Sustainable Green Extraction of Carotenoid Pigments: Innovative Technologies and Bio-Based Solvents

Citation: Morón-Ortiz, Á.; Mapelli-Brahm, P.; Meléndez-Martínez, A.J. Sustainable Green Extraction of Carotenoid Pigments: Innovative Technologies and Bio-Based Solvents. Antioxidants 2024,13, 239. https://doi.org/10.3390/ antiox13020239 Academic Editor: Alessandra Napolitano Received: 5 January 2024 Revised: 9 February 2024 Accepted: 10 February 2024 Published: 15 February 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Review Sustainable Green Extraction of Carotenoid Pigments: Innovative Technologies and Bio-Based Solvents Ángeles Morón-Ortiz, Paula Mapelli-Brahm * and Antonio J. Meléndez-Martínez Food Colour and Quality Laboratory, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (Á.M.-O.); [email protected] (A.J.M.-M.) *Correspondence: [email protected]; Tel.: +34-955-420-973 Abstract: Carotenoids are ubiquitous and versatile isoprenoid compounds. The intake of foods rich in these pigments is often associated with health benefits, attributable to the provitamin A activity of some of them and different mechanisms. The importance of carotenoids and their derivatives for the production of foods and health-promotion through the diet is beyond doubt. In the new circular economy paradigm, the recovery of carotenoids in the biorefinery process is highly desirable, for which greener processes and solvents are being advocated for, considering the many studies being conducted at the laboratory scale. This review summarizes information on different extraction technologies (ultrasound, microwaves, pulsed electric fields, pressurized liquid extraction, sub- and supercritical fluid extraction, and enzyme-assisted extraction) and green solvents (ethyl lactate, 2-methyltetrahydrofuran, natural deep eutectic solvents, and ionic liquids), which are potential substitutes for more toxic and less environmentally friendly solvents. Additionally, it discusses the results of the latest studies on the sustainable green extraction of carotenoids. The conclusions drawn from the review indicate that while laboratory results are often promising, the scalability to real industrial scenarios poses a significant challenge. Furthermore, incorporating life cycle assessment analyses is crucial for a comprehensive evaluation of the sustainability of innovative extraction processes compared to industry-standard methods. Keywords: 2-methyltetrahydrofuran; ethyl lactate; ionic liquids; microwaves; natural deep eutectic solvents; pressurized liquid extraction; pulsed electric fields; subcritical fluid extraction; supercritical fluid extraction; ultrasounds 1. Introduction Carotenoids are versatile compounds naturally found in food sources, including plantderived (e.g., broccoli, tomato, pepper, carrot, or spinach) or animal-derived (e.g., egg yolk, salmon flesh, or mussels) foods. These compounds can also be found in other non-edible plants and animals, as well as in other matrices such as algae, some bacteria, and some fungi. With few exceptions, carotenoids are natural pigments, many of which are involved in many important biological actions [ 1 ]. They play roles in photoprotection, light absorbance, redox reactions, plant resilience to stress, seed dispersal, germination, and pollination, among others [ 2 ]. The diverse range of actions exhibited by carotenoids and their derivatives positions them as crucial compounds in food production, consequently playing a pivotal role in ensuring food security [ 1 ]. These bioactive compounds do not only stand out as natural pigments with many natural functions, but also act as health-promoting compounds through different mechanisms (light absorption, modulation of gene expression, interaction with reactive oxygen species, etc.), resulting in different effects, including antioxidants and anti-inflammatory effects. Certain carotenoids serve as precursors of vitamin A, considered the vitamin with the widest spectrum of actions. They are therefore key to combatting vitamin A deficiency, a major nutritional problem in the world [ 3 ]. In relation to these actions, substantial evidence indicates that these compounds contribute to providing Antioxidants 2024,13, 239. https://doi.org/10.3390/antiox13020239 https://www.mdpi.com/journal/antioxidants Antioxidants 2024,13, 239 2 of 27 various health benefits for humans, including immunity enhancements and potential reductions for the risk of developing several types of cancer, cardiovascular diseases, and bone, skin, and ocular disorders, among others [4,5]. As a consequence of the benefits associated with these compounds, they are of great interest for the production of functional foods nutraceuticals, nutricosmetics, supplements, botanicals, and novel foods [ 1 , 4 ]. To make these products, carotenoids are often extracted from sources where they are at high concentration. The selection of an appropriate solvent depends on the complexity of the matrix and the polarity of each carotenoid, among other factors. Conventionally, carotenoids are extracted using solvents such as hexane, acetone, tetrahydrofuran, petroleum ether, diethyl ether, chloroform, ethyl acetate, and ethanol; however, some of these solvents are considered toxic [ 6 ]. Given the increasing demand for carotenoids on an industrial scale, it is important to ensure economic viability and environmental sustainability in this process [ 6 ]. Thus, the use of green solvents (2-methyloxolane, ethyl lactate, and ionic liquids, among others) and green techniques that help reduce the consumption of energy and other resources (such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), or pressurized liquid extraction (PLE)) has increased in recent years. Addressing the sustainability of the extraction process aligns with broader considerations in food science, health, and eco-friendly practices. 2. Methodology The articles included in this review were retrieved from Scopus. The starting search strategy was as follows: Title: “Carotenoid” AND “each of the technologies/type of solvents” separated with “or”. In some cases, other references included in the works retrieved were also read and commented in the review. In order to keep the work to a reasonable length, when more than 10 references were found for a particular technology or type of solvent, some studies were left out. In such cases, it was procured to include matrices of special interest, usually for their dietary importance or innovative nature. 3. Green Chemistry and Green Extractions: The Concepts Green chemistry is considered the development, design, and implementation of chemical products and processes to decrease or eliminate the usage and generation of hazardous substances [ 7 ]. Green chemistry has established guidelines for modern chemical processes that prioritize sustainability and environmental friendliness. The principles of green chemistry include minimizing waste, using renewable resources, preventing pollution, and designing chemicals and materials that are non-toxic, among others [8]. On the other hand, green extraction involves identifying and creating extraction methods that minimize energy usage, enable the utilization of renewable natural products and alternative solvents, and guarantee the extraction of high-quality and safe products. The principles of green extraction involve innovation through the selection of varieties and the use of renewable plant resources, which emphasizes the use of selecting plant varieties that are abundant and sustainable [ 9 ]. Green extraction promotes the use of alternative solvents, such as water or agro-solvents, and other bio-based solvents as alternatives to toxic and hazardous solvents, such as chlorinated solvents or benzene [ 10 ]. In addition, the reduction of energy consumption during the extraction process through energy recovery and the use of innovative technologies, such as microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), and supercritical fluid extraction (SFE), among others, is also an important goal. Moreover, the principles also comprise the production of coproducts as an alternative to waste, the reduction of the number of experiments, and the obtaining of non-denatured and biodegradable extracts without contaminants [ 7 , 9 ]. Integrating the green extraction practices of natural products with the principles of green chemistry can lead to the development of a “Green Chemistry of Natural Products”, which could promote the use of natural resources and reduce the negative impact of chemical processes on the environment and human health to address global challenges [ 9 ]. Overall, the concepts of green chemistry and green extraction are aimed at promoting Antioxidants 2024,13, 239 3 of 27 sustainable and environmentally friendly practices in chemistry and chemical engineering, with applications in other fields and industries, such as agri-food, pharma, and cosmetics, among others. 