A Perspective on Carotenoids: Z/E-Isomerization, Extraction by Deep Eutectic Solvents and Applications

Abstract

Carotenoids are used commercially for nutraceutical products because of their low toxicity, antioxidant activity, association with a reduction in various diseases and high coloring capacity. However, low stability and bioavailability limited their applications. Alterations in the physicochemical properties of carotenoids by Z-isomerization are beneficial for their extraction and bioavailability. The main strategies applied for enhancing their Z-isomerization include adding a catalyst, especially natural or heterogeneous sulfur-containing compounds. Consumers’ interest in products with carotenoids of natural origin has increased, which has emphasized a need for improved methods for their extraction from food waste. The green extraction methods for carotenoid recovery, especially using natural deep eutectic solvents combined with some novel extraction techniques showed a rapid increase and excellent application prospects. Health problems faced by the older population boost the demand for carotenoid diet supplements for skin health, anti-aging, treating eye disorders, preventing cancer (prostate) and obesity, thereby driving the growth of the carotenoids industry. However, the expansion of the carotenoid worldwide market is hampered by strict regulatory and approval standards. Relevant standards of carotenoids, especially Z-carotenoids, need to be improved.

1. Introduction

Generally, carotenoids mainly exist in the chromoplasts and chloroplasts of various plants and photosynthetic algae [1]. Noticeably, colorless carotenoids phytoene and phytofluene have still been ignored, although they are also major dietary carotenoids that are present in humans at levels comparable or superior to the color carotenoids [2]. Carotenoids (i.e., lycopene and astaxanthin) can reduce the risk of various diseases, such as cardiovascular diseases, and eye-related diseases. In addition, carotenes are also provitamin A [3]. Carotenoids are not synthesized by humans and must be obtained through the diet. The intake of total carotenoids in European countries (median values) varies between ∼9.5 and 16 mg per day, in which the major food sources are vegetables, fruits, and soups. Carotenoids are also widely utilized in food, cosmetic, and pharmaceutical products, primarily due to their bioactive and color properties. These various applications have enhanced the demand for carotenoid-rich products. The global carotenoid market should reach $2.7 billion by 2027 from $2.0 billion in 2022 at a compound annual growth rate (CAGR) of 5.7% for the forecast period of 2022–2027 (The Global Market for Carotenoids, BCC Publishing).

Carotenoids naturally mainly exist in the all-E isomer (>90%), however, Z-carotenoid isomers are abundant in human fluids and plasma, as well as some processed products (Figure 1) [4]. Although the presence of Z-isomers of carotenoids in processed foods can mainly be owing to Z-isomerization reaction induced by thermal treatments or light with/without catalyst, it is also found that some carotenoids can also occur naturally in Z-isomers. For example, for phytoene 15-Z-phytoene (not all-E isomer) is the main isomer in carotenogenic organisms [2]. Notably, some carotenoid Z-isomers (i.e., Z-lycopene and Z-astaxanthin) have been confirmed to exhibit higher bioavailability compared to all-E-isomers [5, 6]. To improve the processing efficiency (i.e., extraction) of carotenoids, Z-isomerization has also been reported to enhance carotenoid solubility [7].

Figure 1.

The chemical structures of main carotenoids and their isomers.

Natural commercial carotenoids are mainly obtained by extracting from various processing by-products of plants and microalgae. Considering safety and environmental protection, some studies have focused on using green solvents to extract carotenoids, including supercritical CO₂, ionic liquids, edible oils, deep eutectic solvents [7]. Especially, natural deep eutectic solvents (DESs) are foreseen as the next generation solvents or solvents for the twenty-first century [8]. To efficiently extract carotenoids by DESs, some processes have been successfully developed by combining hydrophobic DESs with less viscosity such as terpene/fatty acid-based DESs, with thermal treatment and homogenization techniques (i.e., ultrasound-assisted extractions).

In the chapter, carotenoid Z/E-isomerization in different solvents without/with various catalysts, carotenoid extraction by hydrophilic and hydrophobic DESs, DES recyclability from carotenoid extracts, as well as carotenoid applications as a colorant or in nutraceutical products were outlined.

