Oil Extraction from Skipjack Tuna Belly (Katsuwonus pelamis) via Thermomechanical and Supercritical CO₂ Fluid Extraction

Abstract

This study aimed to extract oil from skipjack tuna belly (Katsuwonus pelamis) using thermomechanical extraction and supercritical CO₂ extraction (SFE-CO₂). The SFE-CO₂ process was conducted at 25 MPa and 40 °C for 30 min, 1 h, and 3 h. Thermomechanical extraction yielded 88.1% crude oil, with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contents of 7.6% and 19.2%, respectively. In the SFE-CO₂ process, the shortest extraction time (25 MPa, 40 °C, 30 min) resulted in the highest DHA (12.6%) concentration, as well as a total polyunsaturated fatty acid (PUFA) content of 22.4%. This behavior is attributed to the shorter CO₂–matrix contact time, which favored the selective extraction of target compounds. Conversely, extending the extraction time to 3 h under the same pressure and temperature led to the highest overall yield (26.4%). These findings demonstrate that SFE-CO₂ is a promising green technology for the valorization of fish processing by-products, enabling selective recovery of high-value omega-3 fatty acids while promoting more sustainable production practices.

1. Introduction

According to the Food and Agriculture Organization of the United Nations (FAO), global fish production is estimated to reach 204 million tons by 2030 [1]. Conversely, there has been an increase in waste generation by the fish processing industry, in which approximately 52% of the raw material is discarded in the form of heads, bellies, bones, and viscera [2,3,4,5]. These by-products, however, have high lipid content, making them potential sources for fish oil extraction [6,7,8].

Fish oil is valued for its high concentration of omega-3 polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) [9]. These compounds are widely recognized for their health benefits, including the prevention of cardiovascular and neurodegenerative diseases, the reduction in LDL cholesterol levels, and assistance in treating inflammatory conditions [10,11]. Among the marine species with potential for obtaining this oil, skipjack tuna (Katsuwonus pelamis) stands out, as it is widely distributed in the tropical and subtropical waters of all oceans [12,13,14]. This species has a high reproductive rate and is considered one of the most fished species in the world, although it is smaller than other tuna species, reaching up to 1 m in length and about 20 kg [15].

The most used technique for producing crude fish oil is the thermomechanical extraction method that involves the application of heat and mechanical forces to release oil from fish tissues or by-products [16]. This method is considered environmentally friendly and cost-effective compared to solvent-based techniques, as it does not require chemical solvents and utilizes physical processes to separate oil from other fish components [17]. However, this method employs high temperatures, which may compromise the quality of sensitive compounds [18,19].

In this context, alternative extraction methods have been applied to recover lipid and bioactive compounds from marine species, including ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE), and pressurized liquid extraction (PLE), all of which are widely reported in the literature [20,21,22]. UAE and MAE are characterized by reduced extraction time and lower energy consumption; however, both techniques may lead to the degradation of thermolabile compounds and exhibit limited selectivity [23,24]. EAE operates under milder conditions and typically employs water as a solvent, but its application can be constrained by the high cost of enzymes and challenges associated with industrial scale-up [22,25]. In turn, PLE enhances extraction efficiency and reduces solvent consumption, although it still relies on organic solvents and requires strict control of temperature and pressure conditions [22].

In comparison with these techniques, supercritical fluid extraction using carbon dioxide (SFE-CO₂) stands out due to its higher selectivity, tunable solvating power, and ability to operate at relatively low critical conditions (31 °C and 73 atm), thereby minimizing the thermal degradation of sensitive compounds [26]. Furthermore, supercritical CO₂ exhibits unique physicochemical properties, such as low viscosity and high diffusivity, which enhance mass transfer and contribute to improved extraction efficiency [27,28]. Another important advantage is the easy removal of the solvent by depressurization, resulting in solvent-free extracts with higher purity [24]. Additionally, CO₂ is non-toxic and can be recycled, reinforcing the sustainability of the process. However, SFE-CO₂ also presents some limitations, including high equipment costs, system complexity, and lower efficiency for extracting polar compounds in the absence of co-solvents [29]. Nevertheless, its ability to produce high-quality extracts with enhanced oxidative stability [30,31] makes this technology particularly attractive for high-value applications, such as omega-3-rich fish oil. In addition, the solvent-free nature of the extracts, combined with the possibility of CO₂ recycling and process scalability, highlights the strong potential of SFE-CO₂ for industrial and commercial applications in the food and nutraceutical sectors [32].