4. Technologies Amenable to Green Extractions Green extraction technologies have been employed for carotenoid recovery from diverse matrices, since they enhance carotenoid extraction through various extraction mechanisms (Table 1). Among these methodologies, ultrasound-assisted extraction and supercritical fluid extraction have garnered significant attention in scientific investigations, being extensively explored compared to other alternative sustainable techniques (i.e., microwave-assisted extraction, pulsed electric field, pressurized liquid extraction, subcritical fluid extraction, and enzyme-assisted extraction), as depicted in Figure 1. The main results of the studies on carotenoid recovery with green extraction methodologies discussed in this review are summarized in Table 2. Antioxidants 2024, 13, x FOR PEER REVIEW 3 of 29 nants [7,9]. Integrating the green extraction practices of natural products with the principles of green chemistry can lead to the development of a “Green Chemistry of Natural Products”, which could promote the use of natural resources and reduce the negative impact of chemical processes on the environment and human health to address global challenges [9]. Overall, the concepts of green chemistry and green extraction are aimed at promoting sustainable and environmentally friendly practices in chemistry and chemical engineering, with applications in other fields and industries, such as agri-food, pharma, and cosmetics, among others. 4. Technologies Amenable to Green Extractions Green extraction technologies have been employed for carotenoid recovery from diverse matrices, since they enhance carotenoid extraction through various extraction mechanisms (Table 1). Among these methodologies, ultrasound-assisted extraction and supercritical fluid extraction have garnered significant attention in scientific investigations, being extensively explored compared to other alternative sustainable techniques (i.e., microwave-assisted extraction, pulsed electric field, pressurized liquid extraction, subcritical fluid extraction, and enzyme-assisted extraction), as depicted in Figure 1. The main results of the studies on carotenoid recovery with green extraction methodologies discussed in this review are summarized in Table 2. Figure 1. Number (inside the chart) and percentage (outside the chart) of published research articles related to carotenoid extraction using the respective methodologies. UAE: ultrasound-assisted extraction; MAE: microwave-assisted extraction; PEF: pulsed electric field; PLE: pressurized liquid extraction; SuperFE: supercritical fluid extraction; SubFE: subcritical fluid extraction; EAE: enzymeassisted extraction. Source for article search: Scopus. Table 1. Extraction mechanisms of the green extraction technologies. Extraction Method Mechanism Ref UAE Utilizes sound waves for inducing cavitation in the solution, thereby facilitating the extraction process. [11] MAE Generates heat within the solvent by employing ionic conduction of the dipole rotation and dissolved ions in the polar solvent. [12] Figure 1. Number (inside the chart) and percentage (outside the chart) of published research articles related to carotenoid extraction using the respective methodologies. UAE: ultrasound-assisted extraction; MAE: microwave-assisted extraction; PEF: pulsed electric field; PLE: pressurized liquid extraction; SuperFE: supercritical fluid extraction; SubFE: subcritical fluid extraction; EAE: enzymeassisted extraction. Source for article search: Scopus. Antioxidants 2024,13, 239 4 of 27 Table 1. Extraction mechanisms of the green extraction technologies. Extraction Method Mechanism Ref UAE Utilizes sound waves for inducing cavitation in the solution, thereby facilitating the extraction process. [11] MAE Generates heat within the solvent by employing ionic conduction of the dipole rotation and dissolved ions in the polar solvent. [12] PEF Applies high-voltage microsecond pulses to induce pores in cell membranes, resulting in the disruption of barrier function and leakage of intracellular content. [13] PLE Utilizes organic solvents under high pressures and temperatures, exceeding the boiling point, amplifying the solubility of analytes, and reducing solvent viscosity. This approach minimizes the required time and solvent volume. [14] SuperFE Utilizes CO 2 in supercritical state as extraction solvent, segregating the analytes according to their relative solubility. Supercritical CO 2 has a high density and solvent power, similar to that of a liquid. [15] SubFE Extracts less-polar compounds using water or other fluids in subcritical conditions under high pressures and high temperatures, sustaining subcritical fluids in a liquid state for a brief extraction period. [16] EAE Encompasses the binding of cells to the active site of an enzyme, which induces a transformation of the enzyme form to adapt to the substrate. Consequently, the active components are released from the cells into the extraction medium. [17] Table 2. Main results of studies on carotenoid recovery with green extraction methodologies. Extraction Method Conditions Solvent Matrix Results Ref CSE and UAE (bath) CSE (290 rpm) and UAE (40 kHz, 80 W): time (6, 10, 20, 30, 34 min), SSR (6 mL/59, 80, 130, 180, 201 mg). Acetone, ethanol, petroleum ether, and methanol Cashew apple Optimal conditions: CSE: 38% acetone, 30% ethanol, and 32% petroleum ether, 23 min, and 136 mg; UAE: 44% acetone and 56% methanol, 19 min, and 153 mg. UAE achieved a ~21% faster and higher carotenoid yield in all samples compared to CSE. [18] CSE, MAE, and UAE (probe) CSE: 30 min, RT, SSR: 10 mL/g, 150 rpm; MAE: 100 W, 30 min, 60 ◦C, SSR: 1 g/10 mL. UAE: 40 kHz, 50 W, 60%, 30 min, SSR: 10 mL/g; UAE + MAE (same conditions). Menthol/camphor (1:1, n:n) Orange peel Significantly higher extraction with UAE (~2-fold) compared to CE, no statistical differences between CE and MAE or between UAE and UAE+MAE. [19] Antioxidants 2024,13, 239 5 of 27 Table 2. Cont. Extraction Method Conditions Solvent Matrix Results Ref UAE (probe) 20 kHz, 500 W, amplitude (20, 30, 40, 50, 60, 70%), time (10, 15, 20, 25, 30, 35 min), temperature (55, 60, 65, 70, 75, 80 °C), SSR (10, 20, 30, 40, 50, 60 mL/g). Ethyl lactate, limonene, soybean oil, and sunflower oil Sargassum fusiforme Optimal conditions: ethyl lactate, 20 kHz, 500 W, 53%, 27 min, 75 °C, and SSR: 40 mL/g. [20] CSE and UAE (bath) CSE: 90 min, 40 ◦C, SSR: 40 mL/g; UAE: 45 kHz, ultrasonic power (150, 180, 210 W), time (30, 40, 50 min), and SSR (30, 35, 40 mL/g). Ethanol–petroleum ether mixture (2:1, v/v)Cucurbita moschata Optimal UAE conditions: 45 kHz, 203 W, 30 min, and 31 mL/g. Compared to the CSE, UAE avoided degradation and isomerization, resulting in a higher yield. [21] UAE (probe) 20 kHz, amplitude (20, 40, 60, 80, 100%), time (10, 20, 30, 40 min), SSR (30, 40, 50, 60 mL/g), and hexane/acetone ratio (50/50, 70/30, 90/10). Hexane/acetone mixture Cantaloupe waste Optimal UAE conditions: 20 kHz, 100%, 10 min, 55 mL/g, and hexane/acetone ratio of 80/20. [22] UAE (bath) 40 kHz, 80 W, time (14, 20, 30, 40, 44 min), and SSR (10 mL/10, 30, 80, 130, 150 mg). Acetone/ethanol (3:1) Buriti (Mauritia flexuosa) Optimal UAE conditions: 40 kHz, 80 W, 30 min, 80 mg/10 mL, acetone/ethanol (75:25). [23] UAE (probe) 20 kHz, 70% amplitude, time (3, 10, 20, 30, 37 min), temperature (10, 20, 35, 50, 60 ◦C), SSR (30, 50, 70 mL/g), solvent percentage (13, 30, 55, 80, 97%). Ethanol, methanol, acetone, acetonitirile, and n-hexane Carrot pomace Optimal UAE conditions for total carotenoid content: 17 min, 32 ◦C, 50 mL/g, and 51% ethanol. [24] UAE (probe) UAE: 20 kHz, 30%, 2 min, SSR: 2 mL/0.1 g. Ethanol, methanol, ethyl lactate, MeTHF, and DMSO Chlorella sorokiniana The best solvent depended on the matrix (fresh: ethanol; freeze-dried: methanol; encapsulated: MeTHF and ethyl lactate). [25] CSE and MAE CSE: 30 min, 60 ◦C, 100 mg/5 mL (10 M aqueous KOH with 2.5% ascorbic acid); MAE: time (0, 15, 30, 45, 60, 180, 300, 600 s), 60 ◦C, SSR (1 mL/10, 20, 30, 40, 50, 60 mg), KOH concentration (0, 2, 4, 6, 8, 10, 12 M). Ethanol Chlorella sorokiniana Optimal alkali-assisted MAE pretreatment conditions for lutein: 850 W, 1.47 min, 8.16 M KOH, and SSR of 36.8:1 mg/mL. Lutein yield obtained via MAE was 3.26 folds higher than that obtained via CSE. [26] MAE Power (50, 80, 125, 170, 200 W), time (1, 3.14, 6.3, 9.46, 12 min), and oil-to-waste ratio (5:1, 8:1, 12.5:1, 17:1, 20:1 g/g). Flaxseed oil Carrot juice Optimal conditions: 165 W, 9.39 min, and SSR 8.06:1 g/g. [27] Antioxidants 2024,13, 239 6 of 27 Table 2. Cont. Extraction Method Conditions Solvent Matrix Results Ref MAE and UAE (probe) MAE: power (30, 40, 50 W), time (5, 7, and 9 min), and SSR (10, 20, 30 mL/g); UAE: 20 kHz, power (375, 562.5, 750 W), time (2, 9, 16 min), and SSR (10, 30, 50 mL/g). MAE: acetone, ethanol, petroleum, ether/acetone/ethanol mixture (2:1:1), and nhexane/acetone/ethanol mixture (2:1:1); UAE: acetone, N,N-dimethylformamide, isopropanol/n-hexane mixture (1:1), petroleum ether/acetone mixture (1:1), and petroleum ether/acetone/ethanol mixture (2:1:1) Aristeus antennatus shrimp Optimal MAE conditions: 30 W, 7 min, 20:1 mL/g, and n-hexane/acetone/ethanol 2:1:1 (v/v/v); Optimal UAE conditions: 20 kHz, 600 W, 5 min, 10:1 mL/g, and acetone. No differences between UAE and MAE in carotenoid extraction from the head of the shrimp. UAE resulted in a ~2 times higher total carotenoid extraction compared to that of MAE in the body of the shrimp. [28] PEF 3.5 kW of power (15 kV/cm, 150 µs). Ethanol Rhodotorula glutinis PEF treatment without incubation did not recover carotenoids; however, PEF with 1 h incubation (20 ◦C) permits the extraction of carotenoids from fresh biomass. [29] Bead beating, freeze-thawing, thermal treatment, PEF, and UAE (probe) PEF: 1 Hz, 1 kV/cm, 10 pulses, 50 kJ/Kg; Bead beating: 4800 rpm (5–10 cycles of 60 s), 6 h incubation time; UAE: 450 W, 80%, 10 times for 10 s; Thermal treatment: 1 h, 70 ◦C; Freeze-thawing: in liquid nitrogen and left to melt on ice (repeated 5 times). Ethanol, acetone, and methanol Haematococcus pluvialis and Chlorella vulgaris The best extraction yields were achieved after PEF pretreatment with 6 h incubation and ethanol. Statistically significant differences in the extracted carotenoid yields after PEF pretreatment compared to other treatments in H. pluvialis cells grown in the control BBM medium or in N-free BBM medium supplemented with 6 g/L glucose. [30] CSE and PEF CSE: 25 ◦C, 120 rpm, SSR: 100 mL/5 g, hexane/ethanol/acetone (50:25:25); PEF: 1 Hz, 5 kV/cm, 90 µs, time (20, 160, 300 min), SSR: 5 g/100 mL, hexane/ethanol (25:75, 50:50, 75:25). Acetone, hexane, and ethanol mixtures Tomato waste Optimal PEF conditions: 1 Hz, 5 kV/cm, 90 µ s, 150 min, and 30% hexane. PEF treatment improved the carotenoid extraction by 39% as compared with CSE in a mixture of hexane/ethanol/acetone (50:25:25). [31] Antioxidants 2024,13, 239 7 of 27 Table 2. Cont. Extraction Method Conditions Solvent Matrix Results Ref CSE and PEF CSE: 100 µL sample/1 mL solvent, vortexed; PEF: 0.5 Hz, 20 kV/cm, 75 µ s, with or without 1 h incubation (20 ◦C). Ethanol (96%) Chlorella vulgaris Extraction yield with PEF was significantly higher than that with CSE. Pre-incubation for 1 h after PEF treatment improved extraction yields. [32] ASE, CSE, PEF, and PEF+ASE CSE: 400 rpm, 30 min, RT, SSR (1:10, w/v); PEF: 100 kJ/kg, 74 pulses, 300 mL of tap water/30 g; PEF+ASE: 1 min of preheating period, 5 min of heating period, 60% of flush volume, 10 MPa of extraction pressure (for 15 min) and 60 s of nitrogen purge, 50 ◦C. DMSO and ethanol Shrimp by-products PEF+ASE with DMSO resulted in the highest carotenoid recovery from Aristeus antennatus. The antioxidant capacity varied depending on solvent. [33] CSE and PLE CSE: 15 min, 40 ◦C, SSR (20 mL/0.5 g); PLE: preheating time: 1 min, heating time: 5 min, 40 ◦C, SSR (0.5 g/20 mL). DMSO (0, 30, 50, and 100%) Spirulina, Chlorella and Phaeodactylum tricornutum powder Carotenoid recovery with PLE was significantly higher than that with CSE; 100% DMSO enhanced the extraction yields of carotenoids significantly. [34] CSE, PEF, PLE, and PEF+PLE CSE: 6 h, 40 ◦C, SSR: 20 mL/g (chloroform/methanol (5:2, v/v)); PEF: 3 kV/cm, 44 pulses, 99 kJ/kg energy input, SSR: 200 mL H2O/2 g; PLE: 15 min, 40 ◦C, 1 min preheating period, 5 min heating period, flush volume of 60%, nitrogen purge of 60 s, 103.4 bars,1000–2000 µ S/cm, SSR: 20 mL/0.5 g (H2O, 50% DMSO, 100% DMSO). H2O, 50% DMSO, 100% DMSO Spirulina biomass The extraction yield with PEF + PLE (50% DMSO) was significantly higher than those with PEF, PLE, or CSE. PEF + PLE increased efficiency and reduced the extraction time. [35] PLE 20 min, temperature (50, 100, 150 ◦C), 103.4 bar, nitrogen purge 300 s. Ethanol, ethyl acetate, and n-hexane Chlorella vulgaris and Phaeodactylum tricornutum (wet and dry biomasses) Best conditions for total lipid yield: ethyl acetate and n-hexane (temperature did not affect) for dry C. vulagris; ethyl lactate at 150 ◦C for wet C. vulgaris; ethanol at 100 or 150 ◦C for dry P. tricornutum; and ethanol at 150 ◦C for wet P. tricornutum. [36] Antioxidants 2024,13, 239 8 of 27 Table 2. Cont. Extraction Method Conditions Solvent Matrix Results Ref CSE and SuperFE CSE: 30 min, 40 ◦C, 1 mL/g; SuperFE: 40 − 60 ◦ C, CO 2 flow rate 16.5 g/min, equilibration time (0−30 min), extraction pressure (200 − 500 bar), and ethanol content in supercritical fluid mixture (8−13 wt %), with or without MW pretreatment (time (30–90 s), temperature (41–140 ◦C), up to 300 W). Acetone Brown crab (Cancer pagurus) processing waste Optimal SuperFE conditions: 500 bar, 40 ◦C, and 13 wt % ethanol content after an optimized MW pretreatment (140 ◦C, 90 s, 300 W). [37] CSE, MAE, and SuperFE CSE: 24 h, 30 ◦C, 500 rpm, SSR: 37 mL/g; MAE: time (5–25 min), temperature (40–60 ◦C), power (300–800 W), SSR (20–90 mL/g); SuperFE: 60 ◦C, 250 bar, flow rate 40 g/min, total solvent consumption 100 kg CO2/kg biomass. Ethanol 90% (v/v)Chlorella vulgaris Optimal MAE conditions: 14 min, 60 ◦C, 300 W, and 22 mL/g. CSE presented the highest yield. MAE and SFE led to an antioxidant capacity similar to or better than CSE in a significantly shorter extraction time. [38] SuperFE Extraction time (60, 90, 135, 180, 210 min), temperature (55, 60, 70, 80, 85 ◦C), pressure (250, 300, 375, 450, 500 bar), SSR 1 mL/0.1 g, flow rate CO2(35 g/min), and rice bran oil (3%, w/w). CO2with rice bran oil as co-solvent Citrus paradise Macfad Optimal conditions for lycopene extraction: 143 min, 64 ◦C, and 325 bar. Extraction temperatures higher than 80 ◦C and time lower than 180 min led to lycopene isomerization. [39] SuperFE 80 min, temperature (50, 60, 70 ◦C), pressure (150, 250, 350 bar), CO2flow rate 15 g/min, and co-solvent concentration (5, 10, 15%, v/v). CO2with ethanol as co-solvent Carrot peels Optimal conditions: 59 ◦C, 349 bar, and 15.5% ethanol. [40] SubFE 15 min, temperature (303, 318, 333 K), pressure (5, 11, 17 MPa), co-solvent percentage (2, 4, 6%), 50 min with a constant flow rate of 10 g/min. 1,1,1,2-Tetrafluoroethane with ethanol as co-solvent Laminaria japonica Optimal conditions: 324.13 K, 17 Mpa, and a co-solvent amount of 4.73%. [41] SubFE and UAE (bath) UAE (to select best solvent): 15 min, 35 ◦C, SSR: 10 mL/0.5 g. SubFE: extraction time (30, 60, 90 min), extraction temperature (35, 50, 75 ◦C), pressure of 20 MPa, SSR (200:1, 100:1, 20:1 (v/wet weight). Hexane, DCM, ethanol, methanol, THF, ultrapure water, and THF:DCM (1:1). Phaeodactylum tricornutum Optimal conditions: methanol, 60 min, 35 ◦C, 20 MPa, 120 rpm, and SSR 200:1. [42] Antioxidants 2024,13, 239 9 of 27 Table 2. Cont. Extraction Method Conditions Solvent Matrix Results Ref SOX and SubFE SOX: 360 min, temperature (341.15 K for n-hexane, and 333.15 K for petroleum ether), SSR: 200 mL/ 5 g, solvent removed at 316.15 K; SubFE: 60 min, temperature (293.15, 313.15, 333.15 K), pressure (2, 6, 10 MPa), propane flow rate of ~2 cm3/min. SOX: n-hexane or petroleum ether; SubFE: propane (Maximiliana maripa) pulp oil Optimal SubFE conditions: 293.15 K and 2 MPa. Extraction yield with SOX (n-hexane) was significantly higher than that with SubFE. [43] SOX, SubFE, and SuperFE SuperFE: 52.5 ◦C, 27.50 MPa, 5 mL CO2/min flow rate; SubFE: 12 h, 29 ◦C, 6.8 MPa, 250 extraction cycles; SOX: 12 h, 70 ◦C. SuperFE: CO2; SubFE: ethanol; SOX: n-hexane Saudi date fruit flesh (Sukari, Ambara, Majdool, and Sagai date fruit) The highest carotenoid extraction was found as follows: Sukari (via SubFE) and Ambara, Majdool, and Sagai date fruit (via SuperFE). [44] EAE Pretreatment time (0.5, 2, 3.5, 5, 6.5 h), extraction time (0.5, 1.5, 2.5, 3.5, 4.5 h), extraction temperature (10, 20, 30, 40, 50 ◦C), enzyme solution-to-solid ratio (10, 20, 30, 40, 50 dm3/kg), and enzyme load (0, 0.05, 0.1, 0.15, 0.2 kg/kg); SSR: 30 mL/0.5 g. Hexane, enzyme/Peclyve PR and Cellulyve 50LC (50:50) Tomato waste Optimal conditions: 3.18 h, 30 ◦C, and 0.16 kg/kg enzyme load. [45] EAE Time (12, 18, 24 h), temperature (30, 33.5, 37 ◦C), enzyme dose (0.15, 0.3, 0.45, 0.6, 0.75 mL), pH (4.6, 6, 7.4). 