2. Carotenoid Z/E-isomerization

Z-isomerization technologies of carotenoids have recently been developed to improve their processing efficiency (i.e., extraction) and bioavailability, especially with natural catalysts in different solvents.

2.1 Carotenoid Z/E-isomerization in different solvents

Several methods using different solvents for Z-isomerization of carotenoids, such as organic solvents (i.e., ethyl acetate) and edible oils with thermal, light, and catalytic treatment have been reported [9]. Z-isomerization efficiency of carotenoids greatly depends on solvents and treatment conditions. In general, carotenoids are easily induced to Z-isomerization in dissolved state but this is often accompanied by significant oxidative degradation. How to promote Z-isomerization while inhibiting the oxidative degradation of carotenoids is one of the current research problems.

The kind of solvents has a great effect on the Z-isomerization of carotenoids. Specifically, halogen solvents (i.e., dibromoethane and chloroform) used as reaction medium could promote the Z-isomerization efficiency compared to other common organic solvents (i.e., hexane, methyl tert-butyl ether, and acetone). However, halogen solvents are typically banned to use in food and cosmetic processing owing to their high toxicity. Therefore, Generally Recognized as Safe (GRAS) solvents with low/no toxicity, such as ethanol, ethyl acetate, and vegetable oils, should be used during Z-isomerization for practical industrial application. However, it is quite difficult to achieve high Z-isomerization efficiency using these GRAS solvents with traditional heating treatments, especially processing at high temperatures also induced significant degradation of carotenoids. Therefore, a series of innovative Z-isomerization techniques, combined with thermal treatment, light, and microwave irradiation have been developed to promote carotenoid Z-isomerization. Especially, microwave irradiation treatment has been used as an efficient method for carotenoid Z-isomerization owing to rapid and uniform heating, in which the efficiency of Z-isomerization is higher with microwave irradiation than with traditional heating. In addition, a continuous and efficient carotenoid (lycopene and astaxanthin) Z-isomerization procedure in a flow reactor using supercritical ethanol or subcritical ethyl acetate was also developed [10, 11]. The continuous-flow method based on high-temperature and high-pressure processing is environmentally friendly because of its low energy consumption and the green solvents, and the reaction can also easily be controlled by changing temperature and pressure to achieve high efficiency of Z-isomerization. For example, when all-E-astaxanthin was treated in the continuous-flow reactor at 200°C and 10 MPa, the total Z-isomer ratio of astaxanthin was higher than 60% in only 30 s [11]. Recently, the Z-isomerization and degradation of Lycopene in various hydrophobic natural deep eutectic solvents was also reported. The studies found that HNDES composed of thymol and menthol had the highest lycopene retention, and lycopene Z-isomerization and degradation was promoted by fatty acid-based HNDES [12].

2.2 Carotenoid Z-isomerization with catalysts

Some catalysts have been added to promote the thermal Z-isomerization of carotenoids. It was reported that iron (III) chloride, iodine-doped titanium dioxide, titanium tetrachloride, as well as disulfide compounds, isothiocyanates, and carbon disulfide, promoted Z-isomerization of carotenoids. However, most of these used catalysts, such as iodine and heavy metals, have a negative effect on the human health and the environment. Thus, safety/low toxicity and environmentally friendly catalysts should be used for industrial applications. Recently, efficient isomerization methods with some plant-derived catalysts (i.e., disulfide compounds, isothiocyanates) have received particular attention. The effect of these natural catalysts on the Z-isomerization and stability of carotenoids (i.e., lycopene and astaxanthin) has been recently reported [7, 12, 13, 14]. Especially, the addition of certain food ingredients, such as cabbage and onion, could enhance the Z-isomerization of carotenoids during the thermal processing of tomato products, and isothiocyanates and polysulfides were identified as the causative components [14, 15]. In addition, mustard and roasted sesame oils as reaction media also promoted Z-isomerization of carotenoids (i.e., lycopene and astaxanthin) compared to other vegetable oils because they contained isothiocyanates and polysulfides.

