A sequential design approach for the extraction of bioactive compounds from Callinectes spp. waste using NADES

The consumption of swimming crabs (Callinectes spp.) generates significant amounts of waste, containing valuable bioactive compounds, such as astaxanthin — a keto-carotenoid with high antioxidant potential — that can be extracted using natural deep eutectic solvents (NADES). These solvents are a promising alternative due to their low toxicity, biodegradability and high extraction efficiency. The aim of this study was to optimize the extraction of bioactive compounds from swimming crab residue using experimental design tools. The swimming crabs were collected in Ubatuba, São Paulo, by the Laboratory of Bioecology and Systematics of Crustaceans at the FFCLRP/USP. The effect of solvent type (ethanol, isopropanol, choline chloride:lactic acid (ChCl:LA, 1:2M) and choline chloride:glycerol  (ChCl:GL, 1:2M)) and extraction method (magnetic stirring (752, Fisaton, Brasil), ultrasonic bath (USC-1400, Unique, Brazil), and heating bath (MA-184, Marconi, Brazil) at 45°C with magnetic stirring) on the content of phenolic compounds (TP), carotenoids (TC) and antioxidant activity was initially evaluated. All the extractions were performed for 45 minutes. A Rotational Central Composite Design (RCCD 2³) was subsequently performed to evaluate the effect of extraction time (19.6–140.4 minutes), temperature (23.2–56.8 °C), and the molar ratio of the ChCl:LA solvent (1:1–1:5 M) on the TP, TC and antioxidant activity of the extracts. The response surface methodology was employed to optimize the extraction process. TP of the extracts was determined using the Folin-Ciocalteu method¹. TC was determined by directly reading the extracts in a spectrophotometer² (600-Plus, Femto, Brazil) at 450 nm and antioxidant activity was determined using ABTS assay³. Statistical analyses were performed using the Statistica 14.0.1 software, at a 95% confidence level. The swimming crab residue extracts had a TP, TC and antioxidant activity ranging from 31.53 to 481.45 µg GAE/g, 41.89 to 3,053.13 µg ASX/g and 1,239.53 to 9,134.57 µM TE/g, respectively. Extraction using a heating bath with magnetic stirring in ChCl:LA solvent yielded extracts containing the highest concentration of bioactive compounds. RCCD matrix allowed obtaining extracts with TP, TC and antioxidant activity ranging from 80.60–1,781.32 µg GAE/g, 1,566.75–3,249.43 µg ASX/g and 5,024.07–11,077.55 µM TE/g, respectively. The extraction optimal condition was 140.4 minutes, 56.8 °C, and a solvent ratio of 1:5 M. This condition enabled an extract with a high TC (2,621.37 µg ASX/g), TP (1,883.68 µg GAE/g) and antioxidant activity (11,790.83 µM TE/g) to be obtained. This extract can be used in food, cosmetics and active packaging, according to sustainability and bioeconomy ideals.