95% Ethanol, enzyme/fructozym®MA Carrot Optimal conditions: 24 h, 37 ◦C, pH 7.4, and 0.3 mL enzyme dose. [46] CSE and EAE CSE (to select the best solvent): 30 min, RT, SSR: 20 mL/4 g; EAE: time (1, 2.5, 4 min), temperature (40, 50, 60 ◦ C), SSR (1 mL/5, 17.5, 30 g), enzymatic reaction time (1, 3, 5 h), enzyme/substrate ratio (0.2, 1.1, 2 mL/g), enzyme/enzyme ratio (1, 2, 3). CSE: acetone, ethyl acetate, ethanol, and 1:1 combinations, and ethanol/water mixture (1:1); EAE: ethyl acetate, enzymes: pectinolytic enzyme (P), cellulolytic enzyme (C), and a combination of carbohydrases, including arabanase, cellulase, β -glucanase, hemicellulase, and xylanase (V) Tomato waste Optimal EAE conditions: C-P combination for enzymatic pretreatment, ethyl acetate as solvent, 1 h extraction time, 40 ◦C, 5 h enzymatic reaction time, 0.2 mL/g enzyme/substrate ratio, 5 mL/g solvent/substrate ratio, and 1 enzyme/enzyme ratio. [47] ASE: accelerated solvent extraction; BBM: Bold’s basal medium; CH: Conventional heating; CSE: Conventional solvent extraction; DCM: dichloromethane; DMSO: dimethylsulfoxide; EAE: enzyme-assisted extraction; MAE: microwave-assisted extraction; MeTHF: 2-methyltetrahydrofuran; MW: microwave; PEF: pulsed electric field; PLE: pressurized liquid extraction; RT: room temperature; SOX: Soxhlet extraction; SSR: solvent-to-solid ratio; SubFE: subcritical fluid extraction; SuperFE: supercritical fluid extraction; THF: tetrahydrofuran; UAE: ultrasound-assisted extraction. Antioxidants 2024,13, 239 16 of 27 followed by ethyl acetate extraction has also been optimized through RSM. The optimized parameters for the enzyme pretreatment were determined as celluclast/pectinex (1:1), an enzyme/substrate ratio of 0.2 mL/g, 5 h of enzymatic reaction time, 40 ◦ C, and pH 4.5. The optimized parameters for the extraction with ethyl acetate were as follows: a solvent– substrate ratio of 5 mL/g, 1 h extraction in a shaker, and room temperature. This method results in a recovery of 9.16 mg lycopene/g tomato waste. On the other hand, lycopene could not be detected in the matrix extracted without the enzymatic pretreatment [47]. 5. Extraction of Carotenoids with Bio-Based Solvents The choice of solvent for carotenoid extraction is crucial to achieve an efficient extraction and high yields [ 6 ]. In light of the current environmental challenges, the selection of an appropriate extraction solvent should consider not only its effectiveness, but also its environmental impact. Prioritizing the environmental impacts of extraction solvents is crucial in mitigating the impact of extraction processes on the environment. Bio-based solvents are derived from renewable biomass sources, such as crops, forestry residues, and other plant-based materials. These types of solvents are gaining popularity as an alternative to other traditional solvents derived from petrochemicals, which are non-renewable and have negative environmental impacts [61]. The use of bio-based solvents has several advantages over other traditional solvents. Firstly, bio-based solvents are usually more sustainable and environmentally friendly, as they are derived from renewable resources. In addition, they generally have lower toxicity and are safer for human health. Finally, in some cases, they may have higher solubility and better compatibility with other chemicals. However, bio-based solvents also have some disadvantages, mainly related to the cost of production, which can be higher than that of other traditional solvents due to the higher cost of sourcing and processing renewable biomass [ 62 , 63 ]. The use of bio-based solvents for the extraction of carotenoids has been broadly studied and, overall, offers several advantages over petroleum-derived solvents in terms of environment [ 64 ]. The following bio-based solvents that have been used to extract carotenoids will be evaluated in this review: ethyl lactate, 2-methyltetrahydrofuran (2-MeTHF), natural deep eutectic solvents (NaDESs), deep eutectic solvents (DESs), and ionic liquids (ILs) (Figure 2). The main results of the studies on carotenoid recovery with bio-based solvents discussed in this review are summarized in Table 3. Antioxidants 2024, 13, x FOR PEER REVIEW 15 of 29 5. Extraction of Carotenoids with Bio-Based Solvents The choice of solvent for carotenoid extraction is crucial to achieve an efficient extraction and high yields [6]. In light of the current environmental challenges, the selection of an appropriate extraction solvent should consider not only its effectiveness, but also its environmental impact. Prioritizing the environmental impacts of extraction solvents is crucial in mitigating the impact of extraction processes on the environment. Bio-based solvents are derived from renewable biomass sources, such as crops, forestry residues, and other plant-based materials. These types of solvents are gaining popularity as an alternative to other traditional solvents derived from petrochemicals, which are non-renewable and have negative environmental impacts [61]. The use of bio-based solvents has several advantages over other traditional solvents. Firstly, bio-based solvents are usually more sustainable and environmentally friendly, as they are derived from renewable resources. In addition, they generally have lower toxicity and are safer for human health. Finally, in some cases, they may have higher solubility and better compatibility with other chemicals. However, bio-based solvents also have some disadvantages, mainly related to the cost of production, which can be higher than that of other traditional solvents due to the higher cost of sourcing and processing renewable biomass [62,63]. The use of bio-based solvents for the extraction of carotenoids has been broadly studied and, overall, offers several advantages over petroleum-derived solvents in terms of environment [64]. The following bio-based solvents that have been used to extract carotenoids will be evaluated in this review: ethyl lactate, 2-methyltetrahydro- furan (2-MeTHF), natural deep eutectic solvents (NaDESs), deep eutectic solvents (DESs), and ionic liquids (ILs) (Figure 2). The main results of the studies on carotenoid recovery with bio-based solvents discussed in this review are summarized in Table 3. Figure 2. Number (inside the chart) and percentage (outside the chart) of published research articles related to carotenoid extraction using the respective solvent. 2-MeTHF: 2-Methyltetrahydrofuran; DES: deep eutectic solvent; NaDES: natural deep eutectic solvent; ILs: ionic liquids. Source for article search: Scopus. Figure 2. Number (inside the chart) and percentage (outside the chart) of published research articles related to carotenoid extraction using the respective solvent. 2-MeTHF: 2-Methyltetrahydrofuran; DES: deep eutectic solvent; NaDES: natural deep eutectic solvent; ILs: ionic liquids. Source for article search: Scopus. Antioxidants 2024,13, 239 17 of 27 Table 3. Main results of studies on carotenoid extraction with green solvents. Solvent Extraction Method Conditions Matrix Results Ref Ethyl lactate, acetone, ethyl acetate, hexane, and ethanol CSE SSR: 10 mL/g, extract removed at different time intervals (5–40 min), and temperatures (25, 50, 70 ◦C). Tomato waste Optimal conditions: ethyl lactate, 30 min, and 70 ◦C. [65] Ethyl lactate/ethanol (3:2) CSE Time (10–40 min), temperature (10–30 ◦C), 360 rpm, and crude palm oil proportion (20–60%). Palm olein Optimal conditions: 10 min, 20 ◦C, and 50% of crude palm oil. [66] Hexane, ethyl acetate, ethyl lactate, and ethyl acetate/ethyl lactate (1:3, v/v). UAE (bath) 10 min, 35 ◦ C, and 1 g/20 mL. Tomato by-products The highest lycopene extraction was obtained with ethyl lactate in the wet sample. [67] Ethyl lactate and ethyl lactate/ethanol mixtures (0–100%) CSE Time (0–350 min), temperature (30, 45, and 60 ◦C), and SSR (0.25–1.0 g/10 mL). Tangerine and red tomato, corn, and carrot Optimal conditions: lutein (2 h, 30 ◦C), β-carotene (0.5 h, 30 ◦C), and lycopene (1 h, 45 ◦C). The addition of R-tocopherol or R-lipoic acid improves the extraction efficiency. Ethyl lactate is an excellent solvent for extracting trans- and cis-lycopene isomers from dried tomato powder. [68] Ethanol, acetone, ethyl lactate, sunflower oil, and water UAE (bath) and heating UAE: 35 kHz, 30 min, and 40 ◦C; Heating: 30 and 60 min and 45 and 100 ◦C. Lycium fruits (Goji, naturally dried and freeze-dried) The highest carotenoid levels were obtained via UAE (with water and ethyl lactate) and heating (with ethyl lactate). The lowest content was obtained with sunflower oil, ethanol, and acetone. Water was a better solvent for naturally dried berries and ethyl lactate and sunflower oil for the freeze-dried sample. [69] MeTHF, dimethyl carbonate, cyclopentyl methyl ether, isopropyl alcohol, ethyl acetate, and n-hexane CSE 1 h maceration, 65 ◦C, and 30 g/125 mL. Daucus carota The best green solvents were as follows in descending order: cyclopentylmethyl ether, MeTHF, and ethyl acetate. [70] MeTHF, hexane, tetrahydrofuran, acetone, and 1,4- dioxane CSE First maceration overnight with SSR 1:10 (w/v) and second maceration with SSR 1:2 (w/v). Tagetes erecta L. MeTHF is a potential green alternative