Fish fillet proteolysis is a complex process that involves various biochemical and structural changes post-mortem, along with microbial activity. These changes can result in a decline in fish fillet quality and shelf life, often manifested as muscle softening and the creation of gaps in the myocommata
. Proteases responsible for muscle deterioration, namely, calpains, cathepsins and metalloproteases, can originate from both muscle tissue and the digestive system, and their activity is influenced by factors such as stress, temperature, and handling during harvesting and transportation
. In Mediterranean aquaculture, species such as Sparus aurata , Dicentrarchus labrax , and Pagrus major are harvested using ice slurry that can c ause stress on fish. This stress can exacerbate proteolytic cleavage and muscle softening, leading to further degradation of fillet quality
. The need for a more humane method of fish harvesting has led to the application of electro-stunning , suggested by both EFSA and OIE. However, before proceeding with the wide application of e lectro-stunning in Mediterranean marine fish farming, it is necessary to ensure that fish fillet quality is not compromised
. In this direction, the effects of electro-stunning on fish fillet proteolysis and flesh quality are investigated in the above Mediterranean farmed fish species , the gilthead sea bream, European seabass and the red seabream.
Materials and methods
The fish were harvested at the same fish farm in Astakos , Aitoloakarnania , Greece, during three different temperature periods: Warm (August, 25°C), Moderate (June, 21°C), and Cold (February/March, 15.5°C). Three harvest methods were applied: the conventional method of ice slurry and two electro-stunning settings of higher and lower voltage , with the same flow rate. White muscle samples were excised from each fish on slaughter day (Day 0) and on days 1, 2, 5, 7, and 13 post-harvest and were snap-frozen in liquid nitrogen. The activity of Calpain, Collagenase, Cathepsin B, and L was assayed using the Barrett and Kirschke method with minor refinements, and protein content was quantified using the Bradford method. Activity was expressed as fluorescence units change per minute per mg protein. Additionally, white muscle samples at harvest day (Day 0) and on days 7 and 13 post-harvest underwent histological analysis to assess flesh quality
The post-mortem activity of proteolytic enzymes in European seabass, gilthead seabream, and red seabream at different water temperatures and harvest methods was investigated. The results showed that calpain and collagenase activities were activated early post-mortem, and species-specific variations were observed in enzyme activity levels. Cytoplasmic calpains had the highest average activity among proteolytic enzymes, while collagenase activity shared a similar temporal pattern with calpain. Cathepsin B and L also showed a positively correlated activation regardless of the harvest method. Changes in muscle histology caused by the action of proteolytic enzymes can lead to flesh softening and loss of texture. The myofibrils, which make up most of the muscle fiber volume, are particularly vulnerable to degradation by endogenous proteases
. In our study, we observed an increase in the average single fiber volume density between days 0 and 7, irrespective of the harvest method used, though this increase was significant in electro-stunned groups. The temperature had a significant effect on enzyme activity and the histological phenotype, with the highest activities observed in the moderate temperature period for all species. Harvest method had a milder effect than the water temperature on enzymatic activities and the histological phenotype.
Discussion and conclusion
In the present study the effect of electro-stunning as a harvest method was investigated on the proteolytic activity and flesh quality of three Mediterranean farmed fish species. It is known that electro-stunning can have an impact on pre-slaughter stress and pH levels, which in turn, may affect the activation of proteolytic enzymes
. The observed variations in enzyme activity among different species of fish, water temperatures, and harvest methods can be attributed to differences in muscle composition, physiological status, and post-mortem metabolism. Previous studies have suggested that this increase may be due to osmotic phenomena resulting from changes in intracellular membrane permeability and disruptions in ion balance in the cytoplasm
These findings suggest that water temperature, species and harvest method have an impact in the histological phenotype due to protease activity and they highlight the importance of developing species-specific humane post-harvest strategies to effectively preserve fish fillet quality ensuring animal welfare.
This research was co-financed by Greece and the European Union, the European Maritime and Fisheries Fund, in the context of the implementation of the Greek Operational Programme for Fisheries, Priority Axis “Innovation in Aquaculture”, project title “Development and industrial scale evaluation of an innovative humane slaughter system and assessment of welfare in aquaculture marine fish species” MIS 5010690.