Most of catalysts such as acids (namely H+), isothiocyanates, iodine, and iron (III) chloride generally exhibit great electrophilicity, which promotes Z-isomerization of carotenoids by electrophilic components [16, 17, 18]. However, the E/Z isomerization mechanism of carotenoids induced by polysulfides (i.e., diallyl disulfide DADS) was that sulfur radical (RS•) cleaved by polysulfides plays a role [13, 14]. When lycopene was induced by DADS, formation rates of 5-Z isomers were 3–4 times higher than for 9-Z and 13-Z isomers of lycopene, and lycopene degradation was clearly inhibited [14].

Noticeably, although naturally derived sulfur-containing compounds were thought to be safe, these compounds generally present a strong odor and are homogeneously dissolved, making it difficult to isolate/recycle them from the final carotenoid product. Recently, a heterogeneous (solid) catalyst based on a column packed with isothiocyanate-functionalized silica (Si-NCS) has been developed, with the advantage of easily separating them from the carotenoid product by filtration or centrifugation and being able to be used repeatedly [19]. This catalyst promotes carotenoid Z-isomerization with high efficiency, for example when astaxanthin and lycopene solutions were heated at 50°C in presence of 10 mg/mL Si-NCS, the ratios of their total Z-isomer increased by about 50% and 80%, respectively [19]. In summary, the non-homogeneous reaction has good application prospects with high catalytic efficiency and good safety.

2.3 Z-isomerization for carotenoid extractions

Carotenoid yield could be clearly improved by Z-isomerization pretreatments or adding catalysts during extractions because Z-isomerization of carotenoids increases their solubility. The higher extraction efficiency of lycopene (even a 4–5-fold increase) with a higher ratio of Z-lycopene isomers in the tomato pulp/powder or gar arils were obtained with supercritical CO₂ (SC-CO₂) [20] or with ethanol by microwave irradiation pretreatments [21, 22, 23]. About 4-fold higher yield of β-carotene was also achieved for the powder of algae Dunaliella bardawil rich in 9-Z isomer than that rich in all-E-isomer by extraction of SC-CO2.

The addition of natural catalysts (i.e., polysulfides and isothiocyanates) or foodstuffs with these catalysts could significantly promote the extraction of lycopene from tomato pulp or carotenoids from Paracoccus carotinifaciens [16, 24]. Noticeably, Z-isomerization and degradation of carotenoids were both enhanced by adding isothiocyanates; however, polysulfides could promote the Z-isomerization reaction while markedly inhibiting the degradation of carotenoids [14]. These results suggested that the addition of polysulfides was helpful to improve the extraction yield of carotenoids and Z-isomers content in the extract [13].

2.4 Z-isomerization for carotenoid bioavailability

Z-isomerization reaction can induce an obvious change in the physicochemical properties of carotenoids, leading to improved bioavailability. A series of Z-isomers of carotenoids (i.e., lycopene, astaxanthin, and lutein) have been reported to possess greater bioavailability, and even higher tissue accumulation than corresponding all-E configurations [5, 25, 26].

For example, in rats’ trials fed with the diet rich in astaxanthin Z-isomers, the levels of astaxanthin in blood and some tissues (i.e., eye and skin) were higher than those from the diet containing all-E-isomer. 13Z-astaxanthin was also confirmed to possess higher bioavailability and tissue accumulation efficiency than other isomers of astaxanthin, considering that the content of 13Z-isomer of astaxanthin in blood and tissues was enhanced rather than that of the 9Z-isomer when fed with the Z-isomer-rich diet [26]. However, the 9Z-isomer of astaxanthin exhibited higher cellular-transport efficiency than 13Z- and all-E configurations in the Caco-2 cell monolayers experiment [6]. Different from most colored carotenoids naturally in the all-E configuration, phytoene (PT) and phytofluene (PTF) are found primarily in the Z-isomer. PT is mostly found as 15-Z-PT, and PTF is usually existed as a mixture of various isomers. PT and PTF were confirmed to have high micellization efficiency and also readily absorbed by the human body, probably because of their natural Z-isomers (i.e., 15 Z-isomer) and/or high molecular flexibility [2]. We recently found that the addition of onions (or diallyl disulfide) into tomato purees reduced the ratios of their natural Z-isomers by isomerization reaction of PT and PTF, and further decreased their bioaccessibility during in vitro digestion [27]. In conclusion, most studies have concluded that Z-isomers of carotenoids showed higher bioavailability than their all-E configurations. Therefore, the intake of carotenoid diets rich in Z-isomers, rather than all-E configurations, is recommended in terms of carotenoid bioavailability.