solvent to hexane and tetrahydrofuran for lutein extraction. [71] Antioxidants 2024,13, 239 18 of 27 Table 3. Cont. Solvent Extraction Method Conditions Matrix Results Ref Hexane, dry MeTHF, and MeTHF 95.5% SOX 4.5 h. Olive pomace extracts Dry MeTHF and MeTHF 95.5% recovered carotenoid yields ~11.3 and 12.4 folds higher, respectively, than that of hexane. [72] HBA:HBD (3:1), being HBA ethanol, N,N-dimethylcyclohexylamine, N,N-dimethyloctylamine, and N,N-dimethylbenzylamine and HBD n-butanol GAE, WBE, and UAE GAE: 30 Hz, 40 s, and SSR 1:4; WBE: 120 min, 50 ◦C, and SSR 1:4; UAE: 40 kHz, 15 min, 50 ◦C, and SSR 4 mL/g. Millet DESs extracted a significantly higher carotenoid yield compared to ethanol. GAE resulted in the best extraction method. [73] Acetone, DL-menthol or thymol, and capric acid as HBAs; capric acid and lauric acid as HBDs CSE 60 min, RT, 750 rpm, and SSR 40 mL/g. Tomato The combination of capric acid and lauric acid exhibited extraction capacities comparable to that of acetone. [74] Acetone; cholinium-based ILs: choline bicarbonate (Ch), octanoic acid (Oct), hexanoic acid (Hex); butyric acid (But), and lactic acid (Lac) CSE Time (0, 20, 60, 120, and 144 min), temperature (11.4, 25, 45, 65, and 78.4 ◦C), enzyme concentration (0, 20, 50, 80, and 100 wt %), and SSR: 0.2 g/1 mL. Phaffia rhodozyma Optimal conditions: [Ch][Oct], 60 min, 45 ◦C, and 50 wt % in water. [75] Acetone, methanol, dipropylammonium dipropylcarbamate (DPCARB), diallylammonium diallylcarbamate (DACARB), and dibutylammonium dibutylcarbamate (DBCARB) CSE Time (30, 45, 60, 75, and 90 min), temperature (25, 35, 45, and 55 ◦C), and DPCARB/methanol ratio (9:1, 8:2, 7:3, 6:4). Chlorella sorokiniana Optimal conditions: DPCARB/methanol (9:1), 45 min, and 25 ◦C. [76] Methanol, IL1 [1-decyl 3 methyl imidazolium chloride], IL2 [tetrabutyl phosphonium hydroxide], IL3 [tetrabutyl hexadecyl phosphonium bromide], and IL4 [tetrabutyl ammonium hydroxide] CSE Time (5, 10, and 15 min), temperature (25, 40, and 55 ◦C), IL concentration (5, 22.5, and 40%), SSR (0.1, 0.3, and 0.5 mL/mg). Chlorella saccharophila Optimal conditions: IL2, 5 min, 25 ◦C, 40% IL concentration, and 0.5 mL/mg SSR. [77] CSE: Conventional solvent extraction; GAE: grinding-assisted extraction; HBA: hydrogen bond acceptor; HBD: hydrogen bond donor; ILs: Ionic liquids; MeTHF: 2-methyltetrahydrofuran; RT: room temperature; SOX: Soxhlet extraction; SSR: solvent-to-solid ratio; UAE: ultrasound-assisted extraction; WBE: water bath extraction. Antioxidants 2024,13, 239 19 of 27 5.1. Ethyl Lactate Ethyl lactate is an organic compound that occurs naturally in small amounts in certain foods, such as poultry, fruits, and wine. It can be obtained by esterifying lactic acid and ethanol at high temperatures (80–120 ◦ C) and low pressure and using a catalyst, such as sodium hydroxide. Ethyl lactate is separated from the resulting mixture via distillation. As the production has a low environmental impact and the lactic acid used can be obtained through the fermentation of sugars by certain microorganisms, such as lactic acid bacteria, ethyl lactate is considered a renewable alternative to petroleum-derived solvents [ 78 , 79 ]. Ethyl lactate is biodegradable, which means that it can be broken down by water or microorganisms in the environment. In addition, it is considered a non-toxic solvent [ 80 ]. Its physical and chemical properties make it desirable for carotenoid extraction (Table 4). Ethyl lactate has been used for carotenoid extraction from different matrices such as dry tomato waste, crude palm olein (CPO), and C. sorokiniana, among others [ 25 , 65 , 66 ]. In particular, 17 research articles have evaluated the extraction of carotenoids with this solvent (Figure 2). This number represents 20% of the studies in which carotenoids are extracted with the bio-based solvents evaluated in this review (NaDESs, DESs, ethyl lactate, ILs, and MeTHF). Taking into account that NaDESs, DESs, and ILs represent a set of solvents, it is likely that ethyl lactate is the most researched bio-based solvent (among those evaluated in this review) for the extraction of carotenoids (Figure 2). Table 4. Physical and chemical properties of 2-methyltetrahydrofuran and ethyl lactate. Solvent 2-Methyltetrahydrofuran Ethyl Lactate Molecular formula C5H10O C5H10O3 Molecular weight (g/mol) 86.132 118.131 Appearance Colourless Colourless Stability Stable, but highly flammable. Not suitable for use with oxidizing agents, potent acids, or strong bases. There is a risk of developing explosive peroxides during storage, which is why it is frequently supplied with an inhibitor as a precaution. Stable, flammable, and not compatible with powerful oxidizing agents. Toxicity LD50 orally in Rabbit: >300–2000 mg/kg. LD50 dermal Rat > 2000 mg/kg LD50 orally in Rat (female): >2.000 mg/kg. LD50 dermal Rabbit: >5.000 mg/kg Safety Flammable, corrosive, and irritant. Acute toxicity. It requires safety glasses, good ventilation, a test for the presence of peroxides before use, and the removal of ignition sources from the working area. Flammable, corrosive, and irritant for eyes and skin. Melting point (◦C) −136 −26 Boiling point (◦C) 82 154 Organic solvent solubility Miscible Miscible Water solubility Immiscible Miscible (with partial decomposition) Density (g/mL) 0.855 1.03 Refraction index 1.407 1.415 Dielectric constant 6.97 13.1 Molar refraction (mL/mol) 24.8 28.72 Dipole moment (D) 1.38 2.55 LD50, median lethal dose. Data obtained from ChemSpider, ChemicalBook, and Stenutz [81–83]. It was observed that in a conventional carotenoid extraction (30 min, 70 ◦ C) of dry tomato waste, the carotenoid yield obtained with ethyl lactate (243.00 mg/kg) was much higher than those obtained with acetone (51.90 mg/kg), ethyl acetate (46.21 mg/kg), hexane (34.45 mg/kg), and ethanol (17.57 mg/kg) [ 65 ]. In CPO, the extraction of carotenoids and tocols was optimized using a mixture of ethyl lactate and ethanol (3:2, v/v), setting Antioxidants 2024,13, 239 20 of 27 the optimal parameters as 20 ◦ C, 10 min of mixing (at 360 rpm), and 1 mL CPO/mL extraction solvent [ 66 ]. Finally, in encapsulated C. sorokiniana, ethyl lactate recovered a higher total carotenoid content compared to 2-MeTHF, ethanol, methanol, and DMSO, although significant differences were found only with ethanol and DMSO [25]. Szabo et al. (2022) found that in the lycopene extraction using an ultrasonic bath (10 min at 35 ◦ C), the use of ethyl lactate as a solvent resulted in 1.4- and 2.5-fold increases in the extraction yield compared to hexane in fresh and freeze-dried tomatoes, respectively [ 67 ]. Furthermore, it has been suggested that ethyl lactate is a suitable solvent for extracting both trans- and cis-lycopene from lyophilized tomatoes using a temperaturecontrolled water bath (45 ◦ C for 1 h) [ 68 ]. In another study, the total carotenoid content extracted from freeze-dried goji berries via UAE (40 ◦ C, 30 min, 35 kHz) using ethyl lactate as the extraction solvent was found to be significantly higher than those extracted with ethanol (2.4–7.0 folds higher), acetone (2.6–8.7 folds higher), and sunflower oil ( 1.2–1.6 folds higher) [ 69 ]. These findings highlight the potential of ethyl lactate as a promising solvent for the extraction of carotenoids from different sources. 5.2. 2-Methyltetahydrofuran 2-Methyltetrahydrofuran (2-MeTHF) has recently gained attention, as its synthesis pathway is produced from inedible renewable resources. It is known to degrade when exposed to air and sunlight, forming cellulose precursors such as xylose and glucose, although the mechanism of degradation is poorly understood [ 84 ]. 2-MeTHF has different uses in biolubricants, fuel additives, reagents, polymer precursors, foam materials, resins, environmentally friendly solvents, plasticizers, flavour and food additives, coatings, pharmaceuticals, cosmetics, surfactants, and intermediates for the manufacturing of herbicides and polycarbonates. Its diverse properties and broad applications make it a valuable compound for research and development in the chemical industry (Table 4) [ 85 ]. Its high boiling point (82 ◦ C) can be advantageous for certain purposes, as it reduces the reaction time and enables reactions that require higher temperatures and its separation from water via distillation without adding drying agents. In addition, its low miscibility allows a clean workup. Furthermore, the production of 2-MeTHF from renewable sources has a low carbon footprint and has no known genotoxic or mutagenic effects [ 84 , 86 ]. This green solvent has been recently assessed by the European Food Safety Authority (EFSA) in terms of toxicity, concluding that a daily intake lower than 1 mg 2-MeTHF/kg body weight has no safety concerns [87]. 