LC-QTOF-MS/MS for tentative identification of antioxidant peptides in defatted Spirulina platensis cakes obtained by supercritical CO₂ extraction

Abstract

This work tackles a supercritical fluid extraction (SFE) of microalga spirulina (Arthrospira platensis) to recover potential products from a nutritional and bioactive point of view. The execution of a response-surface model (RSO) through Box-Behnken design yielded 86% defatting, which significantly contrasts traditional Soxhlet extraction (9%). Contrarily to most of the reported works in the literature that focused on the analysis of the recovered oil, this study points out the added value of the remaining cakes after processing (spirulina residues). Namely, protein content determined by the DUMAS method concluded that a noted increase in the SFE cake was produced with respect to the raw material (from 69.2 to 74.4 g/100 g). In this regard, a tentative identification of antioxidant peptides through LC-QTOF-MS/MS was carried out. More deeply, 14 peptide sequences containing specific amino acids with potential antioxidant capacity were found in the SFE remaining cakes, of which the peptide sequences “SIVNADAEARYLSPGELDRIK” and “RYLSPGELDRIKSFVT” were only detected in these cakes and not in the Soxhlet solids. In addition, 4 short amino acid groups associated with potential antioxidant capacity were also identified in both cases (“EL”, “RY”, “RYL”, and “YL”). ICP-MS served to determine the mineral profile in these materials, with most of them remaining in the cakes after SFE and Soxhlet extractions. With respect to oils, they were rich in Mg, P, K, and Fe minerals. The SFE oil extract had a much higher antioxidant capacity, phenolic content, chlorophyll A, and carotenoid content than the Soxhlet extract. Overall, this study presents an advanced extraction technique that efficiently recovers bioactive compounds and nutrients with potential food applications.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00216-025-06073-x.

Introduction

One of the most widespread strategies at the European and global levels is the search for new foods and ingredients, such as nutrients and bioactive compounds, from non-animal matrices, such as microalgae. Microalgae have a high nutrient content, with lipid values ranging from 20 to 50% of the dry weight [1] and proteins between 30 and 70% of the dry weight [2]. In addition, they have a very interesting profile of carbohydrates, vitamins, and minerals, together with a significant amount of antioxidant bioactive compounds, which can be used as food additives and/or for the development of nutraceuticals [3–5]. Moreover, microalgae also have a high fiber content, which can be used in the development of new food products, thus responding to society’s demand for new foods that reduce chronic diseases and achieve optimal health. Obviously, the content of each of the compounds of interest mentioned above, as well as their nutritional and bioactive profile, varies according to the type of microalgae species studied.

The extraction of lipids from microalgae is of double interest since, on the one hand, it allows obtaining an oil, which in some cases can have a high concentration of unsaturated fatty acids, including polyunsaturated fatty acids such as omega-3 and omega-6 [5, 6] and on the other hand, it allows obtaining a defatted microalgae cake and, therefore, with an even higher protein content [7].

However, despite their great interest from the point of view of environmental sustainability, microalgae are not a cheap raw material so far and present some barriers when it comes to being used in food, such as the presence of contaminants, both chemical and microbial, possible allergenicity/toxicity, lack of studies regarding their nutritional impact (e.g., protein digestibility), as well as the lack of acceptance by the consumer. It is worth mentioning that previously there was a legislative barrier in terms of microalgae use, which has been solved after the acceptance of different microalgae as food by EFSA in recent years [8]. In this sense, one of the strategies to be followed to overcome the existing barriers could be to carry out pre-treatments with eco-innovative technologies, either to reduce the microbial load and mitigate contaminants, or to obtain the different extracts/compounds that can be used as ingredients, or even both strategies at the same time. There are several previous studies evaluating the use of eco-innovative technologies such as pulsed electric fields (PEF), pressurized liquid extraction (PLE), and supercritical fluid extraction (SFE) for the development of new sustainable processes of preservation, extraction, dehydration, etc. of microalgae [9, 10].

Different conventional extraction techniques have been used, like Soxhlet, Folch, and Bligh-Dyer, for the extraction of lipids from microalgae, but while these methods are usually simple to operate and low budget, what promotes their use, they also have disadvantages like large consumption of organic reagents and environmental pollution, in addition to the prolonged extraction times (several hours) [11]. Likewise, the use of hexane has been questioned due to environmental and occupational safety problems [12]. Moreover, solid–liquid and liquid–liquid extraction has been commonly used for the recovery of high-added-value compounds (e.g., phenolic compounds, carotenoids, etc.). However, these conventional methods are characterized by low yields and long extraction times, as well as high solvent and energy consumption and potential health risks, involving extraction conditions (pH, temperature, extraction time, solvent type, concentration, etc.) that can alter the functional properties of potentially valuable compounds. The shortcomings of these conventional methods have stimulated interest in eco-innovative and sustainable technologies, which better preserve the compounds of interest, such as nutrients and bioactive compounds, since they work at lower temperatures and avoid the use of solvents as much as possible [13].

Among the various alternatives, eco-innovative technologies for recovering extracts rich in high value-added compounds with potential applications as ingredients are particularly noteworthy. One such technology is supercritical fluid extraction (SFE), which enables more ecological, sustainable, and viable processes in line with the concept of “green” or “eco-friendly” extraction [13]. For instance, SFE allows obtaining unaltered extracts without solvent residues and without degradation of quality due to high temperatures or oxidation; use of non-toxic solvents such as carbon dioxide; variation of solubility through solvent characteristics; and high selectivity for the extraction of non-polar compounds [14]. However, according to the target compound to be extracted, it is possible that long extraction times can be required compared to other techniques. The use of modifiers (also called cosolvents) to improve extraction and selectivity is frequent [13]. In addition, there is a necessity for valorisation of cakes or residues that remain after SFE treatment, in the context of a biorefinery, trying to make the most of the material submitted to processing.