3. Carotenoid extractions by deep eutectic solvents

Solvents of petrochemical origin such as hexane and acetone have been used to extract carotenoids. However, these solvents can produce toxic volatile compounds, and raise some environmental issues, as well as public health and safety concerns. Therefore, the green chemistry concept pushes some researchers to find alternative green solvents for carotenoid extraction. Supercritical CO₂, edible oils, ionic liquids, and deep eutectic solvents have emerged for carotenoid extractions, free of volatile petrochemical solvents. Especially, there is an explosion in the latest 5 years in using deep eutectic solvents (DESs) for carotenoid extractions by combining with some novel extraction techniques (i.e., bead-milling, homogenization, ultrasound/microwave-assisted, and high-pressure processing) (Table 1).

DES (mole ratio)	Extraction methods	Source of natural products	Target compounds (highest yield)	References
Choline-based DES
Choline chloride: tartaric acid (2:1) with methanol (80:20)	Ultrasound (600 W, 5 or 10 min, 30–35°C), and Microwave-assisted (120 W, 20 min; 30 W, 7 min) extraction	Apricot and shrimp wastes	Carotenoids (β-carotene/astaxanthin 761/267 μg/g dry residue)	[28]
Choline chloride: lactic acid (1:1); Choline chloride: lactic acid (1:2); Choline chloride: butyric acid (1:1); Choline chloride: butyric acid (1:2) aqueous solutions (at 80% w/w)	Stirring at 300 rpm and 65°C for 60 min;	Wet biomass of P. rhodozyma (at a concentration of 0.2 g/mL)	β-carotene/astaxanthin (46/48 μg/mL wet biomass)	[29]
Choline chloride: oxalic acid (1:1); Choline chloride: oxalic acid (1:1) with water added (20% v/v); Choline chloride: urea (1:2); Choline chloride: urea (1:2) with water added (20% v/v); Choline chloride: glycerol (1:2); Choline chloride: glycerol (1:2) with water added (20% v/v)	Stirring at 150 rpm and 50°C for 2 h	Dunaliella salina microalgae	Carotenoid (89.5%)	[30]
Lactic acid: glucose (5:1); Betaine: ethylene glycol (1:2); Choline chloride: citric acid (2:1); Choline chloride: glycerol (1:2); Proline: malic acid (1:1); Choline chloride: urea (1:1); DL-menthol: camphor (1:1); DL-menthol: eucalyptol (1:1); Lauric acid: octanoic acid (1:3)	Stirring at 150 rpm and room temperature for 30 min	Orange peel	Carotenoid (168.7 mg/100 g fresh peel)	[31]
Choline chloride: triethylene glycol (1: 3) with ethanol (6: 4)	Ultrasonic-assisted extraction (300 W, 40 min)	Pumpkin peel	Pumpkin peel pigment (2.460%)	[32]
Terpene/fatty acid-based DESs
Choline-chloride: levulinic acid (2:1, 1:1, 1:2); menthol: lactic acid (2:1, 1:1, 1:2); Menthol: lactic acid (2:1, 5:1, 8:1)	Shaking at 60°C for 30 min; ultrasound-assisted extraction (100 W, 10–50 min, 30–70°C)	Tomato processing by-product	Lycopene (1463 μg/g powder)	[33]