2-MeTHF has recently emerged as a potential alternative to conventional organic solvents, such as n-hexane, for the extraction of various bioactive compounds, including carotenoids, from natural sources [ 70 ]. There are limited studies on the extraction of carotenoids with 2-MeTHF, representing 6% of the studies in which carotenoids are extracted with the bio-based solvents evaluated in this review (NaDESs, DESs, ethyl lactate, and ILs) (Figure 2). Kashyap et al. (2022) concluded that 2-MeTHF is a promising alternative to n-hexane and tetrahydrofuran for the extraction of lutein throughout maceration extraction (three washes: 1:10 (w/v) 3 h, 1:5 (w/v) 3 h; 1:5 (w/v) 24 h) from flowers of Tagetes erecta [ 71 ]. In a recent study, it was reported that the extraction of olive pomace using the Soxhlet extraction technique (4.5 h) with aqueous 2-MeTHF (95.5%) and dry 2-MeTHF (recovered via distillation), resulted in 11.2 folds and 12.5 folds higher carotenoid concentrations, respectively, compared to those obtained using n-hexane [ 72 ]. Yara-Varon et al. reported that a conventional solid–liquid extraction via maceration (65 ◦ C, 1 h, solid- to-liquid ratio of 3:12.5 (g/mL)) in carrots with 2-MeTHF resulted in a 12% higher total carotenoid content compared to that obtained with n-hexane [ 70 ]. As already commented, 2-MeTHF has also been proven to be efficient for the extraction of carotenoids from wildtype and phytoene-accumulating Chlorella sorokiniana [ 25 ]. Overall, these studies suggest that 2-MeTHF could serve as a promising alternative to conventional organic solvents for the extraction of carotenoids from natural sources. Antioxidants 2024,13, 239 21 of 27 5.3. Natural Deep Eutectic Solvents and Deep Eutectic Solvents There are several definitions for describing natural deep eutectic solvents (NaDESs, or commonly named as NADES) and deep eutectic solvents (DESs), the most general being a mixture of two or more compounds associating through hydrogen bonding. The specific interactions among these constituents (the formation of a hydrogen bonding network, which increases the system’s stability and, in the case of ionic constituents, allows the charge delocalization) generally enable a significantly lower melting point of these solvents compared to those of their constituents. Thus, the melting points of some NaDESs (mainly those containing amides, carboxylic acids, and sugar-derived polyols with organic salts) are even below room temperature. Some factors also result in decreases in the melting points of these solvents. The asymmetry of the cationic constituents lowers the melting point due to lower lattice energy, while increasing the electron affinity of the anionic constituents also lowers the melting point as it results in stronger hydrogen bonds between the two components. Furthermore, the addition of a ternary component to the system or stronger interactions between the components can also lead to a decrease in the melting point. It has been observed that NaDES components with lower molecular weight have a greater melting point depression [ 88 ]. NaDESs are a type of DES that are synthesized from natural components, such as sugars, making them more environmentally friendly than DESs [ 89 ]. Both solvents have unique properties, such as their polarity, ionic conductivity, and phase behaviour, that make them promising alternatives to traditional solvents in various extraction and processing applications. NaDESs are characterized by a high viscosity, which can be a limitation for industrial applications, although this can be addressed by adjusting the water content or solvent temperature. DESs, on the other hand, are characterized by low volatility and non-flammability, which make them ideal for lignocellulosic biorefinery applications [90,91]. DESs are classified into four different types (type I–IV) depending on the components used in their synthesis, water miscibility, and pH conditions. However, a new type V DES has recently been proposed. These V-type DESs can be easily recovered and revitalized through an evaporation process. This attribute makes them a more practical option for use in certain industrial bioprocesses, unlike other DES varieties [90]. DESs and NaDESs have emerged as promising alternatives to conventional solvents for the extraction of natural compounds, such as carotenoids [ 92 ]. To date, there are 17 and 22 published scientific articles in which carotenoids were extracted with NaDESs and DESs, respectively (Figure 2). Recently, DESs have been tested for the extraction of β -carotene from millet via grinding-assisted extraction (40 s, 1:4 g/mL of solid–liquid ratio, 2,6-di-tert-butyl- 4-methylphenol-assisted (3 mg/mL)), using N,N-dimethylcyclohexylamine (DMCHA-B), N,N-dimethyloctylamine (DMOA-B), and N,N-dimethylbenzylamine (DMBA-B) as hydrogen bond acceptors and n-butanol as a hydrogen bond donor, in a 3:1 proportion. The study reported that the contents of β -carotene extracted using these DMCHA-B, DMOA-B, and DMBA-B were 2.7, 3.1, and 3.3 times higher than that obtained with ethanol, respectively [ 73 ]. Similarly, in a study with tomato powder, lycopene extraction was carried out via conventional extraction (maceration with magnetic stirring, 62 min, a solvent-to-solid ratio of 64:1 (v/w)) using a NaDES based on terpenes and fatty acid mixture (capric acid/lauric acid, 1:2). The study stated that the carotenoid extraction efficiency of the NaDES was either equal to or higher than that of acetone (up to 1.08 times greater) [ 74 ]. Finally, in a recent study performed by Viñas-Ospino et al. (2023), the UAEs (120 W ultrasound intensity, 1:20 (w/v) solid–solvent ratio, 20 min) of carotenoids from orange peel using several NaDESs (choline chloride/urea, proline/malic acid, l-menthol/d,l-camphor, l-menthol/eucalyptol, and lauric acid/octanoid acid) were compared. Among these NaDESs, menthol/camphor (1:1) was the most promising solvent regarding its stability and yield [ 19 ]. Overall, these studies highlight the potential of DESs and NaDESs as a viable alternatives to conventional solvents for the extraction of carotenoids. Antioxidants 2024,13, 239 22 of 27 5.4. Ionic Liquids Ionic liquids (ILs), also known as Newton’s liquids, are composed of an organic cation and an organic or inorganic anion with a melting point below 100 ◦C, which are normally liquid at room temperature [93]. Due to their low vapour pressure, ILs have a low probability of releasing toxic substances into the atmosphere; nonetheless, ILs can still contaminate soil and groundwater, and the degree of adsorption depends on their lipophilicity/hydrophilicity and the length of the cationic alkyl chain. Their chemical and thermal stability make them highly resistant to decomposition, resulting in long-term persistence in the environment. In any case, the biodegradability of ILs varies depending on their constituent cations and anions. Water-soluble ILs, such as cholinium or carboxylates, facilitate biodegradation as they can dissolve in water and interact more easily with microorganisms. However, lipophilic ILs aid in transports across cell membranes of microorganisms, which may make them more persistent in the environment. Thus, the components of ILs should be carefully selected, as their properties can be adjusted to improve their persistence and degradability [ 94 ]. In summary, although some properties of ILs make them attractive from the point of view of green chemistry, there are currently doubts about whether they could be considered eco-friendly solvents [93]. In recent years, ILs have emerged as promising solvents for the extraction of carotenoids from various sources, including microalgae, plants, and food waste, resulting, in some cases, in higher yields and selectivity compared to conventional solvents, such as acetone or methanol [ 95 ]. Research on the extraction of carotenoids with ILs represents 28% of the articles published on the extraction of carotenoids with the solvents evaluated in this review (Figure 2). This percentage is higher than those published on NaDESs (20%) and DESs (26%) (Figure 2). In a recent study, the recovery of β -carotene and astaxanthin from Phaffia rhodozyma (wet biomass) via solid–liquid extraction (0.2:1 (w/v) solid-to-liquid ratio, 60 min, 65 ◦ C, stirring speed of 300 rpm) using a cholinium-based IL resulted in 1.9 and 1.7 times higher yields than that with acetone, respectively. However, this study suggested that concentrated ILs pose a challenge due to their high viscosity and the requirement for further techno-economic assessment before industrial implementation [ 75 ]. In another study, the extraction of lutein from C. sorokiniana via conventional extraction (maceration, 45 min, 20 ◦ C) was effective using an alkyl carbamate IL (synthesized by mixing dipropylammonium dipropylcarbamate (DPCARB) and liquefied