Comparison of Astaxanthin Pigment Extraction from Shrimp Shells and Gammarus Crustacean Using Microwave and Soaking in Ionic Liquid Microemulsion in Water

Background: The diversity and anticancer properties of carotenoid pigments have attracted much attention. Carotenoids are yellow and red pigments found in bacteria, fungi, plants, and animals (Fidor and Borda, 2014). Carotenoids inactivate the initiators of harmful chemical reactions, such as free radicals. On the other hand, due to their strong antioxidant properties, they prevent the oxidation of unsaturated fatty acids and have shown important functions in the pharmaceutical, food, and cosmetic industries (Sachindra and Mahendrakar, 2005). Many studies have been conducted on the extraction of carotenoid pigments from crustaceans such as crabs and shrimps (Parjiklai, Al-Houri, Ferte, & Christensen, 2015; Soumya & Sachindra, 2015), of which astaxanthin and its esters are the most abundant (Sachindra, Bhaskar, & Mahendrakar, 2006). Among the important shrimp species, banana shrimp is the second most important commercial species found in Hormozgan waters. Among the crustaceans, Gammarus is an apodous crustacean and an important commercial species in some countries and has a high concentration of carotenoids, protein, various types of enzymes, and essential unsaturated fatty acids. This crustacean plays an important role in cleaning the aquatic environment of fish such as salmon, and their presence in the fish diet increases the rate of digestion and absorption of food, resulting in increased growth performance (Escobar-Lux, Parsons, Samuelson, & Agnalt, 2020). In recent studies, this Atlantic crustacean has been studied and investigated as a rich source of carotenoid pigments (Namati, Shokri, & Pazouki, 2015). There are various methods for extracting pigments, including chemical methods (Hooshmand, Shabanpour, Mousavinasab, & Golmakani, 2017), microbial methods (Das et al., 2007), and the use of enzymatic digestion by proteolytic enzymes (N-Sachindra & Mahendrakar, 2011). Each of the mentioned extraction methods has advantages and disadvantages. The conventional soaking method for the extraction of carotenoid pigments is usually time-consuming and involves high cost and large volumes of solvent. Also, in most cases, these methods have lower efficiency compared to new methods such as microwave-assisted extraction and ultrasound (Li, Fabiano-Tixir, Tomao, Krautow, & Chamet, 2013). Microwave-assisted extraction is based on the absorption of microwave energy by polar molecules of chemical compounds (Rotary and Orsat, 2012). In recent years, in order to reduce the effects of volatile and toxic solvents, hydrophilic ionic liquids have been used as dispersing solvents and hydrophobic ionic liquids as extracting solvents. Ionic liquids are a salt in liquid form that is considered among green solvents due to its tunable physicochemical properties, high chemical and thermal stability, and negligible vapor pressure at room temperature, and has a molecular structure consisting of different cations and anions (Khu et al., 2019). However, the viscosity of most ionic liquids is higher than that of organic solvents, which results in a decrease in the mass transfer rate. Microemulsion is a promising method that enables selective extraction of biomolecules in the food and chemical industries (Amiri-Rigi and Abbasi, 2019). Ionic liquid microemulsion provides a suitable environment for the release and extraction of astaxanthin due to its adjustable polarity, low surface tension and ability to solubilize hydrophobic compounds. Also, its high stability and ability to penetrate the biological matrix increase the extraction efficiency compared to traditional solvents. To date, microemulsions have been used to extract proteins, pigments and trace elements. Since a high percentage of aquatic waste consists of wastes that contain many value-added compounds, their extraction not only contributes to the economic prosperity of the fisheries industry but also contributes significantly to environmental protection. Gao et al. (2020) extracted astaxanthin from shrimp waste using ultrasound and ionic liquid microemulsion. The microemulsion containing tributyl octylphosphonium bromide significantly increased the extraction of astaxanthin due to stronger electrostatic interactions and hydrogen bonding (Gao et al., 2020). In this regard, Nunes et al. (2021) extracted astaxanthin as a carotenoid with high antioxidant capacity from crab exoskeleton waste for use in food products. This extraction involved a microwave pretreatment step (with hydroalcoholic solvents with 0-50% water by volume at different temperatures from 40 to 140°C) and supercritical fluid extraction (at a pressure of 200-500 bar, a temperature of 40-60°C, and an ethanol content of 8-13% by weight). The extracted astaxanthin content was reported to be 12 times higher than that of the traditional Soxhlet extraction method, indicating that this proposed method significantly improves the extraction efficiency (Nunez et al., 2021).

Objective: The aim of this study was to compare astaxanthin extraction from shrimp (Fenneropenaeus merguiensis) and gammarus crustacean (Pontogammarus maeoticus) using soaking and microwave-assisted methods in the presence of ionic liquid microemulsion.

Methods: To extract astaxanthin from lyophilized powder of banana and hard shell shrimp (Gammarus spp.) skin, the traditional method of soaking in a microemulsion solvent of ionic liquid in water (5:1, v/v) at ambient temperature for 24 hours was used. Microwave extraction was also performed under pre-optimized conditions with a frequency of 2.45 GHz in a 5-fold solvent to sample ratio, a power of 100 W, and a time of 91.81 seconds (Faizi et al., 2025). After extraction, the obtained extract was diluted with ethanol and then filtered with a 0.45 μm syringe filter to prepare for analysis (Fan et al., 2019). Next, in order to compare the extraction of astaxanthin using two methods, soaking and microwave, tests were used for the amount of total carotenoids, astaxanthin, recovery percentage, and antioxidant properties.

Results: According to the results, the highest yield of astaxanthin was obtained from shrimp (80.39 ± 1.09 µg/ml) using the microwave method. Therefore, shrimp was identified as a better source of astaxanthin compared to gammarus. Furthermore, the recovery percentage and total carotenoid content for shrimp were 93% and 59%, and 83.54 ± 0.56 ml/g and 77.98 ± 1.33 ml/g for the microwave and soaking methods, respectively, indicating the superiority of the microwave-assisted extraction. In addition, the antioxidant activity of astaxanthin extracted by the soaking method was higher than that of the microwave method. Compared to the synthetic antioxidant BHT, the antioxidant activity of astaxanthin was always greater, and increased with concentration.

Conclusion: Crustacea waste can be used as the cheapest raw material for the extraction of carotenoid pigments. On the other hand, considering that the usual methods of carotenoid extraction are time-consuming and require a lot of solvent, the use of modern extraction methods such as microwave has become common today. Overall, based on the findings of this study, shrimp waste can be considered a suitable and effective source for astaxanthin extraction using microwave-assisted methods.