Although supercritical CO₂ extraction (SFE-CO₂) has been successfully applied to recover fish oils from different by-products, such as catfish and tuna heads [13,33], sardine and shrimp waste [30,34], and mackerel skin [30], its application to skipjack tuna (Katsuwonus pelamis) belly has not yet been reported in the scientific literature. Therefore, the behavior of this specific matrix under SFE-CO₂ conditions remains largely unexplored. Moreover, the influence of SFE on oil yield, physicochemical quality, and fatty acid selectivity in this specific lipid-rich matrix remains unclear. Therefore, this study aimed to evaluate and compare thermomechanical extraction and SFE-CO₂ for oil recovery from skipjack tuna belly. The extracted oils were characterized in terms of yield, physicochemical quality parameters, and fatty acid profile under SFE-CO₂ operating conditions of 25 MPa and 40 °C at extraction times of 30 min, 1 h, and 3 h, in order to assess the selectivity and potential of this green technology for the valorization of tuna processing residues.

2. Materials and Methods

The raw material used was skipjack tuna belly (Katsuwonus pelamis), obtained from fish processing at a local industry in Rio Grande, RS, Brazil, and subsequently stored at −20 °C until use.

2.1. Raw Material and Oil Characterizations

The moisture content of the raw material was determined according to the AOAC (1995) methodology [35], and lipid content by Bligh and Dyer [36]. The crude and bleached oils were characterized according to the standard methods of the American Oil Chemists’ Society (AOCS) [37], including free fatty acid content expressed as oleic acid (FFA, method Ca 5a-40), peroxide value (PV, method Cd 8-53), and p-anisidine value (AnV, method Cd 18-90). To evaluate the primary and secondary oxidation products, the TOTOX value (total oxidation state of the oil) was calculated as TOTOX = 2 PV + AnV. The saponification value (SV) and iodine value (IV) of the crude and bleached oils were determined by nuclear magnetic resonance (NMR).

The oils extracted by supercritical extraction (SFE-CO₂) were characterized according to the methodology described by Carneiro et al. [38], evaluating the saponification value, iodine value, free fatty acid content, and oxidation state (Roa), calculated from the integration of the NMR spectrum peaks and application in the respective equations. The samples were analyzed by nuclear magnetic resonance (NMR) (Bruker High Field, model 400 MHz Ascend, Rheinstetten, Germany), using a 9.4 T magnet corresponding to 400 MHz for ¹H, with a 5 mm diameter probe. For obtaining the NMR spectra, 20 mg of each lipid was dissolved in 0.7 mL of CDCl₃ and placed into the equipment, using a standard ¹H sequence consisting of a 90° pulse, with an acquisition time of 9109 s, 2000 scans, and a spectral width of 24.03 kHz.

The methodology proposed by Crexi et al. [39] was used to determine the fatty acid profiles of the oil samples. Methyl esters were prepared so that the samples could then be injected into a gas chromatography system (GC-FID) (Shimadzu, GCMS 2010 Plus, Tokyo, Japan), which used a 100 m silica capillary column with a 0.25 mm diameter. The fatty acids were quantified and identified by peak integration and comparison of the retention times of the samples with those of the fatty acid methyl ester standards (Supelco 37 Component FAME MIX—Sigma-Aldrich Products, St. Louis, MO, USA).