Given these above lectures, a supercritical CO₂ extraction of Spirulina sp. was tackled in this study. Contrarily to most of the reported works that encompass a chemical assessment of the recovered extract and usually rule out the post-extraction remaining residue, this research addresses an in-depth characterization of this unexplored material that remarks its nutritional value by assessing mineral and bioactive peptides profiles through advanced analytical tools such as ICP-MS or LC-QTOF-MS/MS, respectively, also highlighting an enrichment in the protein content compared to the untreated matrix. In addition, the recovered oil was analyzed for a complete upcycling of the raw material, pointing out the importance of scCO₂ technology above traditional Soxhlet extraction in an attempt to establish novel valorisation protocols that boost process efficiency and sustainability.

Materials and methods

Samples, chemicals, and reagents

Spirulina (Arthrospira platensis) was acquired in powder form from China. It was stored in a dry place and protected from light during the execution of the experiments. Ethanol (99%) was purchased from Merck (Darmstadt, Germany); CO₂ was provided by Carburos Metálicos S.L. in 50 L cartridges to be used in supercritical conditions; n-hexane (> 95%) was supplied by Scharlab; sodium carbonate (Na₂CO₃) and methanol were purchased from VWR (Saint-Prix, France); Folin-Ciocalteu reagent, gallic acid, ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), potassium persulfate (K₂S₂O₈), and Trolox (6-hydroxy-2,5,7,8-tetramethylcroman-2-carboxylic acid) were acquired from Sigma-Aldrich.

Extraction methods

Supercritical fluid extraction (SFE)

All SFEs were carried out in a supercritical fluid equipment of the company Jasco Analítica Spain, S.L. located at the laboratory of the ALISOST group, in the Faculty of Pharmacy and Food Sciences of the Universitat de València (València, Spain).

In a typical experiment, 4 g of raw microalgae powder was introduced in a 5 mL stainless steel reactor and subjected to different experimental conditions based on a response-surface optimization (RSO) through a Box-Behnken design for a total of 15 experiments. Three factors were set, namely pressure (MPa), temperature (°C) and time (min).

In a previous investigation conducted by our research team [15], experimental conditions for SFE were as follows: 50 °C, 25 MPa, and 60 min. In order to widen the operation range, the selected conditions for this study are as follows: 40–60 °C for T, 20–40 MPa for P, and 30–120 min for extraction time. The central point and the two ends of these intervals were considered for producing RSO through Box-Behnken design. Conditions of each run to be executed are defined in Table Supporting Material 1 (SM1). Central points of the design were conducted by triplicate (Runs 3, 9, and 13, Table SM1) to confirm the reproducibility of the RSO, as well as optimal conditions derived from the execution of this model. Experiments were defined through Statgraphics Centurion XV ® software.

For each run, a supercritical mixture composed of CO₂ and a low proportion of a doping cosolvent (ethanol) was passed through the Spirulina powder. Absolute flow was set at 16 mL/min, supposing ethanol 10% of the total stream. These conditions were set attending to previous conditions [15]. The extract (targeted compounds solubilized in ethanol) was collected under continuous flow conditions in an amber glass container (Pyrex) to protect the extracted compounds from light. To perform each experiment, the methods were created in the ChromNav Control Center program. Once the extraction duration has been reached, the depressurization of the equipment was activated in order to remove the extraction vessel.

Two extraction products were obtained: a post-remaining cake, in the extraction vessel, and an oil dissolved in ethanol, in the Pyrex bottle. The cake was stored in plastic cups protected from light with aluminum foil and kept at room temperature until complete drying. For the oil extracts, ethanol was removed under reduced pressure at 40 °C in a rotary evaporator (Büchi rotavapour R-200) and stored in an amber glass vial.

Soxhlet extraction

A more traditional method (Soxhlet) was used for conventional extraction. Lipids were extracted from 4 g of biomass utilizing hexane as a solvent in a 1:50 ratio (m/v). Experiments were conducted by triplicate.

Soxhlet extraction pursued two main objectives in this study: (i) to reproduce the optimal conditions obtained with the SFE treatment (120 min), and (ii) determine the total fat content present in spirulina, for which 6 h of extraction were required to adhere to adequate standard protocols.

In both cases, the recovered solvent was distilled under reduced pressure in a rotational evaporation at 40 °C to isolate the oil that will serve for further experiments. The remaining solid residue was dried at room temperature and weighed to determine extraction percentage (objective i) or total fat content (purpose ii).

Extraction performance

Process efficiency was determined through oil yield, which estimates the mass of oil recovered after SFE or Soxhlet extraction in relation to the initial amount of spirulina (Eq. 1). In addition, for optimal conditions, defatting yield, which relates the total amount of oil recovered to the total fat present in the raw material, was also calculated (Eq. 2).

$$\text{Oil yield}\left(\%\right)=\frac{m\text{oil recovered}(\text{g})}{m\text{raw spirulina}(\text{g})}\times 100$$ 1

$$\text{Defatting performance}(\%)=\frac{m\text{oil recovered}(\text{g})}{m\text{total fat involved in the process}(\text{g})}\times 100$$ 2

Protein determination

For proteins determination, the CHNS elemental analysis was performed [16]. This analysis was conducted on three samples: (i) raw microalgae without being subjected to any type of treatment, (ii) Spirulina cake after Soxhlet extraction, and (iii) Spirulina cake after SFE treatment.

