Supercritical Extraction Technology of Obtaining Polyunsaturated Acids from Starfish (Lysastrosoma anthosticta Fisher, 1922)

Introduction. Starfish (Asteroidea) are marine echinoderms with more than 160 species. Starfish are a valuable source of protein and fats. The present research featured the chemical composition of starfish, which can be used as a commercial source of lipids. Study objects and methods. The study defined the optimal parameters for extracting the lipid fraction of Lysastrosoma anthosticta with supercritical carbon dioxide, as well as the qualitative composition of the obtained extracts. Results and discussion. The yield of fatty acids obtained with supercritical carbon dioxide co-solvent was 1.8 times higher than that obtained with standard extraction according to the Folch method. The content of impurities was lower than in the samples with chloroform-methanol system. The polyunsaturated fatty acids isolated from L. anthosticta mainly belonged to ω-3 (18.0%), ω-6 (11.7%), ω-7 (21.2%), ω-9 (10.1%), and ω-11 (6.5%). The rest was saturated fatty acids, mainly palmitic (14%) and myristic (6%). The qualitative composition of the lipid fraction did not depend significantly from the isolation method. However, the supercritical extraction increased the product yield, extraction rate, and the quality of the extraction residue. Supercritical carbon dioxide left a dry residue, which had no typical smell and was brittle enough for grinding. Such residue can presumably be used to produce protein concentrate. Conclusion. Supercritical extraction with chloroform can be recommended to isolate fatty acids from marine organisms at 60°C and 400 bar.


Introduction
The population of the earth is growing every year, which makes technologies for food obtaining and processing very important for humanity. Efficient processing technologies produce more useful products while doing less harm to the environment. The oceans are the least explored part of the earth. Every year, dozens of new compounds are isolated from marine aquatic organisms around the world. Many of them possess various beneficial biological properties that can be used, for instance, in pharmacology. Every year, new secondary metabolites of great practical and fundamental interest are extracted from echinoderms.
Starfish (Asteroidea) are widespread marine echinoderms of more than 160 species. Starfish are predators that damage shellfish plantations and coral reefs. In the XX-XXI centuries, world fisheries have been busy looking for new sources of nutrients, especially for marine carriers of biologically active substances that can be used to obtain highly effective medicines [1]. The present research featured Lysastrosoma anthosticta; the research objective was to select a promising method of supercritical extraction.
Supercritical fluid extraction and supercritical fluid chromatography have been used since the late 1970s in food analysis for determining lipids and toxicants. Supercritical fluid extraction is an effective means of natural product extraction. The supercritical extraction process has potential advantages over conventional extraction processes, such as shorter extraction time, reduced organic solvent volume, and more selective extraction [2].

Study objects and methods
Samples of Lysastrosoma anthosticta Fisher, 1922 were harvested in the Peter the Great Bay (Russia, Sea of Japan) in 2020. Starfish with a total weight of 42 000 g were gutted, cut with scissors into small pieces (about 1 cm long), and stored in a plastic bag at -70°C. Frozen samples were lyophilized and crushed. The dry weight was 3630 g. After that, 30 g of aliquots was used for extraction. All experiments were done in triplicates. Extraction with supercritical CO 2 was performed using THAR SFC 500 (USA). Figure 1 shows the technological scheme of the supercritical extraction unit. Extraction was carried out using supercritical CO 2 : pressure -200, 400, and 600 bar per square inch; t -30, 40, 50, 60, and 70°C; flow rate -20 g/min. At the second step, the experiment extraction was carried out using CO 2 and 5% solvent (chloroform): pressure -200, 400, and 600 bar per square inch; t -30, 40, 50, 60, and 70°C; flow rate -20 g/min. The control extraction of 30 g of dried starfish was carried out using the Folch method with a mix of chloroform-methanol (2:1) at the rate of 20 parts of the extraction mixture per one part of the tissue at 30, 40, 50, 60, and 70°C [3].
The extracts were analyzed by high-performance liquid chromatography (HPLC) with tandem mass spectrometry