CO 2 , at a molar ratio of 2:1). An approximately three-fold higher lutein yield was observed with this CO 2 -based IL (DPCARB) compared to that of acetone, and an approximately 1.2 times higher yield was observed with DPCARB compared to that of methanol [ 76 ]. Moreover, in another study, tetrabutyl phosphonium hydroxide was used for the extraction of lutein from Chlorella saccharophila (40% (w/v) of ionic liquid concentration, 5 min extraction time, 0.5 mL/mg of solvent/biomass ratio, and 25 ◦ C temperature), and the extraction yield was approximately 20.5 times higher compared to that of methanol [ 77 ]. In addition, the carotenoid extraction from orange peel via UAE (200 W, 20 kHz, 80% amplitude, 5 min, 1:3 solid/liquid ratio) was compared using acetone and different ILs (1-butyl-3-methylimidazolium chloride ([BMIM][Cl]), 1-n-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), and 1-hexyl-3-methylimidazolium chloride ([HMIM][Cl])). The total carotenoid contents of the extracts obtained with [BMIM][Cl], [BMIM][BF4], and [HMIM][Cl] were 2.3, 2.9, and 1.5 times higher than that obtained with acetone, respectively [ 96 ]. Although ILs offer potential as efficient and sustainable extraction solvents for natural carotenoids, addressing the associated challenges and optimizing extraction conditions for different carotenoid sources through further research is crucial. This will enable the production of high-value natural carotenoid products with applications in various industries [95]. Antioxidants 2024,13, 239 23 of 27 6. Conclusions Carotenoids are extracted with different organic solvents, including hexane, methanol, and DMSO, which can pose harm to health and the environment depending on their use and exposure. On the other hand, many conventional techniques for carotenoid extraction involve a high amount of solvent and energy consumption. Thus, there is increasing interest in improving the extraction of carotenoids to make it more sustainable. Different techniques (such as ultrasound, microwaves, enzyme treatment, pulsed electric fields, pressurized liquid extraction, and sub- and supercritical fluid extraction) are being tested, as well as innovative solvents including ionic liquids, deep eutectic solvents (i.e., either natural or not), and other green solvents deriving from renewable feedstocks (2-methyltetrahydrofuran, ethyl lactate). Among these methodologies, ultrasound-assisted extraction and extraction with supercritical fluids are those that have generated the greatest number of original scientific publications in relation to the extraction of carotenoids. In any case, there are still few studies that test these bio-based solvents for carotenoid extraction. In any case, much research is still needed, as well as technological, economic, and environmental assessments. Some aspects that require more attention are the purity and the composition of the extracts. Being highly lipophilic, carotenoids are extracted together with other lipophilic compounds. In addition, in general, there is not much selectivity in the type of carotenoid extracted when the source contains several of them. The extraction of several carotenoids and lipophilic compounds could be desirable or not depending on the research objective or industrial application. For instance, the fact that a carotenoid-rich extract exhibits certain properties or actions does not necessarily mean that they are attributable to them. On the other hand, there are occasions in which the extraction of a certain carotenoid or type of carotenoid is sought but cannot be achieved easily. One example would be the extraction of colourless carotenoids from sources in which they occur together with other coloured carotenoids. Maybe the customizable designs of NaDESs and DESs can offer advantages in these directions in the near future. In any case, although laboratory results are in many cases very positive, their scalability to real industrial scenarios remains, in many cases, an important obstacle, mainly due to techno-economic reasons (for instance, an insufficient production capacity, the cost of infrastructure, the cost of the final product, etc.). On the other hand, it is important to include life cycle assessment analyses to better assess whether innovative extraction processes are more sustainable than the processes that are applied in the industry. Author Contributions: A.J.M.-M.: conceptualization and design of the idea, literature research, writing—original draft preparation, and supervision. P.M.-B.: supervision, original draft preparation, writing—review and editing, and figure preparation. Á.M.-O.: writing—original draft preparation and figure preparation. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grant PID2019-110438RBC21 (NEWCARFOODS) funded by MCIN/AEI/10.13039/501100011033. AMO was supported by a grant associated with grant PID2019-110438RB-C21 (NEWCARFOODS). PMB was supported by a postdoc fellowship from the Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía (PAIDI 2020). The authors are members of the Spanish Carotenoid Network (CaRed), grant RED2022-134577-T, funded by MCIN/AEI/10.13039/501100011033. Conflicts of Interest: AJMM carries out consultancy work for diverse companies. PMB and AMO declare no conflicts of interest. References 1. Meléndez-Martínez, A.J.; Böhm, V.; Borge, G.I.; Cano, M.P.; Fikselová, M.; Gruskiene, R.; Lavelli, V.; Loizzo, M.R.; Mandi´c, A.; Mapelli-Brahm, P.; et al. Carotenoids: Considerations for their Use in Functional Foods, Nutraceuticals, Nutricosmetics, Supplements, Botanicals and Novel Foods in the Context of Sustainability, Circular Economy and Climate Change. Annu. Rev. Food Sci. Technol. 2021,12, 433–460. [CrossRef] [PubMed] 2. Kolašinac, S.M.; Stevanovi´c, Z.P.D.; Kilibarda, S.N.; Kosti´c, A. Carotenoids: New applications of “old” pigments. Phyton 2021,90, 1041–1062. [CrossRef] Antioxidants 2024,13, 239 24 of 27 3. Meléndez-Martínez, A.J. An Overview of Carotenoids, Apocarotenoids, and Vitamin A in Agro-Food, Nutrition, Health, and Disease. Mol. Nutr. Food Res. 2019,63, 1801045. [CrossRef] [PubMed] 4. Mapelli-Brahm, P.; Barba, F.J.; Remize, F.; Garcia, C.; Fessard, A.; Mousavi Khaneghah, A.; Sant’Ana, A.S.; Lorenzo, J.M.; Montesano, D.; Meléndez-Martínez, A.J. The impact of fermentation processes on the production, retention and bioavailability of carotenoids: An overview. Trends Food Sci. Technol. 2020,99, 389–401. [CrossRef] 5. Meléndez-Martínez, A.J.; Stinco, C.M.; Mapelli-Brahm, P. Skin Carotenoids in Public Health and Nutricosmetics: The Emerging Roles and Applications of the UV Radiation-Absorbing Colourless Carotenoids Phytoene and Phytofluene. Nutrients 2019,11, 1093. [CrossRef] [PubMed] 6. Meléndez-Martínez, A.J.; Mandi´c, A.I.; Bantis, F.; Böhm, V.; Borge, G.I.A.; Brnˇci´c, M.; Bysted, A.; Cano, M.P.; Dias, M.G.; Elgersma, A.; et al. A comprehensive review on carotenoids in foods and feeds: Status quo, applications, patents, and research needs. Crit. Rev. Food Sci. Nutr. 2022,62, 1999–2049. [CrossRef] [PubMed] 7. Chemat, F.; Vian, M.A.; Cravotto, G. Green extraction of natural products: Concept and principles. Int. J. Mol. Sci. 2012,13, 8615–8627. [CrossRef] [PubMed] 8. Chemat, F.; Vian, M.A.; Ravi, H.K.; Khadhraoui, B.; Hilali, S.; Perino, S.; Tixier, A.S.F. Review of alternative solvents for green extraction of food and natural products: Panorama, principles, applications and prospects. Molecules 2019,24, 3007. [CrossRef] 9. Chemat, F.; Abert-Vian, M.; Fabiano-Tixier, A.S.; Strube, J.; Uhlenbrock, L.; Gunjevic, V.; Cravotto, G. Green extraction of natural products. Origins, current status, and future challenges. TrAC—Trends Anal. Chem. 2019,118, 248–263. [CrossRef] 10. Nanda, B.; Sailaja, M.; Mohapatra, P.; Pradhan, R.K.; Nanda, B.B. Green solvents: A suitable alternative for sustainable chemistry. Mater. Today Proc. 2021,47, 1234–1240. [CrossRef] 11. Soro, A.B.; Garcia-Vaquero, M.; Tiwari, B.K. Equipment and recent advances in ultrasound technology. In Innovative and Emerging Technologies in the Bio-Marine Food Sector: Applications, Regulations, and Prospects; Elsevier Inc.: Amsterdam, The Netherlands, 2021. [CrossRef] 12. Bagade, S.B.; Patil, M. Recent Advances in Microwave Assisted Extraction of Bioactive Compounds from Complex Herbal Samples: A Review. Crit. Rev. Anal. Chem. 2021,51, 138–149. [CrossRef] [PubMed] 13. Ranjha, M.M.A.N.; Kanwal, R.; Shafique, B.; Arshad, R.N.; Irfan, S.; Kieliszek, M.; Kowalczewski, P.