Bioactive peptides analysis: samples’ preparation and determination

Prior to the determination of bioactive peptides in the resulting SFE and Soxhlet Spirulina remaining cakes, samples were prepared as follows: 50 mg of sample was weighed and dissolved in 500 µL of water. The samples were then vortexed for 1 h and centrifuged at 11,500 r.p.m. for 5 min. The supernatant was collected and pure acetonitrile (1:1) was added and vortexed. Samples were then allowed to precipitate overnight at 4 °C and centrifuged. Finally, the supernatants were dried in a speedvac to remove all acetonitrile, and the pellets were resuspended in a solution of 0.1% formic acid (FA). After quantification by nanodrop, the samples were diluted with 0.1% FA to the appropriate concentration. A volume of 20 µL was then loaded onto an EVO tip (EvoSep).

Bioactive peptides were analyzed by liquid chromatography (EvoSep One, Evosep Biosystems) coupled to tandem mass spectrometry (timsTOF Pro, Bruker) [17]. For this, samples loaded in the Evotip pure were eluted on an analytical column (Performance 15 cm × 150 µm, 1.5 µm; Evosep) using the Evosep One system and resolved using the 30 SPD chromatographic method. The eluted peptides were ionized in a captive spray at 1700 V at 200 °C and analyzed in a ddaPASEF mode. The TIMS settings were set as follows: Mode custom; 1/K0: 0.7–1.76 V s/cm²; Ramp Time: 100 ms; Duty Cycle: 100%; Ramp rate: 9.42 Hz; MS averaging: 1; auto calibration: off. The MS settings used were Scan 100–1700 M/Z, positive ion polarity and Scan Mode: PASEF, the number of PASEF ramps was 4, the total cycle time was 0.5 s, the charge minimum was 0 and the maximum was 5, the target intensity was 12,500, the intensity threshold was 1000 and active exclusion was on. System sensitivity was checked with 50 ng of HELA digested proteins. 4025 proteins were identified using the 30 SPD gradient.

NOVOR Cloud was used for peptide identification using the Swiss Prot All database. Finally, similar previously identified sequences with antioxidant capacity were evaluated using the BIOPEP-UWM database. The search option “profiles of potential biological activity” was then used to select antioxidant activity.

Determination of total phenolics, antioxidant capacity, and pigments

To determine the total soluble phenols in spirulina, the oil extract obtained under supercritical (optimal) conditions, as well as the extract obtained using conventional Soxhlet treatment, were analyzed. For this purpose, the Singleton and Rossi (1965) protocol, also known as the Folin-Ciocalteu method, was applied. 100 µL of the aqueous extract from the samples was prepared and mixed with 3 mL of 4% Na₂CO₃ and 100 μL of 50% Folin-Ciocalteu reagent. It was shaken and incubated for an hour. Absorbance was then measured at 765 nm on the PerkinElmer UV/Vis Lambda 2 spectrophotometer (PerkinElmer, Jügesheim, Germany). Quantitative determination was made by interpolation of values with the calibration curve with gallic acid as a standard. Total phenolic content was expressed as mg gallic acid equivalents (GAE)/mL of the extract. Analyses were carried out in triplicate.

Total antioxidant capacity was measured by the Trolox Equivalent Antioxidant Capacity (TEAC) method. This test is based on the decolorization of the ABTS radical, which is blue green in colour, so that as the antioxidant capacity of the sample increases, the opacity is lost. In this work, both Spirulina extracts obtained after SFE treatment and Soxhlet (oils) were analysed. Briefly, a 7 mM solution of ABTS was mixed with 440 µL of 140 mM K₂S₂O₈. This stock solution was stored in the dark for 12–16 h and then diluted with ethanol until the absorbance was 0.70 ± 0.02, giving the working solution. When measuring the absorbance, ethanol was used as a blank. In addition, a reference cuvette (A0) containing 100 µL of distilled water and 2.5 mL of the working solution was prepared.

For the samples (Af), 100 µL of the same and 2.5 mL of the solution containing the radical were added to each cuvette. They were incubated for 10 min (avoiding light and oxygen) and the absorbance was measured at 734 nm and 30 °C in a PerkinElmer UV/Vis Lambda 2 spectrophotometer (PerkinElmer, Jügesheim, Germany). Trolox was used as a reference. To know the percentage inhibition of the samples, Eq. 3 was applied. Analyses were carried out in triplicate.

$$\%\mathrm{inhibition}=\left(1,-,\frac{A_f}{A_0}\right)·100$$ 3

To analyze the pigment concentration of Spirulina extracts (oil) after treatment with SFE and conventional Soxhlet extraction, spectrophotometry determination was used. The content of Chlorophyll A (Chl_a) and carotenoids (Car) was calculated based on the procedure described by Lichtenthaler et al. [18] with the modifications of Lima et al. [19] for the microalgae Spirulina. The different equations used were:

$${\text{Chl}}_{\text{a}}=15.65·A_{666}-0.766·A_{644}$$ 4

$$\text{Car}=\frac{1000·{\text{A}}_{470}-2.86·{\text{Chl}}_A}{245}$$ 5

For this purpose, the oil extracts were diluted in ethanol and the absorbance was measured in the PerkinElmer UV/Vis Lambda 2 spectrophotometer (PerkinElmer, Jügesheim, Germany) at 470, 644, and 666 nm. Measurements for pigment determination were performed in triplicate.

