Introduction
With the ban on ethoxyquin and concerns about the use BHT and BHA in the European Union, novel alternatives of antioxidants for aquaculture have become an issue. The most promising replacements for these synthetic antioxidants are natural antioxidants primarily present in plants. A source of high amounts of natural antioxidants can be found within by-products from the olive-mill and winery industries, with are prominent in the EU. However, antioxidants bioavailability may be compromised due to the lignocellulosic matrix of these by-products. Solid-state fermentation (SSF) is a sustainable bioprocess that can utilize agro-industrial by-products as a substrate for microbial growth in low water activity conditions, producing enzymes that hydrolyze the cell wall and release antioxidants into the substrate. After SSF, these important bioactive compounds are recovered with different applications, including feed additives for aquaculture. Dietary fortification with antioxidants can be beneficial in reducing fish’s oxidative stress status and delaying lipid peroxidation. This work aimed to study the antioxidant potential of extracts from olive mill and winery by-products, unfermented or fermented, in European Sea Bass.
Materials and methods
An optimized mixture of olive mill and winery wastes (Filipe et al., 2019) was fermented by SSF with Aspergillus ibericus (MUM-01.29; Micoteca od the University of Minho (UM). After fermentation, an aqueous extraction was performed (1:5 solid/water, w/v) for 30 minutes with constant stirring. Another extract was also prepared from the same mixture but without the fermentation process. The recovered extracts were then lyophilized (FWO and UWO, fermented and unfermented extracts, respectively). Four isolipidic and isoproteic diets (18% lipids, 50% protein) were formulated with 0 (control diet), 0.34, and 0.68% of FWO or with 0.19% of UWO to reach an antioxidant activity of 0 (control diet), 683 (FWO1 diet), 1365 (FWO2 diet) and 683 (UWO diet) µmol Trolox kg-1 of diet. A growth trial with European sea bass (IBW= 21.5g) testing each experimental diet in triplicate was performed for 66 days. At the end, the liver and intestine of 9 fish from each diet were sampled, immediately frozen in liquid nitrogen, and stored at -80º C. Samples were then homogenized in Tris–HCl buffer at a pH of 7.8, centrifuged at 30,000×g for 30 min at 4 °C and supernatants were recovered. Superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and thiobarbituric acid reactive substances were analyzed in both tissues.
Results and discussion:
In the liver (Table 1), there is a general decrease in specific oxidative stress-related enzyme activity without affecting liver peroxidation with the dietary supplementation with FWO or UWO extracts. Antioxidant compounds present in these diets may have had a direct action on preventing or neutralizing the reactive oxygen species (ROS) production, modulating the activation of oxidative stress enzymes.
In the intestine (Table 2), a general increase in specific oxidative stress-related enzyme activity, together with a decrease in lipid peroxidation, was observed with the dietary inclusion of FWO1 or UWO extract. Besides their direct ROS scavenging activity, dietary inclusion of FWO1 and UWO extracts also activated the antioxidant enzymes system. Compared to the FWO1 diet, the FWO2 diet induced lower activity of the antioxidant enzymes and increased intestinal lipid peroxidation.
Conclusion
Present results showed that dietary inclusion of FWO and UWO extracts at a level corresponding to the antioxidant activity of 683 µmol Trolox kg-1 diet improved the intestine’s antioxidant status, increasing its ability to cope with ROS production and decreasing the lipid peroxidation. In the liver, these extracts reduced the overall antioxidant enzymes activity without compromising the hepatic peroxidation
Acknowledgements: This work was supported by the Ocean3R project (ref NORTE-01-0145-FEDER-000064), founded by the Northern Regional Operational Programme (NORTE2020) through the European Regional Development Fund (ERDF).
Bibliography: Filipe, D., Fernandes, H., Castro, C., Peres, H., Oliva‐Teles, A., Belo, I., & Salgado, J. M. (2019). Biofuels, Bioproducts and Biorefining, 14(1), 78–91. https://doi.org/10.1002/bbb.2073