2.2. Tuna Oil by the Thermomechanical Extraction and Chemical Refining

Figure 1 presents the experimental procedure to obtain the refined oil by thermomechanical extraction. The crude oil extraction involves the steps of grinding, cooking, pressing, and centrifugation [39,40]. For this procedure, 10 kg of skipjack tuna belly were used, previously thawed at room temperature and ground with the aid of a meat grinder. The ground material was then subjected to a cooking step in a steam-heated autoclave-type vessel at temperatures between 95 and 100 °C for 30 min. During this stage, the material was continuously agitated, and temperature was monitored at different points to ensure uniform heating throughout the mass. The use of steam heating enabled homogeneous heat transfer, even at this processing scale. After this process, screening was performed using a Tyler no. 14 sieve, followed by hydraulic pressing to obtain the liquid fraction. Finally, the separation of the oil phase was carried out by centrifugation at 7000× g for 20 min. The yield was determined based on the percentage of crude oil recovered in relation to the crude oil content present in the skipjack tuna bellies, as described in Equation (1).

$$\mathrm{\eta }(\%)={\displaystyle \frac{(m_{OBR})}{m_{OBD}}}100$$

(1)

where η is the yield of crude oil extracted (%, ww⁻¹), m_OBR is the mass of oil recovered after extraction (g), and m_OBD is the mass of oil extracted by the Bligh and Dyer method (g).

The crude oil was refined through the steps of degumming, neutralization, washing/drying, and bleaching [39,41,42]. The degumming step was carried out for 30 min, at 80 °C, stirring at 500 rpm, under reduced pressure (6.7 kPa, absolute) and with the addition of 1.0% phosphoric acid solution (85% vv⁻¹). The neutralization step took place for 20 min at 40 °C and stirred at 500 rpm under reduced pressure (6.7 kPa, absolute), with the addition of sodium hydroxide solution (20% ww⁻¹). After each of the previous operations, the samples were centrifuged for 20 min at 7000× g to obtain oil separation. Then, the oil was washed and dried. The bleaching step was carried out at 70 °C and a reduced pressure of 6.7 kPa, absolute, stirred at 40 rpm, and with the addition of 1% adsorbent (95% activated earth mixture and 5% activated carbon, ww⁻¹), for a contact time of 20 min; after, the oil was filtrated in a Büchnner funnel, with a pre-layer of diatomaceous earth (Figure 1). The bleached oil yield was calculated as the ratio between the mass of bleached oil obtained after the refining process and the total extractable lipid content of the raw material determined by the Bligh and Dyer method, as described in Equation (2).

$$\mathrm{\eta }(\%)={\displaystyle \frac{(m_{OBL})}{m_{OBD}}}100$$

(2)

where η is the yield of bleached oil (%, ww⁻¹), m_OBL is the mass of bleached oil obtained after the refining process (g), and m_OBD is the mass of oil determined by the Bligh and Dyer method (g).

2.3. Supercritical Extraction of Skipjack Tuna Bellies

The preparation of skipjack tuna belly samples followed the methodologies described by Da Silva et al. [33] and Kuvendziev et al. [43]. Drying was carried out in a reduced pressure oven at 60 °C and 13.3 kPa, absolute, for 14 h. After drying, the solid was inserted into the extractor. Figure 2 shows the equipment used for the SFE-CO₂.

The SFE-CO₂ conditions applied in this study were based on the optimization previously reported by da Silva et al. [33], who established 25 MPa and 40 °C as the optimal pressure and temperature for fish oil recovery. Using these predefined parameters, the present work focused on evaluating the influence of extraction time (30 min, 1 h, and 3 h) on oil yield (η), fatty acid profile, and the relative contents of EPA and DHA. For each extraction run, 30 g of sample were loaded into the extraction vessel, and the remaining void volume was filled with glass beads to ensure uniform packing and solvent distribution. CO₂ was then introduced into the system, and the temperature was raised to the predetermined value. Once the set conditions were reached, a 30 min stabilization period was allowed to promote adequate contact between the sample and supercritical CO₂ and to ensure homogeneous thermal distribution within the extractor. After stabilization, the extraction proceeded, and the collected extracts were subsequently analyzed. The SFE-CO₂ yield was determined according to Equation (2) and expressed as the ratio between the mass of oil obtained under different SFE-CO₂ conditions and the total lipid content of the raw material, determined by the Bligh and Dyer method.