ICP-MS mineral determination

The mineral profile was determined in the following samples: (i) raw spirulina, (ii) extraction products (remaining cakes and extracts) obtained via SFE, and (iii) extraction products (cakes and extracts) resulting from Soxhlet extraction. The minerals analysed were Mg, P, K, Ca, Fe, and Zn, as these are the most interesting minerals from a nutritional point of view, being essential for several biological processes.

For solid samples, 300 mg of samples (initial samples and remaining cakes) was weighed into a Teflon digester and subjected to previous digestion with 250 µL of H₂O₂ and 1 mL of HNO₃ in a microwave system (MARS, CEM, Vertex, Spain) (800 W and 180 °C for 15 min). The samples were then filtered and diluted to an appropriate volume using ultrapure water. For liquids (i.e., SFE and Soxhlet extracts), 1 mL of these crudes at a concentration of 1 mg/mL was subjected to digestion as described above. In both cases, mineral content was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Agilent model 7900). ICP-MS conditions were set as follows: carrier gas flow (1.07 L min⁻¹), helium (He) as reactant gas, high-frequency emission power (1550 W), Ar gas flow (15.0 L min⁻¹), nebulizer pump speed (0.10 rps), and radio frequency matching (1.80 V). The experiments were carried out in triplicate. Mineral content was analyzed using 45Sc and 72Ge as internal standards, and a 0–1000 μg/L standard curve was used for quantitative analysis. The value of the correlation coefficient was R ≥ 0.9999 and each calibration point had an RSD value of ≤ 5%. At the end of the sample sequence analysis, a calibration sample was analyzed, obtaining an average between the reference and the obtained value around RSD ≤ 5%.

Statistical analyses

A Box-Behnken design, a type of response-surface methodology, was selected to optimize oil recovery conditions under Supercritical Fluid Extraction. Moreover, all tests were conducted in triplicate, and the average values are presented as mean ± SD. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple range test to identify significant differences (p < 0.05). Graphs were created using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA).

Results and discussion

Supercritical fluid extraction (SFE) efficiency through response-surface optimization (RSO)

SFE extraction process was optimized based on the oil extraction performance. Results are shown in Table 1.

Table 1.

Experimental results of the oil extraction performance of Spirulina sp. after supercritical fluid extraction (SFE)

Run	T (°C)	t (min)	P (MPa)	Oil weight (g)	Defatting performance (%)
1	40	75	20	0.20	4.99
2	60	120	30	0.23	5.74
3	50	75	30	0.20	4.98
4	50	120	20	0.25	6.25
5	60	75	20	0.21	5.26
6	50	30	20	0.09	2.27
7	50	120	40	0.24	5.96
8	40	75	40	0.21	5.25
9	50	75	30	0.20	4.42
10	50	30	40	0.07	1.75
11	60	30	30	0.13	3.28
12	40	30	30	0.09	2.24
13	50	75	30	0.16	4.00
14	60	75	40	0.16	3.99
15	40	120	30	0.17	4.27

T, temperature; t, time; P, pressure

Briefly, an increased oil yield was observed with the elapse of extraction time: in the 30-min treatments, oil yields of 2.27%, 1.75%, 3.28%, and 2.24% were obtained (Table 1, entries 6 and 10–12, respectively), while 75-min treatments showed oil yields ranging from 3.99% to 5.26%. Finally, after 120 min, the values of oil extracted were found between 4.27% and 6.25%.

In this point, it should be remarked that albeit a kinetic study would have been the most ideal solution to determine the optimal extraction time, this step was not considered in this study due to various reasons. Firstly, because of the nature of the material: microalgae possess a strong cell wall that sometimes requires harsher conditions to be broken down, for instance, high temperatures, pressure, or processing time. For that, we decided to operate in an acceptable range of conditions for these variables defined in the RSO in view of enhancing process efficiency, considering as the starting point our previous study [15]. Thus, pressure was applied from 20 to 40 MPa, the maximum value allowed by our supercritical equipment; 40–60 °C were considered for temperature, principally trying to avoid the degradation of certain bioactive molecules. Finally, time was optimized from 30 to 120 min since when a process intensification technology (SFE) is used, excessive time above 120 min can result contradictory.

Concerning again the kinetic study, kinetic curve can significantly vary depending on the temperature and pressure employed, so that different conclusions could have been obtained.

Furthermore, the reproducibility of the model can be assured attending to the oil performance obtained in the central points of the design (experiments 3, 9, and 13, Table 1), with proximate yields of 4.98, 5.05, and 4.00%, respectively.

Once the RSO was executed (15 experiments), the data recovered (i.e., oil performance) was again introduced in the Statgraphics Centurion XV® software, that provides the optimal extraction conditions derived from this model, as well as a thorough ANOVA analysis with regression coefficients for the optimized variable (see Supporting Material, section 2).

According to the extraction yields obtained, the optimal conditions for this design were 21.37 MPa, 120 min, and 60 °C, as displayed in Table 2 and Fig. 1. Figure 1a also shows these optimal conditions in the form of a graphical representation. It shows how the extraction yields increased with the elapse of extraction time, and that the change in pressure hardly influenced these results, slightly noting the maximum yield at 20 MPa.

Table 2.

Optimal response for Spirulina with supercritical fluid extraction (SFE) treatment through response-surface model

		P (MPa)	T (°C)	t (min)	Oil performance (%)	Defatting yield (%)
Optimal SFE	Experimental	21.37	60	120	5.03 ± 0.66	86.78 ± 9.27
	Predicted value	21.37	60	120	6.5
Soxhlet		-	(refluxed hexane)	120	0.53 ± 0.03	9.18 ± 0.57

Fig. 1.