Ł.; Irfan, M.; Khalid, M.Z.; Roobab, U.; et al. A critical review on pulsed electric field: A novel technology for the extraction of phytoconstituents. Molecules 2021,26, 4893. [CrossRef] [PubMed] 14. Alvarez-Rivera, G.; Bueno, M.; Ballesteros-Vivas, D.; Mendiola, J.A.; Ibañez, E. Pressurized liquid extraction. In Liquid-Phase Extraction; Elsevier Inc.: Amsterdam, The Netherlands, 2019. [CrossRef] 15. Ahmad, T.; Masoodi, F.A.; Rather, S.A.; Wani, S.M.; Gull, A. Supercritical Fluid Extraction: A Review. J. Biol. Chem. Chronicles 2019,5, 114–122. [CrossRef] 16. Cheng, Y.; Xue, F.; Yu, S.; Du, S.; Yang, Y. Subcritical water extraction of natural products. Molecules 2021,26, 4004. [CrossRef] [PubMed] 17. Majik, M.S.; Gawas, U.B. Recent Advances in Extraction of Natural Compounds. In New Horizons in Natural Compound Research; Elsevier Inc.: Amsterdam, The Netherlands, 2023. [CrossRef] 18. Coelho, T.L.S.; Silva, D.S.N.; dos Santos, J.M., Jr.; Dantas, C.; Nogueira, A.R.d.A.; Lopes Júnior, C.A.; Vieira, E.C. Multivariate optimization and comparison between conventional extraction (CE) and ultrasonic-assisted extraction (UAE) of carotenoid extraction from cashew apple. Ultrason. Sonochem. 2022,84, 105980. [CrossRef] [PubMed] 19. Pani, M.; Radoj, I. Using novel hydrophobic deep eutectic solvents to improve a sustainable carotenoid extraction from orange peels. Food Biosci. 2023,53, 102570. [CrossRef] 20. Nie, J.; Chen, D.; Ye, J.; Lu, Y.; Dai, Z. Optimization and kinetic modeling of ultrasonic-assisted extraction of fucoxanthin from edible brown algae Sargassum fusiforme using green solvents. Ultrason. Sonochem. 2021,77, 105671. [CrossRef] 21. Song, J.; Yang, Q.; Huang, W.; Xiao, Y.; Li, D.; Liu, C. Optimization of trans lutein from pumpkin (Cucurbita moschata) peel by ultrasound-assisted extraction. Food Bioprod. Process. 2018,107, 104–112. [CrossRef] 22. Benmeziane, A.; Boulekbache-Makhlouf, L.; Mapelli-Brahm, P.; Khaled Khodja, N.; Remini, H.; Madani, K.; Meléndez-Martínez, A.J. Extraction of carotenoids from cantaloupe waste and determination of its mineral composition. Food Res. Int. 2018,111, 391–398. [CrossRef] [PubMed] 23. Silva, D.S.N.; Silva, M.d.S.; Coelho, T.L.S.; Dantas, C.; Lopes Júnior, C.A.; Caldas, N.M.; Vieira, E.C. Combining high intensity ultrasound and experimental design to improve carotenoid extraction efficiency from Buriti (Mauritia flexuosa). Ultrason. Sonochem. 2022,88, 106076. [CrossRef] [PubMed] 24. Umair, M.; Jabbar, S.; Nasiru, M.M.; Lu, Z.; Zhang, J.; Abid, M.; Murtaza, M.A.; Kieliszek, M.; Zhao, L. Ultrasound-assisted extraction of carotenoids from carrot pomace and their optimization through response surface methodology. Molecules 2021,26, 6763. [CrossRef] [PubMed] 25. Morón-Ortiz, A.; Mapelli-brahm, P.; León-Vaz, A.; Benítez-González, A.M.; Meléndez-Martínez, A.J.; León, R. Ultrasound-assisted extraction of carotenoids from phytoene-accumulating Chlorella sorokiniana microalgae: Effect of milling and performance of the green biosolvents 2-methyltetrahydrofuran and ethyl lactate. Food Chem. 2024,434, 137437. [CrossRef] 26. Mary Leema, J.T.; Persia Jothy, T.; Dharani, G. Rapid green microwave assisted extraction of lutein from Chlorella sorokiniana (NIOT-2)—Process optimization. Food Chem. 2022,372, 131151. [CrossRef] [PubMed] Antioxidants 2024,13, 239 25 of 27 27. Elik, A.; Yanık, D.K.; Gö˘gü¸s, F. Microwave-assisted extraction of carotenoids from carrot juice processing waste using flaxseed oil as a solvent. Lwt 2020,123, 109100. [CrossRef] 28. Tsiaka, T.; Zoumpoulakis, P.; Sinanoglou, V.J.; Makris, C.; Heropoulos, G.A.; Calokerinos, A.C. Response surface methodology toward the optimization of high-energy carotenoid extraction from Aristeus antennatus shrimp. Anal. Chim. Acta 2015,877, 100–110. [CrossRef] [PubMed] 29. Martínez, J.M.; Delso, C.; Angulo, J.; Álvarez, I.; Raso, J. Pulsed electric field-assisted extraction of carotenoids from fresh biomass of Rhodotorula glutinis.Innov. Food Sci. Emerg. Technol. 2018,47, 421–427. [CrossRef] 30. Martínez, J.M.; Gojkovic, Z.; Ferro, L.; Maza, M.; Álvarez, I.; Raso, J.; Funk, C. Use of pulsed electric field permeabilization to extract astaxanthin from the Nordic microalga Haematococcus pluvialis.Bioresour. Technol. 2019,289, 121694. [CrossRef] 31. Luengo, E.; Álvarez, I.; Raso, J. Improving Carotenoid Extraction from Tomato Waste by Pulsed Electric Fields. Front. Nutr. 2014,1, 12. [CrossRef] [PubMed] 32. Luengo, E.; Condón-Abanto, S.; Álvarez, I.; Raso, J. Effect of Pulsed Electric Field Treatments on Permeabilization and Extraction of Pigments from Chlorella vulgaris.J. Membr. Biol. 2014,247, 1269–1277. [CrossRef] 33. De Aguiar Saldanha Pinheiro, A.C.; Martí-Quijal, F.J.; Barba, F.J.; Benítez-González, A.M.; Meléndez-Martínez, A.J.; Castagnini, J.M.; Tappi, S.; Rocculi, P. Pulsed Electric Fields (PEF) and Accelerated Solvent Extraction (ASE) for Valorization of Red (Aristeus antennatus) and Camarote (Melicertus kerathurus) Shrimp Side Streams: Antioxidant and HPLC Evaluation of the Carotenoid Astaxanthin Recovery. Antioxidants 2023,12, 406. [CrossRef] 34. Wang, M.; Morón-ortiz, Á.; Zhou, J.; Benítez-gonzález, A.; Mapelli-brahm, P.; Meléndez-martínez, A.J.; Barba, F.J. Effects of Pressurized Liquid Extraction with dimethyl sulfoxide on the recovery of carotenoids and other dietary valuable compounds from the microalgae Spirulina,Chlorella and Phaeodactylum tricornutum.Food Chem. 2022,405, 134885. [CrossRef] [PubMed] 35. Zhou, J.; Wang, M.; Berrada, H.; Zhu, Z.; Grimi, N.; Barba, F.J. Pulsed electric fields (PEF), pressurized liquid extraction (PLE) and combined PEF + PLE process evaluation: Effects on Spirulina microstructure, biomolecules recovery and Triple TOF-LC-MS-MS polyphenol composition. Innov. Food Sci. Emerg. Technol. 2022,77, 102989. [CrossRef] 36. Derwenskus, F.; Metz, F.; Gille, A.; Schmid-Staiger, U.; Briviba, K.; Schließmann, U.; Hirth, T. Pressurized extraction of unsaturated fatty acids and carotenoids from wet Chlorella vulgaris and Phaeodactylum tricornutum biomass using subcritical liquids. GCB Bioenergy 2019,11, 335–344. [CrossRef] 37. Nunes, A.N.; Roda, A.; Gouveia, L.F.; Fernández, N.; Bronze, M.R.; Matias, A.A. Astaxanthin Extraction from Marine Crustacean Waste Streams: An Integrate Approach between Microwaves and Supercritical Fluids. ACS Sustain. Chem. Eng. 2021,9, 3050–3059. [CrossRef] 38. Georgiopoulou, I.; Tzima, S. applied sciences Process Optimization of Microwave-Assisted Extraction of Chlorophyll, Carotenoid and Phenolic Compounds from Chlorella vulgaris and Comparison with Conventional and Supercritical Fluid Extraction. Appl. Sci. 2023,13, 2740. [CrossRef] 39. Lad, J.D.; Kar, A. Supercritical CO 2 extraction of lycopene from pink grapefruit (Citrus paradise Macfad) and its degradation studies during storage. Food Chem. 2021,361, 130113. [CrossRef] 40. de Andrade Lima, M.; Charalampopoulos, D.; Chatzifragkou, A. Optimisation and modelling of supercritical CO 2 extraction process of carotenoids from carrot peels. J. Supercrit. Fluids 2018,133, 94–102. [CrossRef] 41. Lu, J.; Feng, X.; Han, Y.; Xue, C. Optimization of subcritical fluid extraction of carotenoids and chlorophyll a from Laminaria japonica Aresch by response surface methodology. J. Sci. Food Agric. 2014,94, 139–145. [CrossRef] 42. Aslanbay Guler, B.; Deniz, I.; Demirel, Z.; Yesil-Celiktas, O.; Imamoglu, E. A novel subcritical fucoxanthin extraction with a biorefinery approach. Biochem. Eng. J. 2020,153, 107403. [CrossRef] 43. Turola Barbi, R.C.; de Souza, A.R.C.; Hamerski, F.; Lopes Teixeira, G.; Corazza, M.L.; Hoffmann Ribani, R. Subcritical propane extraction of high-quality inajá(Maximiliana maripa) pulp oil. J. Supercrit. Fluids 2019,153, 104576. [CrossRef] 44. Ghafoor, K.; Sarker, M.Z.I.; Al-Juhaimi, F.Y.; Babiker, E.E.; Alkaltham, M.S.; Almubarak, A.K.; Ahmed, I.A.M. Innovative and Green Extraction Techniques for the Optimal Recovery of Phytochemicals from Saudi Date Fruit Flesh. Processes 2022,10, 2224. [CrossRef] 45. Zuorro, A.; Fidaleo, M.; Lavecchia, R. Enzyme-assisted extraction of lycopene from tomato processing waste. Enzyme Microb. Technol. 2011,49, 567–573. [CrossRef] [PubMed] 46. Adadi, P.; Barakova, N.V.; Adadi, P. Application of surface response methodology for an enzyme-assisted extraction of carotenoids. J. Microbiol. Biotechnol. Food Sci. 2019,9, 195. [CrossRef] 47. Catalkaya, G.; Kahveci, D. Optimization of enzyme assisted extraction of lycopene from industrial tomato waste. Sep. Purif. Technol. 2019,219, 55–63. [CrossRef] 48. Kumar, K.; Srivastav, S.; Sharanagat, V.S. Ultrasound assisted extraction (UAE) of bioactive compounds from fruit and vegetable processing by-products: A review. Ultrason. Sonochem. 2021,70, 105325. [CrossRef] 49. Banakar, V.V.; Sabnis, S.S.; Gogate, P.R.; Raha, A.; Saurabh. Ultrasound assisted continuous processing in microreactors with focus on crystallization and chemical synthesis: A critical review. Chem. Eng. Res. Des. 2022,182, 273–289. [CrossRef] 50. Geow, C.H.; Tan, M.C.; Yeap, S.P.; Chin, N.L. A Review on Extraction Techniques and Its Future Applications in Industry. Eur. J. Lipid Sci. Technol. 2021,123, 2000302. [CrossRef]