2.4. Statistical Analysis

All experiments were carried out in duplicate. Analysis of variance (ANOVA) was applied to verify the significance of the experimental factors on the response variables, adopting a significance level of 95% (p ˂ 0.05). Comparisons between the results obtained from the characterization of crude oil, bleached oil, and SFE-CO₂ oil were performed using Tukey’s test for mean comparison, with the Statistica 7.0 software (StatSoft, Tulsa, OK, USA).

3. Results and Discussion

3.1. Raw Material Characterization

The moisture contents of fresh and dried skipjack tuna bellies were 69.4 ± 0.4% and 7.0 ± 0.3%, respectively, while the lipid contents were 5.8 ± 0.5% and 21.2 ± 0.3%, respectively. The increase in lipid content in the dried samples is attributed to a concentration effect resulting from water removal during drying, which proportionally increases the constituents of the dry matter [44]. The reduction in moisture content (in the dried raw material) is important, especially for the SFE-CO₂ process, as the presence of water can hinder extraction and affect the solubility of the oil in supercritical CO₂ [45]. Moreover, the increase in lipid concentration demonstrates the effectiveness of the drying step in concentrating the components of interest, making the raw material more suitable for extraction [46].

The variation observed in moisture and lipid contents, as well as the potential fluctuation of these parameters in fish, highlights the importance of detailed raw material characterization. Factors such as the fish’s age, diet, and environmental conditions directly affect its chemical composition, with particular relevance to the lipid fraction, which is the primary target of this study [47,48].

3.2. Characterization of Skipjack Tuna Belly Oil Extracted via Thermomechanical Process and SFE-CO₂

Table 1. Results of skipjack tuna belly oil characterization via thermomechanical extraction and supercritical fluid extraction (SFE-CO₂).

	Crude Oil *	Bleached Oil *	SFE-CO2 (25 MPa, 40 °C, 30 min) *	SFE-CO2 (25 MPa, 40 °C, 1 h) *	SFE-CO2 (25 MPa, 40 °C, 3 h) *
%FFA	2.81 ± 0.02 a	0.45 ± 0.03 b	0.05 ± 0.01 c	0.06 ± 0.02 c	0.10 ± 0.02 c
IV (gI2 100 g−1)	170 ± 2 a	166 ± 1 a	137 ± 1 b	137 ± 1 b	120 ± 2 c
SV (mgKOH g−1)	183 ± 1 a	187 ± 2 a	168 ± 1 b	166 ± 2 b	178 ± 2 a
Roa	-	-	1.13 ± 0.01 a	1.14 ± 0.01 a	0.83 ± 0.17 a
PV (meqO2 kg−1)	16.9 ± 0.2	2.4 ± 0.1	-	-	-
AnV	7.1 ± 0.1	10.1 ± 0.1	-	-	-
Totox	40.9 ± 0.1	14.9 ± 0.1	-	-	-
Yield (%)	88.1	67.8	17.0	20.8	26.4

* Mean value ± standard deviation (n = two replicates); %FFA: free fatty acids; PV: peroxide value; IV: iodine value; SV: saponification value; Roa: olefinic/aliphatic ratio; AnV: P-anisidine value. Letters with different superscripts within the same row show significant differences (p ≤ 0.05).

presents the results of the physicochemical characterization of skipjack tuna belly oils, comparing the crude and refined oils obtained by thermomechanical extraction with the oils extracted by SFE-CO₂ (25 MPa, 40 °C) at different times (30 min, 1 h, and 3 h).

