A rational approach for estimating antioxidant capacity: from dietary fish formulations to bioreactor water samples

Abstract

Concern about the harmful properties of synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), or ethoxyquin, in fish feed has led to the banning of ethoxyquin by the European Union (EU) in 2017 and an increased interest in antioxidants from natural sources. Hundreds of natural antioxidants are found in plants, including polyphenols, and there is therefore a need for proposing and validating efficient analytical methods to establish their antioxidant capacity (AC) and consequently, the best ingredients for fish feed.

The four primary tasks of this thesis were i) to select/propose a suitable assay to be established at the Institute of Marine Research (IMR) as the in-house method for monitoring the AC of different feed and ingredients used in fish trials; ii) to validate the selected AC assay according to the regulations of the accredited organizations, iii) to apply the validated AC assay to aquaculture samples, and iv) to develop a novel approach for determining the AC assay of aquaculture samples based on an already established method.

After a comprehensive evaluation of the existing AC methods, the cupric ion reducing antioxidant capacity (CUPRAC) assay was selected for further validation. However, there were some unexpected and serious experimental features in the original CUPRAC assay (e.g., lack of solubility and replication) that have been overlooked in the current literature and that undermined the assay’s reliability. The drawbacks were overcome by applying multivariate strategies to address the lack of solubility problem and using replicates in all the steps of the study. The analytical characteristics of the method (e.g., concentration range, precision, accuracy, limit of detection and limit of quantification) were validated and the assay implemented to determine the AC of premix fish feed ingredients containing polyphenols from rosemary and olive oil, fish feed at different production stages (e.g., before extrusion, before drying, final feed product) with different inclusion levels of polyphenols (0.00, 0.01, and 0.05%), and to grade water samples from different microalgae industrial bioreactors in terms of their phenolic content. Finally, a novel approach to the traditional CUPRAC assay was proposed to control the quality of the commercial feed ingredients.