Optimal conditions estimated by the response-surface design (a) and main effects of pressure, treatment time, and temperature on oil extraction performance (b)

The same is also shown in Fig. 1b, where the influence of the different variables (pressure, time and temperature) to recover the oil extraction performance is evident.

More specifically, it is observed that the pressure has the maximum performance at 21.37 MPa, and an increase in pressure slightly decreases the extraction performance. On the other hand, an increase in the duration of SFE treatment leads to a significant increase in performance, with 120 min being the optimal duration for this study. Finally, the extraction yield increases as the temperature increases, so that the highest results of this variable are obtained at 60 °C.

It is estimated that at these conditions, a predicted oil performance of 6.5% should be obtained (according to the data provided by Statgraphics Centurion XV®). Nonetheless, the execution of a SFE in these conditions to validate the RS model led to an oil performance of 5.03%, which corresponds with a defatting yield of 86.75% (Table 2). It can be thus concluded that there is no significant difference between the average of these yields and the expected optimal value. These results agree with those reported previously in the available literature, which states that the pressure of 4000 psi (~ 27.5 MPa) is the one that allows a higher extraction yield for two different species of microalgae: Arthrospira sp. and Schizochytrium sp. In the same study, cosolvent was also used to enhance scCO₂ extraction, although methanol was used instead of ethanol [20]. These authors also observed an increase in lipid extraction with the elapse of the extraction time, and after 6 h of extraction (the maximum time spent), the maximum yield was obtained [20].

In respect to oil extraction yields obtained by the conventional Soxhlet treatment for 2 h, significantly lower outcomes were reported compared to those obtained via SFE during that time (0.53% oil yield, 9.18% defatting). This difference is clearly observed in the stains of the extracts obtained with SFE and the Soxhlet extractions (Fig. 2).

Fig. 2.

Difference in the colour of the extracted oil. A Supercritical fluid extraction (20 MPa/60 °C/120 min). B Soxhlet 120 min

Taking into account the reviewed literature, Soalana et al. [21] obtained a superior performance in Soxhlet extraction (29%) versus recovery of oil extracted by SFE (~ 25%). However, it is worth mentioning that in that study, a difference of 16.5 h between the extraction time of the different methods was stated, with the Soxhlet being the one that was maintained for the longest time (18 h), while the SFE extraction was performed in 1.5 h. This difference in treatment conditions shows that Soxhlet requires too long extraction times, which has a greater economic and environmental impact.

Oil analysis

SFE allows the recovery of two extraction products. Most of the literature is focused on the analysis of the oil extract, with different assays for antioxidant activity, lipidic profile, etc. Herein, the main target is the post-extraction solid and its potential as a source of proteins and minerals, as well as bioactive peptides. Nonetheless, oil quality was also assessed and contrasted with some works reported in the literature.

Determination of antioxidant compounds

The total polyphenol content (TPC) and antioxidant activity (TEAC) obtained in the oil extracted from Arthrospira platensis by SFE and Soxhlet (conventional) are shown in Fig. 3. Regarding the total antioxidant capacity of the oil extracted by the TEAC assay, as shown in Fig. 3a, a large difference in TEAC values can also be observed between SFE (10.681 μmol TE/g DW) and the conventional Soxhlet extraction (0.987 μmol TE/g DW), with a significant difference between treatments once again (p < 0.0001). With the SFE treatment, it was possible to increase the extraction of antioxidant compounds 10 times compared to conventional treatment. Dejsunkranont et al. [22] reported a higher antioxidant capacity by performing several supercritical fluid extraction (SFE) experiments on a Spirulina maxima sample. These authors observed an antioxidant activity of 118.62 μmol TE/g under conditions of 31 MPa, 60 °C, and 120 min. However, in this case, the extraction was carried out without a cosolvent [22].

Fig. 3.

Comparison of TEAC (Trolox Equivalent Antioxidant Capacity) (a) and TPC (total polyphenol content) (b) analyses in oil extracted from Spirulina sp. by supercritical fluid extraction (SFE) and Soxhlet as a control. Results expressed as mean ± SD. p < 0.0001. μmol TE/g DW, μmol Trolox equivalents per gram dry weight; mg GAE/g DW, mg gallic acid equivalents per gram dry weight

As can be observed in Fig. 3b, the value of TPC is clearly influenced by SFE treatment (4.600 mg GAE/g DW), which is significantly higher (p < 0.0001) with respect to the control (i.e., the conventional Soxhlet extraction) (0.156 mg GAE/g DW.) That is logical since it is not expected a high concentration of phenolic compounds in a crude recovered through a low polar environment (i.e., pure hexane). Therefore, it is shown how that innovative technologies, such as SFE, are considered more promising than conventional methods in terms of the recovery of bioactive compounds in microalgae [23].

It has been shown that the use of polar solvents favors the extraction of certain phenolic compounds. In the present study, ethanol was used as a cosolvent trying to maximize process efficiency and recovery of bioactive compounds, using the optimal conditions related to the highest extraction yield. A 10% stream is considered for the cosolvent based on our previous work [15] with no optimization addressed in this study.