The yields of thermomechanical extraction and refining obtained from crude and bleached skipjack tuna belly oil were 88.1% and 67.8%, respectively. These results were similar to those reported by Engelman et al. [49], who observed yields of 88% in the thermomechanical extraction step (crude oil) and 75% after refining (bleached oil) using skipjack tuna heads. However, reported yields vary considerably depending on the raw material and the basis of calculation. In sardine, a process involving cooking at 30 °C followed by hydraulic pressing and centrifugation resulted in a yield of 39.5 wt% when expressed relative to the total oil content, but only 6.1 wt% when calculated on a whole-fish basis [50]. Similarly, wet rendering of catfish heads at 80 °C for 25 min produced 6.37 g oil per 100 g of raw material, corresponding to a recovery rate of 54.16% [51].

For SFE-CO₂, the highest yield of 26.4% was obtained in the experiment with the longest extraction time (25 MPa, 40 °C, 3 h). It is important to note that this yield, although lower than that of the thermomechanical method, can be explained by the higher selectivity of the supercritical process, which does not extract all lipids indiscriminately but focuses on specific fractions. The study by da Silva et al. [33] for catfish oil extraction also highlighted that the highest yield in SFE-CO₂ (83.8%) was achieved under conditions of 25 MPa, 40 °C, and 3 h, a much higher result that may point to differences in the raw material matrix (catfish head vs. skipjack tuna belly) or in the experimental methodology itself.

Other studies report a wide range of yields for fish oil extraction using SFE-CO₂, depending on the species and processing conditions. Reported values vary from approximately 20% to as high as 98% [52]. For instance, Fang et al. [53] conducted a study on different extraction methods for obtaining tuna liver oil using SFE-CO₂. The operating conditions were 35 MPa, 40 °C, and 4 h, resulting in a yield of 98.4%, indicating that it would be possible to extract virtually all the oil from tuna liver, while avoiding lipid oxidation, and suggesting that the liver matrix is particularly rich and more accessible to the solvent. Santos et al. [54] conducted a factorial experimental design for the extraction of blue shark (Prionace glauca) liver oil using SFE-CO₂ technology. Based on the results obtained, experiment no. 9, carried out under conditions of 30 MPa, 50 °C, and 6 h, resulted in a yield of 60%. Shin et al. [13] used skipjack tuna (Katsuwonus pelamis) as raw material for oil extraction via SFE-CO₂, analyzing different parts of the fish (head, viscera, and skin) under operating conditions of 30 MPa and 55 °C for 2 h. The highest extraction yields were obtained from the head (14.2%) and the skin (13.8%). Together, these studies demonstrate that SFE-CO₂ performance is highly dependent on the biological matrix and extraction conditions, which explains the comparatively lower yield observed for skipjack tuna belly in the present study.

Although thermomechanical extraction showed a higher crude oil yield (88.1%), this value includes the co-extraction of phospholipids, free fatty acids, moisture, and other non-lipid compounds [39]. After refining, the bleached oil yield decreased to 68%, more accurately representing the effective fraction of purified oil. In this context, comparison with supercritical fluid extraction (SFE-CO₂) becomes more appropriate when based on refined oil, since the supercritical process is recognized for its high selectivity and for producing extracts with a higher degree of purity, in addition to avoiding the use of toxic organic solvents [22]. Thus, the yields obtained for bleached oil (67.8%) and SFE-CO₂ (17.0–26.4%) more consistently reflect the fraction of lipids effectively recovered in each process.

