXT we have developed a concept where protein patterns are studied by electrophoresis (SDS-PAGE) using both intact muscle and model systems.

Using this concept we have tried to find out to what extent the 3 different proteases, cathepsin B, cathepsin L and cathepsin D, all present in rainbow trout muscle, contribute to the softening process. First we have analysed the muscle from fish of different texture by electrophoresis and found proteins whose presence in the muscle relates positively or negatively to the texture. Then we have added purified proteases to muscle extracts from rainbow trout to find out how the different cathepsins influence the texture-related proteins found in the intact muscle. In this way we showed that the cathepsins actually affected many proteins of the muscle. Among those several proteins correlating to texture were affected by either cathepsin B, cathepsin D or both. Although cathepsin L affected many proteins in the muscle only few of the texture-related were among them and this protease therefore it only seems to have a minor influence on the texture of rainbow trout filets.

We could conclude that the balance between the activities of cathepsin B and cathepsin D was important for the textural appearance. Dominance of cathepsin B seemed to preserve texture better than dominance of cathepsin D which led to softening.