Menthol: acetic acid (1:1) with ethanol addition; Menthol: lauric Acid (2:1) with ethanol addition	Stirring (200 rpm) for 180 min at room temperature	Crude palm oil	Carotene/β-carotene (213/0.052 ppm in oil)	[34]
Menthol: acetic acid (2:1); Menthol: lactic acid (2:1); Menthol: lauric acid (2:1)	Stirring at 200 rpm for 120 min at 40°C with and without enzyme	Sunflower wastes (petals and florets)	Carotenoids (1449 mg/100 g biomass)	[35]
Perillyl alcohol: camphor (1:1); Menthol: perillyl alcohol (1:1); Menthol: camphor (1:1); Menthol: eucalyptol (1:1); Menthol: myristic acid (8:1)	Stirring at 30, 45, and 60°C) for 120, 360, and 1440 min	Brown crab shell residues (shrimp shells, mussels, and Haematococcus pluvialis)	Astaxanthin (∼1000 μg/g dry residue)	[36]
Caprylic acid: capric acid (2:1, 3:1, 4:1); Caprylic acid: lauric acid (3:1); Pelargonic acid: lauric acid (3:1); Capric acid: lauric acid (2:1); Pelargonic acid: capric acid: lauric acid (3:1:1); Menthol: capric acid (2:1); Menthol: caprylic acid (1:1); Menthol: lauric acid (2:1)	Ultrasound-assisted extraction (26.25–78.75 W, 10 min, 20–70°C, matrix to solvent ratio (7–23 mg/mL))	Pumpkin (Cucurbita maxima)	β-carotene (151 μg/mL solvent)	[37]
Menthol: capric acid (1:1/1:2/2:1); Menthol: lauric acid (1:1/1:2/2:1); Thymol: capric acid (1:1/1:2/2:1); Thymol: lauric acid (1:1/1:2/2:1); Capric acid: lauric acid (1:1/1:2/2:1)	Stirring at 750 rpm and room temperature for 60 min	Tomato	Lycopene (14.92 mg/100 g Fresh Weight); β-carotene (0.91 mg/100 g Fresh Weight); Carotenoid (15.98 mg/100 g Fresh Weight)	[38]
Menthol: camphor (0.25–5:1); α-Terpineol: camphor (0.25–5:1); α-Terpineol: thymol (0.25–5:1); Camphor: thymol (0.25–5:1); α-Terpineol: menthol (0.25–5:1); Fenchyl alcohol: menthol (0.25–5:1); Coumarin: thymol (0.25–5:1); Fenchyl alcohol: thymol (0.25–5:1); Menthol: thymol (0.25–5:1); Fenchyl alcohol: α-terpineol (0.25–5:1)	Stirring at 1200 rpm and 30°C for 70 min	Microalgae	Lutein (6.26 mg/g)	[39]
DL-menthol: oleic acid (2:1); Thymol: oleic acid (3:1); Geraniol: oleic acid (13:1)	Stirring at 50–100 rpm and room temperature for 6 h	Haematococcus pluvialis	Astaxanthin (84.9%)	[40]
Thymol: dodecanoic acid (1.25:1); Thymol: decanoic acid (1:1); Thymol: octanoic acid (1:1.38); Tetrabutylammonium chloride: decanoic acid (1:2); Tetrabutylammonium chloride: octanoic acid (1:2); Tetrabutylammonium chloride: acetic acid (1:2)	Stirring at 1000 rpm and 25°C for 1 h	The Alga Tisochrysis lutea	Fucoxanthin (22.03 mg/g dry weight)	[41]
DL-menthol: lactic acid (8:1)	Ultrasonic-assisted extraction (60°C, 20 min)	Tomato pomace	Lycopene (82.86 μg/g); β-carotene (2923.02μg/g)	[42]
Choline chloride: urea (1:2); Proline: malic acid (1:1); L-menthol: D, L-camphor (1:1); L-Menthol: eucalyptol (1:1); Lauric acid: octanoid acid (1:3)	Ultrasonic-assisted extraction (120 W, 20 min)	Orange peel	Carotenoid (653.51 mg/ 100 g fresh peel)	[31]

Table 1.

Carotenoid extractions by deep eutectic solvents (DESs).