The free fatty acid (FFA) content is a critical parameter for assessing the quality and safety of edible oils, as elevated FFA levels can promote oxidation reactions, degrade oil quality, and reduce sensory and nutritional value [55,56]. According to the Food and Drug Administration, the free fatty acid content (%FFA) in edible oils should be below 1%. In the present study, the free acidity of the crude oil exceeded the established limit (Table 1), possibly due to the extraction process being conducted at approximately 100 °C, which may have favored the hydrolysis of triacylglycerols, releasing free fatty acids [57]. However, after chemical refining, the %FFA of the bleached oil (around 0.45%) was significantly reduced (p ≤ 0.05), meeting the parameters required by the FDA and demonstrating the effectiveness of the refining steps in purifying the oil. Similarly, all oils extracted by SFE-CO₂, under all evaluated conditions (25 MPa, 40 °C, 30 min; 25 MPa, 40 °C, 1 h; 25 MPa, 40 °C, 3 h), showed acidity levels within legal limits [58]. This is a noteworthy aspect of SFE-CO₂, indicating that the lower operating temperature and the absence of chemical solvents reduce the hydrolytic degradation of lipids.

It was also possible to observe the efficiency of the refining process in improving oil quality (Table 1), notably the stability of the p-anisidine value (AnV), the significant reduction in peroxide value, and the TOTOX value (from 40.9 to 14.9) from crude to bleached oil. These results remained below the limits established by the FDA, which are 10 meqO₂kg⁻¹ for PV and 26 for TOTOX, indicating that the refined oil showed excellent preservation status and low primary and secondary oxidation. This highlights the ability of chemical refining to improve the oxidative quality of the oil.

The Roa parameter is a sensitive indicator of the oxidative state of oils, with values above 0.66 indicating low oxidation [38,59]. In this study, the Roa values obtained for the oils extracted by supercritical extraction (SFE-CO₂, 25 MPa, 40 °C) were from 0.83 to 1.13, demonstrating that all oils fall within the range considered adequate, reflecting higher quality and the potential use for human consumption. Furthermore, higher Roa values indicate a greater proportion of intact triacylglycerol structures, suggesting lower oxidative degradation [60]. Thus, all oils obtained showed good quality, with Roa indicating good preservation of lipid integrity under the three conditions evaluated. In combination with the low %FFA values, the results indicate that it is possible to obtain oil with characteristics similar to those of refined oil solely through SFE-CO₂, without the need for conventional refining steps.

Figure 3 presents the NMR spectra with their respective peak integration values. The iodine values (IVs) and saponification values (SVs) of the crude and bleached oils showed no significant changes (p > 0.05) after thermomechanical refining. This result is consistent with the literature [39,42,61], which indicates that chemical refining primarily acts on the removal of impurities and degradation products without altering the fatty acid profile of triacylglycerols.

However, a significant difference (p ≤ 0.05) was observed in SFE-CO₂ under the condition of 25 MPa and 40 °C for 3 h, compared to the other conditions, attributed to extraction time. According to Da Silva et al. [33], the longer the contact time between the raw material and CO₂, the lower the concentration of polyunsaturated fatty acids extracted, indicating that time is a determining factor in the selectivity of the process, directly influencing the extraction of the compounds of interest.

3.3. Fatty Acid Profiles and Oil Yield of Skipjack Tuna Bellies Obtained via Thermomechanical Process and SFE-CO₂

Table 2. Fatty acid profiles of skipjack tuna belly oil via thermomechanical extraction and supercritical fluid extraction (SFE-CO₂).