Outcomes derived from this work can be contrasted with the study by Georgiopoulou et al. [23], who obtained the highest phenolic content in Chorella vulgaris by SFE + 10% ethanol (17.3 mg GAE/g extr.) compared to a conventional solid–liquid extraction (SLE) (11.02 mg GAE/g extr.). This difference is probably due to the recovery of phenolic compounds of different polarities. The results obtained in the present study are also related to the study by Wang et al. [24], in which microalgae, including Spirulina, were subjected to pressurized liquid extraction (PLE) and whose TPC is 12.5 mg GAE/g DW. In any case, the protocol herein applied led to a significantly lower TPC compared to these two works.

Determination of chlorophyll A and carotenoids

Spirulina contains distinctive natural pigments of orange, green, and blue (carotenoids, chlorophylls, and phycocyanins, respectively). In the present work, chlorophyll A (Chl_a) and carotenoids (Car) were analyzed (Fig. 4). Chlorophyll B has not been calculated in this study since, according to Lima et al. [19], the microalgae Spirulina lacks Chl_b, and chlorophyll C is only present in brown algae.

Fig. 4.

Content of chlorophylls and total carotenoids obtained from the microalgae Spirulina for supercritical fluid extraction (SFE) and conventional (Soxhlet) extraction. Results expressed as Mean ± SD. p < 0.0001. μg/g DW, μg per gram dry weight

Chlorophyll A, apart from the role it plays in photosynthesis, has demonstrated a strong antioxidant capacity [4]. In addition, it can be used as a natural colouring for some foods. On the other hand, carotenoids also contribute to the elimination of free radicals and are precursors of vitamin A [25].

For SFE, an increase in chlorophyll A extraction was observed compared to soxhlet. On average, a total of 114.985 μg Chl_a/g DW was obtained in the conventional Soxhlet extraction compared to 2650.484 μg Chl_a/g DW when applying the SFE extraction (i.e., up to 20 times higher). Statistical analysis indicates that the results obtained for chlorophyll A were statistically significant (p < 0.0001). This difference is also observable in Fig. 2, where the SFE extraction has a greener coloration than the conventional one, due to the greater presence of chlorophyll A.

These values can be contrasted with the work of Tong et al. [26], who focused on the extraction of chlorophyll A from the microalgae Arthrospira platensis by SFE; therefore, the conditions were optimized according to chlorophyll A. The optimal conditions used were 40 MPa, 323.2 K (around 50 °C), 1 h of treatment duration, 8 g/min of CO₂ flux, and 10 mL of different cosolvents, including ethanol. The results of chlorophyll A obtained were almost 2.5 mg/g. Moreover, compared to conventional treatment, about 3 times more chlorophyll A was extracted during the same extraction time by these authors. In addition, it was observed that the presence of a cosolvent facilitates the extraction of chlorophylls, since the treatment with scCO₂ without any cosolvent was also analyzed and hardly any chlorophylls were extracted [26].

On the other hand, SFE treatment also significantly increased carotenoid extraction. On average, 532.430 μg Car/g DW was extracted after SFE and 76.061 μg Car/g DW after conventional extraction, so that SFE treatment increased carotenoid extraction by 7 times compared to soxhlet extraction, which was statistically significant (p < 0.0001).

These results can be compared with the study by Marzorati et al. [4], in which scCO₂ was used for carotenoid extraction under the following conditions: 300 bar (30 MPa), CO₂ flux of 15 mL/min, 45 °C, and 1 h of extraction (without any organic cosolvent, which is more selective for carotenoid extraction). These authors obtained 3.5 mg of Car/g DW in the extract obtained under the above conditions.

Determination of minerals

Essential minerals are vital for maintaining various bodily functions, including building materials for human bones, muscle contraction, nerve function, and regulating the body’s water balance. They are also components of hormones, enzymes, and other biologically active compounds. Some minerals also play an important role in the optimal functioning of the immune system [27]. Spirulina contains a considerable amount of potassium (K). Moreover, it provides major minerals (calcium (Ca), phosphorus (P), and magnesium (Mg)) at ratios similar to those present in milk. It is also important to note that Spirulina is an exceptional source of iron (Fe), containing approximately ten times more than other iron-rich foods [28, 29].

Concerning mineral content in the Spirulina extracts obtained via SFE and Soxhlet (Fig. 5), significantly higher values were found in the SFE extracts for Mg (5.3 vs 0.21 mg/L), P (1.3 vs 0.5 mg/L), K (4.1 vs 0.8 mg/L), and Fe (122 vs 39.6 µg/L), being the Mg and K contents of the SFE extracts remarkable. However, non-significant differences were observed for Ca (0.9 vs 1.25 mg/L), Cu (8.2 vs 16.4 µg/L), and Zn (83.3 vs 86.6 µg/L) between the SFE and Soxhlet extracts.

Fig. 5.

Major (Mg, P, K, Ca) and minor (Fe, Cu, Zn) minerals present in the recovered oils after SFE and conventional extraction of spirulina

In any case, it is important to note that in all cases the mineral content of the SFE extracts was higher compared to conventional extraction, highlighting the role of SFE technology in the extraction of essential minerals. The same trend was observed in a previous study where SFE improved mineral extraction, increasing from 49.33 to 91.50 µg/g DM for Mg and from 0.87 to 2.00 µg/g DM for Fe compared to conventional extraction, while conventional was more effective in recovering P and Ca [15]. Regarding other innovative extraction technologies, pressurized liquid extraction (PLE) allows an extraction rate close to 100% for Zn, while Mg, Ca, and Fe were extracted from Phaeodactylum tricornutum at rates of 12.4%, 0.6%, and 4.8%, respectively [15, 24].

