The use of natural antioxidants (NAOX) for preventing fish feed oxidation and reducing oxidative stress on fish is gaining popularity in the fish feed sector. Nowadays, NAOX have been proposed as a sustainable alternative to synthetic antioxidants such as the banned ethoxyquin or the highly questioned BHT and BHA. Hence, it is imperative to study the behavior of those compounds with natural antioxidant activity (e.g., polyphenols) during the whole fish feed production (from ingredients to final feed) and fish organs. This project studied the effects of a commercial ingredient containing NAOX from two different sources, added at two levels to a commercial fish feed. Thus, the objective was to analyze quantitatively selected polyphenols in various stages of a fish farming process for salmon. Simple extraction methods for the selected polyphenols were developed for different matrices: antioxidant ingredient , fish feed and fish tissue, and further quantification by LC-MS/MS was conducted, allowing the monitoring of the content of polyphenols from the antioxidant ingredient to the fish tissue. Additionally, for studying the effect of the NAOX in the process, several oxidation and oxidative stress indicators have been measured; in feed ingredients, in feed at different stages of production and storage, and fish fed the two diets, compared to a control diet.
Selected polyphenols from four stages (ingredients, before extrusion, before drying and final feed) of the fish feed production cycle and from salmon organs (liver and muscle) were analyzed (Figure 1.) The main polyphenols quantified in fish feed ingredients, fish diets at different production stages, and fish organs were hydroxytyrosol, carnosol, gallic acid and carnosic acid . Additionally, other related components , presumably 12-O-methyl carnosic acid, rosmanol and rosmarinic acid, were detected and semi-quantified by using the standards that closely resemble their chemical structure. The NAOX’s content was not stable, an important lo ss of polyphenols was evidenced, especially during the extrusion process. However, the analysis of liver and muscle samples, dissected from salmon fed the final feed of the production process, revealed that two polyphenols were transferred into these tissues, namely, carnosic acid, methyl carnosic acid.
Interestingly, and despite of the loss of NAOX, results from oxidative parameters suggest thast d ietary NAOX affected MDA and glutathione metabolism in both the muscle and liver of Atlantic salmon: MDA was lowered already at 0.01% NAOX in the muscle, while 0.05% was necessary to significantly lower liver MDA. This could indicate that dietary NAOX prevented in vivo lipid oxidation in the fish tissues.GSH and GSSG were lowered in the liver while Eh was constant. In the muscle, GSH increased while GSSG was constant, leading to a more reduced Eh. This shows that redox metabolism was affected by dietary NAOX. No effects of the dietary NAOX on growth, condition factor, body indices or cataract were detected. The results obtained were promising and further studies are needed for advancing in the understanding of NAOX in redox biology of fish.