Results and discussion
An analysis of yields of lipids fraction from Lysastrosoma anthosticta under various conditions showed that the extraction with supercritical carbon dioxide with no co-solvent was less effective than the standard extraction according to the Folch method (Fig. 3). However, when extra 5% chloroform was added to the extraction system as a co-solvent, it significantly increased the yields of the total fatty acid fraction. The choice of the solvent and the extraction range parameters was based on the results of previous works where starfish material was treated with a plant matrix [4,5]. Despite the fact that the main target components are soluble in liquid CO 2 only above 200 bar, it is possible to separate significant amounts of the lipid fraction [6,7]. The lipid fraction obtained by supercritical extraction with chloroform was 1.8 times higher than in the standard Folch method. The extraction was found to be quite effective only when a co-solvent was used. The optimal parameters for extraction with a co-solvent included a temperature of 60°C and a pressure of 400 bar. The data obtained correlated with the most frequently selected parameters. Most often, when using this technology, the authors chose a pressure of 300-350 bar as the optimal one [8][9][10][11]. With these parameters, a good yield of lipids can be obtained as quickly as possible; a further increase in the temperature and extraction pressure did not lead to a significant increase in the yield.
Obtaining chemical profiles is an extremely important result for any biological analysis system. In this work, we used the HPLC-ESI-MS/MS method with additional ionization and analysis of fragmented ions. High accuracy mass spectrometric data were recorded on an ion trap amaZon SL BRUKER DALTONIKS equipped with an ESI source in the mode of negative and positive ions. The experiment used a four-stage ion separation mode (MS/MS mode). A qualitative analysis showed that the ratio of polyunsaturated to saturated fatty acids did not depend on the extraction method (Table 1). An analysis of polyunsaturated fatty acids isolated from L. anthosticta showed that they mainly belonged to ω-3 (18.0%), ω-6 (11.7%), ω-7 (21.2%), ω-9 (10.1%), and ω-11 (6.5%). The rest was saturated fatty acids, mainly palmitic (up to 14%) and myristic (up to 6%). The predominance of polyunsaturated acids is typical of starfish [12,13]. A review of scientific publications showed that starfish can be considered as a valuable source of feed additives for agricultural animals and birds. For instance, Danish compound feed producers are considering the possibility of industrial use of starfish to produce additives for compound feeds. According to recent studies, such compound feed can reduce the excretion of nitrogen in pigs. Scientists from the Center for Aquatic Animals in Denmark proved that starfish can be an effective alternative to traditional sources of feed protein, e.g. soybeans. Starfish meal could replace the most commonly used protein sources and increase the weight gain in piglets. In fact, starfish-based animal feed may be more economically rational than traditional protein sources. In addition, starfish are being rigorously tested as a possible protein source for bird nutrition. If the results are confirmed, starfish will become a rich source of protein, which will create high demand from egg producers, who are constantly searching for new sources of protein [14].

Conclusion
Starfish are a valuable raw material for protein and lipid production. The chemical composition of starfish makes it possible to use it in food and feed industry. The content of proteins was 9.5-14.0%, lipids -0.5-3.5%, minerals -1.5-32.0%. In comparison with the integumentary tissue, the internal organs of starfish have a higher content of potassium and iron [15,16].
The yields of fatty acids obtained under conditions of supercritical carbon dioxide co-solvent were 1.8 times higher than those obtained with the standard Folch method, while the content of impurities was lower than when the extraction was performed using a chloroformmethanol system. The analysis of polyunsaturated fatty acids isolated from Lysastrosoma anthosticta showed that they mainly belonged to ω-3 (18.0%), ω-6 (11.7%), ω-7 (21.2%), ω-9 (10.1%), and ω-11 (6.5%). The rest was saturated fatty acids: palmitic (14%) and myristic (6%). Thus, isolating fatty acids from marine organisms using supercritical extraction with chloroform can be recommended as an effective commercial method.
In addition, supercritical carbon dioxide with a solvent left a dry residue, brittle enough for further grinding and without typical smell. Such a residue can presumably be used to produce protein concentrate.

Contribution
All the authors contributed equally to the study and bear equal responsibility for information published in this article.

Conflict of interest
The authors declare that there is no conflict of interests regarding the publication of this article.