	Crude Oil *	Bleached Oil *	SFE-CO2 (25 MPa, 40 °C, 30 min) *	SFE-CO2 (25 MPa, 40 °C, 1 h) *	SFE-CO2 (25 MPa, 40 °C, 3 h) *
C14:0	4.48 ± 0.07 b	4.43 ± 0.07 b	7.00 ± 0.02 a	7.05 ± 0.28 a	6.84 ± 0.08 a
C16:0	21.30 ± 0.13 b	20.39 ± 0.20 b	26.13 ± 0.10 a	25.31 ± 0.53 a	26.16 ± 0.17 a
C16:1	4.99 ± 0.06 b	4.90 ± 0.12 b	6.98 ± 0.10 a	7.02 ± 0.10 a	6.80 ± 0.25 a
C18:0	4.30 ± 0.09 a	4.21 ± 0.17 a	4.46 ± 0.06 a	4.43 ± 0.20 a	4.18 ± 0.01 a
C18:1 ɷ9 cis	16.49 ± 0.58 bc	16.93 ± 0.17 bc	15.46 ± 0.63 c	18.09 ± 0.61 ab	18.87 ± 0.21 a
C18:2 ɷ6	2.30 ± 0.24 a	2.28 ± 0.03 a	2.83 ± 0.21 a	2.68 ± 0.34 a	2.36 ± 0.07 a
C18:3 ɷ3	1.08 ± 0.04 a	1.16 ± 0.10 a	1.18 ± 0.03 a	1.07 ± 0.06 a	1.07 ± 0.07 a
C20:1 ɷ9	4.24 ± 0.17 ab	4.64 ± 0.46 a	3.29 ± 0.03 bc	2.91 ± 0.09 c	3.36 ± 0.27 bc
C24:0	1.63 ± 0.05 b	2.30 ± 0.13 a	2.01 ± 0.05 ab	2.21 ± 0.20 a	2.08 ± 0.03 a
C20:5 ɷ3(EPA)	7.58 ± 0.05 a	7.39 ± 0.39 ab	5.78 ± 0.06 c	6.16 ± 0.22 bc	5.38 ± 0.56 c
C22:6 ɷ3(DHA)	19.24 ± 0.24 a	19.07 ± 0.06 a	12.64 ± 0.26 b	11.48 ± 0.09 c	11.72 ± 0.02 c
Unidentified	0.07 ± 0.01 c	0.07 ± 0.01 c	0.52 ± 0.01 a	0.56 ± 0.02 a	0.23 ± 0.03 b
Total Monounsaturated	25.71 ± 0.34 b	26.47 ± 0.70 ab	25.72 ± 0.76 b	28.03 ± 0.80 ab	29.04 ± 0.72 a
Total Polyunsaturated	30.20 ± 0.69 a	29.90 ± 0.33 a	22.43 ± 0.56 b	21.40 ± 0.53 b	20.53 ± 0.54 b
Total Saturated	31.70 ± 0.57 c	31.34 ± 0.25 c	39.61 ± 0.23 a	37.83 ± 0.45 b	39.25 ± 0.30 a
Total (SFA + MUFA + PUFA)	87.61	87.71	87.76	87.26	88.82

* Mean value ± standard deviation (n = two replicates); the results were expressed as percentage (%), relative to the total identified fatty acids. EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids; letters with different superscripts on the same line show significant differences (p ≤ 0.05).

details the fatty acid profiles of skipjack tuna belly oils, comparing the compositions obtained via thermomechanical extraction (crude and bleached oil) and SFE-CO₂ under the different extraction time conditions.

Table 2 shows that a total of 11 types of fatty acids were identified in all analyzed samples, including palmitic (C16:0), palmitoleic (C16:1 cis), stearic (C18:0), and oleic (C18:1 cis) acids, and, of fundamental nutritional importance, eicosapentaenoic acid (C20:5, EPA) and docosahexaenoic acid (C22:6, DHA).

The skipjack tuna belly oil samples obtained via the thermomechanical process showed, on average, the following fractions: 31.5% saturated fatty acids, 26.1% monounsaturated fatty acids, and 30.1% polyunsaturated fatty acids. The maintenance of EPA and DHA concentrations (crude oil with 7.58% EPA and 19.24% DHA, and bleached oil with 7.39% EPA and 19.07% DHA) after chemical refining (approximately 30% polyunsaturated fatty acids in both) supports the conclusion that refining does not significantly alter the fatty acid profile but rather purifies the oil from impurities (Table 2). These EPA and DHA values in the refined oil are close to those reported by Engelman et al. [49], who used a thermomechanical extraction method and found 9.4% EPA and 22.5% DHA. The slight variations can be attributed to factors intrinsic to the raw material, such as species, seasonality, and fish diet.