Assessment of nutritional properties of solid residues: protein content, bioactive peptides, and mineral profile

As has been previously referred, the analysis of spirulina residues (i.e., post-extraction solid that remains with no further utilisation) for further upcycling is the main attainment in this work with respect to most of the literature reporting SFE of spirulina, in which this solid is treated as a waste, with no considerations for its recycling and any previous analysis. In this regard, preliminary assessment of this material addressed to determine its nutritional profile, namely protein content, bioactive peptides, and mineral profile.

Protein content and bioactive peptides

Outcomes derived from the total protein content analysis in raw Spirulina, as well as in the cakes obtained by Soxhlet and SFE methods, are shown in Table 3.

Table 3.

Effect of supercritical fluid extraction (SFE) on Spirulina proteins

Sample	Proteins (g/100 g)
Untreated microalgae	69.2 ± 0.2
Conventional Soxhlet cake	68.4 ± 0.3
Optimal SFE cake	74.4 ± 1.1

No significant differences were observed in the protein contents of untreated microalgae samples (69.2 g/100 g) and the remaining cakes obtained after conventional Soxhlet extraction, while SFE-treated Spirulina samples showed a slightly increased (74.4 g/100 g). That can be explained since a similar extraction time was applied to both procedures (120 min) to process the material. Therefore, the intensification mechanisms involved in the supercritical CO₂ technology (i.e., high pressures, higher diffusivity, solubilization, etc.) would facilitate a more efficient extraction of oil than conventional systems, thus leading to a higher enhancement in the post-extraction recovered material, as has been observed.

Similar results were reported in the study conducted by Qiuhui et al. [30], in which the different nutrients present in Spirulina were evaluated by carrying out a control treatment and another with scCO₂. The authors obtained 69.75 g protein/100 g in the control, while the protein values found after scCO₂ treatment were 69.79 g/100 g.

Moreover, a relevant analysis that involves a tentative identification of antioxidant peptides was also performed, once again focused on the remaining cakes obtained by SFE and Soxhlet extraction (Tables 4 and 5).

Table 4.

Antioxidant peptides found in the Spirulina remaining cake after supercritical fluid extraction (SFE) with potential antioxidant capacity and the group responsible for this bioactivity

Score	Sequence	Involved amino acids	Position
117.029	AYNGDPFVGHLSTPISDSAFTR	HL	[10-11]
		AY	[1-2]
112.499	MKTPLTEAVSIADSQGRFLSST	LT	[5-6]
107.894	NDAAGDGTTTATVLAHAMVKE	AH	[15-16]
102.612	AASKTNDAAGDGTTTATVLAHAM	AH	[20-21]
101.345	NGDPFVGHLSTPISDSAFTR	HL	[8-9]
99.331	SPKQIEDVAAYVVDQAEKGW	AY	[10-11]
		GW	[19-20]
		PK	[2-3]
98.596	SIVNADAEARYLSPGELDRIK	EL	[16-17]
		RY	[10-11]
		RYL	[10-12]
		YL	[11-12]
98.192	ASKTNDAAGDGTTTATVLAH	AH	[19-20]
97.024	DAAGDGTTTATVLAHAMVKE	AH	[14-15]
96.155	ASKTNDAAGDGTTTATVLAHAM	AH	[19-20]
94.038	MKTPLTEAVSIADSQGRFL	LT	[5-6]
92.208	TNDAAGDGTTTATVLAHAMVKE	AH	[16-17]
90.566	RYLSPGELDRIKSFVT	EL	[7-8]
		RY	[1-2]
		RYL	[1-3]
		YL	[2-3]
90.211	TSKADSLISGAAQAVYNKFP	VY	[15-16]
		GAA	[10-12]
		GA	[10-11]

Table 5.

Antioxidant peptides found in the Spirulina remaining cake after Soxhlet extraction with potential antioxidant capacity and the group responsible for this bioactivity

Score	Sequence	Involved amino acids	Position
110.563	MKTPLTEAVSIADSQGRFLSST	LT	[5-6]
103.673	NDAAGDGTTTATVLAHAMVKE	AH	[15-16]
102.329	AYNGDPFVGHLSTPISDSAFTR	HL	[10-11]
		AY	[1-2]
99.598	ALQDDWQQSHLERRQVLAE	HL	[10-11]
97.423	TSKADSLISGAAQAVYNKFP	VY	[15-16]
		GAA	[10-12]
		GA	[10-11]
94.656	AYNGDPFVGHLSTPISDSAFTR	HL	[10-11]
		AY	[1-2]
94.595	DAAGDGTTTATVLAHAMVKEGLR	AH	[14-15]
93.841	DAAGDGTTTATVLAHAM(O)VKE	AH	[14-15]
91.271	NGDPFVGHLSTPISDSAFTR	HL	[8-9]

For this purpose, the bioactivity and potential bioactivity of the identified peptides were checked using the BIOPEP-UWM database. Only peptides with a score of more than 90% were considered. The database did not show any correlation between the identified peptides and antioxidant activity. However, the presence of several short amino acid sequences with antioxidant properties within these peptides was revealed. As can be observed in Tables 4 and 5, 14 peptide sequences were found including specific amino acids with potential antioxidant capacity in SFE residue cakes compared to the 9 observed in Soxhlet remaining cakes. The following peptide sequences including specific groups with potential antioxidant activity “AYNGDPFVGHLSTPISDSAFTR” (HL, AY), “MKTPLTEAVSIADSQGRFLSST” (LT), “NDAAGDGTTATVLAHAMVKE” (AH), “NGDPFVGHLSTPISDSAFTR” (HL), and “TSKADSLISGAAQAVYNKFP” (VY, GAA, GA) were tentatively identified in both SFE and Soxhlet remaining cakes. It is important to note that the peptide sequences “SIVNADAEARYLSPGELDRIK” and “RYLSPGELDRIKSFVT” were only detected in the remaining SFE cakes, having a significant antioxidant potential, with 4 short amino acid groups associated with potential antioxidant capacity, in both cases “EL”, “RY”, “RYL”, and “YL”.