For the oils extracted via SFE-CO₂, maintaining a pressure of 25 MPa and a temperature of 40 °C, a significant variation in the fatty acid profile was observed as a function of extraction time (Table 2). The shortest extraction time (30 min) yielded the highest combined EPA + DHA content (18.4%, corresponding to 5.78% EPA and 12.64% DHA) and a total PUFA content of 22.4%. These values are consistent with those reported by da Silva et al. [33] for catfish heads under similar conditions (25 MPa, 40 °C, 1 h), which showed 18.3% EPA + DHA and 23.7% PUFAs. Increasing the extraction time to 1 h and 3 h resulted in a slight decrease in EPA + DHA (17.6% and 17.1%, respectively). This behavior indicates a modification in the relative composition of the extracted oil over time, rather than the extraction of new lipid fractions. No additional fatty acids were identified with increasing extraction time; instead, small variations were observed in the relative proportions of existing compounds. These values are comparable to those reported by da Silva et al. [33] in experiment no. 5 (25 MPa, 40 °C, 1 h) with catfish heads, which showed 18.3% for EPA + DHA and 23.7% for polyunsaturated fatty acids. In the other SFE-CO₂ operation variations (25 MPa, 40 °C, 1 h and 25 MPa, 40 °C, 3 h), there was a slight reduction in EPA and DHA concentrations (17.6% and 17.1%, respectively) and in total polyunsaturated fatty acids (21.4% and 20.5%).

Overall, these findings highlight the trade-off between yield and selectivity in SFE-CO₂. While the longest extraction time (3 h) maximized total oil yield, the shortest time (30 min) enhanced the highest relative concentration of DHA. Therefore, optimizing SFE-CO₂ conditions should consider not only total yield but also the selective enrichment of nutritionally valuable fatty acids.

4. Conclusions

The oil was extracted from skipjack tuna belly using both thermomechanical methods and supercritical fluid extraction with CO₂ (SFE-CO₂), demonstrating the potential of SFE-CO₂ as an alternative technique for fish oil recovery. The refining process improved oil quality by reducing free fatty acid (%FFA) content and the peroxide value (PV), while maintaining a fatty acid profile comparable to that of crude oil, with no significant losses of EPA and DHA. Although thermomechanical extraction resulted in a higher crude oil yield (88.1%), the yield of bleached oil (67.8%) more accurately represents the effectively purified fraction. Therefore, comparison with SFE-CO₂ yields (17.0–26.4%) is more meaningful when based on refined oil, particularly considering the higher selectivity of the supercritical process, which is also reflected in quality indicators such as low %FFA and suitable olefinic/aliphatic ratio (Roa). Oils obtained via SFE-CO₂ showed significantly higher proportions of saturated fatty acids (SFAs) and lower levels of polyunsaturated fatty acids (PUFAs), including reduced DHA contents, compared to thermomechanically extracted and refined oils. These results indicate that the supercritical process exhibits preferential extraction behavior, favoring less unsaturated lipid fractions under the evaluated conditions. Furthermore, increasing extraction time in SFE-CO₂ promoted changes in the relative composition of fatty acids rather than the formation of new lipid fractions. The slight decrease in PUFA content, particularly DHA, over time suggests a shift in extraction selectivity driven by kinetic differences among lipid components, with more readily extractable fractions being recovered earlier in the process. Although both extraction approaches yield oils with similar qualitative fatty acid profiles, SFE-CO₂ stands out for its ability to modulate extraction selectivity as a function of processing time and operating conditions. In addition, SFE-CO₂ aligns with the principles of green chemistry and sustainable technologies, highlighting its potential for the valorization of fish processing by-products through cleaner and more efficient processes, enabling the production of tailored, high value-added oils and contributing to the development of alternative nutrient sources.