Previous studies performed in algae biomass have reported that peptides containing hydrophobic amino acids (Leu, Val, Trp, Ala, Phe, and Tyr) in their structure could exhibit higher free radical scavenging activity by helping the peptides to pass through membrane lipid bilayers. In addition, acidic or basic amino acids (Glu, Asp, and Lys) could act as metal ion chelators, while aromatic amino acids (Tyr, Trp, and Phe) could quench free radicals by electron transfer [31, 32]. Nonetheless, as far as our knowledge goes, this is the first study that tackles a tentative analysis of the bioactive peptides present in the spirulina SFE residues, which points out the value of this work.

With this tentative identification, the potential of spirulina residues as antioxidant materials is corroborated, albeit further studies or investigations would be required in order to assess the real antioxidant power of these materials.

Mineral profile

In respect to minerals’ content evaluated in untreated spirulina and remaining cakes obtained after SFE extraction and Soxhlet (Fig. 6), the major minerals (Ca, P, Mg) were detected without significant differences in the raw samples and the remaining cakes, with values ranging from 3.9 to 4.2 mg/g for Mg, 10.5 to 10.7 mg/g for P, and 2.7 to 2.8 mg/g for Ca. Potassium was the mineral quantified at higher levels, with a significant difference between SFE (12.83 mg/g) and Soxhlet (12.03 mg/g) residual cakes. Regarding minor minerals, no significant differences were observed for Cu and Zn, which were found in the raw material and remaining cakes at levels of 0.28–0.51 mg/kg and 9.3–11 mg/kg, respectively. The mineral Fe was detected in an interesting amount between 838 and 861 mg/kg. In this case, the level was significantly lower in the SFE residual cake compared to the Soxhlet cake; however, as has been mentioned before, a higher amount of Fe was detected in SFE extract compared to the conventional Soxhlet extract (122 µg/L vs 39.6 µg/L). A previous study reported similar levels of Mg and P in Spirulina (3.2 mg/g dw) and (9.3 mg/g), respectively. However, these authors reported higher levels of Zn (44 mg/kg) [24]. The results obtained in the present study suggest that most of the minerals remain in the cakes after extraction with SFE and conventional Soxhlet extraction; however, the extracts obtained with SFE are richer in all the minerals analysed in this study compared to conventional extracts, highlighting their Mg, K, and Fe contents.

Fig. 6.

Major (Mg, P, K, Ca) and minor (Fe, Cu, Zn) minerals present in the untreated material and the remaining cakes obtained after SFE and conventional extraction

Conclusions

Trying to ensure the current demand for foods, this study has presented a sustainable valorisation of microalga spirulina through SFE to recover nutrients and materials with bioactive potential. Supercritical fluids demonstrated to be more efficient for the recovery of compounds since they afford a higher defatting while enable the use of non-toxic substances such as CO₂ or ethanol. In parallel to oil recovered, which presented an acceptable antioxidant capacity, mineral, chlorophyll, and carotenoid content, the resulting SFE cake showed an enhancement in terms of protein content (74.4 g/100 g) compared to the raw material (69.2 g/100 g). It also contains peptide sequences, such as “SIVNADAEARYLSPGELDRIK” and “RYLSPGELDRIKSFVT”, with specific amino acids that could have potential antioxidant capacity. In addition, most of the minerals present in spirulina remained in this valuable cake obtained after SFE in a higher proportion compared to the extracts. In summary, this work has highlighted the added value of spirulina residues obtained after supercritical CO₂ extraction, pointing out their nutritional and bioactive potential, and exploiting new forms of upcycling that, as far as our knowledge goes, have not been deeply explored until now.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

Lucía Cuesta-Ramos and Nerea Sánchez-Moreno: writing—original draft, investigation, formal analysis, data curation. Noelia Pallarés and Manuel Salgado-Ramos: conceptualization, writing—original draft, investigation, formal analysis, data curation, project administration, writing—review and editing. Francisco J. Martí Quijal and Emilia Ferrer: writing—review and editing, validation, supervision, resources, project administration, funding acquisition, conceptualization. Juan M. Castagnini: writing—review and editing, validation, supervision, methodology, software, conceptualization. Francisco J. Barba: writing—review and editing, methodology, validation, supervision, resources, project administration, funding acquisition, conceptualization.

Funding

Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study forms part of the AGROALNEXT program (AGROALNEXT/2022/060-Desarrollo y optimización de procesos innovadores y sostenibles de extracción de aceite y proteínas a partir de microalgas, insectos, residuos y subproductos agroalimentarios: Evaluación de propiedades biológicas (EXTRAOLIOPRO)) and was supported by MICIU with funding from European Union Next Generation EU (PRTR-C17.I1) and by Generalitat Valenciana. Manuel Salgado-Ramos thanks Generalitat Valenciana for his postdoctoral contract supported by Subvenciones para la contratación de personal investigador en fase postdoctoral (APOSTD) fellowship program (Ref. CIAPOS/2023/236), co-funded by European Union).

Data availability

The data that support the findings of this study are available on request.

Declarations

Conflict of interest

The authors declare no competing interests.