DESs as a kind of supramolecular green solvents, are a combination of hydrogen bond acceptors (i.e., choline salts), and hydrogen bond donors (i.e., organic acids). Compared with ionic liquid and traditional organic solvents, DESs, especially natural deep eutectic solvents, are valued for their biodegradability/low impact on the environment, economical, and ease of manufacture, and fully represent green chemistry criteria. The ability to easily customize DESs by adjusting raw materials and proportions to suit the extraction matrix is another biggest advantage. In addition, the ability to improve the stability of carotenoids combined with the intrinsic safety and edibility of natural DES components makes the mixture attractive to the food industry. These intrinsic excellent characteristics have caused a rapid increase in the extraction of carotenoids using DESs.

3.1 Hydrophilic DESs with co-solvents for carotenoid extractions

Most of the traditional DESs are polar and have high viscosity, and therefore they are not suitable for extracting carotenoids. Water and alcohols as co-solvents have been added in DESs to facilitate carotenoid extraction by lowering the viscosity of DESs [43, 44]. Compared to DMSO, yields of carotenoids (β-carotene and astaxanthin) from P. rhodozyma biomass were increased to about 10% when using the DES choline chloride: butyric acid (1:2) water solution (at 80% w/w) [29].

Alcohols, especially methanol and ethanol are also often used as DES co-solvent [43]. The DES choline chloride: tartaric acid (2:1) with the addition of methanol has been used for carotenoid extraction from apricot by-product (β-carotene) and shrimp heads (astaxanthin) by ultrasonic-assisted and microwave-assisted extraction [28]. The yield of β-carotene from apricot by-product with the DES was higher than astaxanthin from shrimp heads in both extraction methods. The yield of carotenoid (astaxanthin) extracted by choline chloride: tartaric acid (2:1) was also similar to that of hexane: acetone: ethanol (2:1:1) in microwave-assisted extraction. A two-aqueous phase system consisting of DESs and alcohol was prepared to extract carotenoids from pumpkin peel with cellulase by ultrasound-assisted extraction. The extraction solvent composed of DES (choline chloride/triethylene glycol (1:3)) and ethanol (6:4) showed the highest yield of 2.46% at the optimized ultrasonic extraction conditions, namely powder of 300 W for 40 min, solid/liquid ratio of 1: 40 g/mL, cellulase addition amount of 2.10% [32]. Noticeably, the yield of carotenoids from pumpkin peel decreased with the DES water content, as carotenoids are insoluble in water.

3.2 Carotenoid extraction by hydrophobic DESs

Hydrophobic DESs with less viscosity such as terpene/fatty acid-based DESs have been developed for carotenoid extraction [36, 45, 46]. Three hydrophobic DESs prepared by menthol: camphor, menthol: eucalyptol, lauric acid: octanoic acid and two hydrophilic proline: malic acid and choline chloride: urea was compared to extract carotenoids from orange peels, in which the extracts obtained with these hydrophobic DESs showed the highest values of total carotenoids, and there was no significant difference for the three hydrophobic DESs [31, 47]. DESs composed of menthol and different organic acids (lauric, acetic, and lactic) were also used for carotenoid extraction from sunflower wastes [35]. In addition, carotenoids (especially lycopene and β-carotene) from tomato pomace were also extracted by two DESs (ethyl acetate: ethyl lactate and Men-Lac) and an organic solvent mixture (n-hexane: acetone), in which the DES (ethyl acetate: ethyl lactate) with non-thermal air-drying treatment achieved the highest yields of lycopene and β-carotene [42].

DESs composed of menthol and lactic acid (Men-Lac) were reported to increase lycopene yields by ultrasound-assisted extraction due to the strong hydrophobicity, compared with choline-chloride: levulinic acid, in which Men-Lac with the ratio of 8:1 had the highest extraction efficiency [33]. This probably was due to the lower viscosity of the menthol-based DES [48]. The DES decanoic acid: dodecanoic acid was found to exhibit comparable extraction capacity for lycopene from freeze-dried tomato fruits to acetone [38]. The hydrophobic DESs based on N, N-dime-thylcyclohexylamine, and n-butanol were used to extract β-carotene from millet by grinding-assisted extraction, and phenolic antioxidants were also added to improve the stability of β-carotene [49]. Furthermore, a hydrophobic DES composed of C8 and C10 fatty acids (3:1) showed high β-carotene recovery from pumpkin [37].

The recovery efficiency of different DESs toward lutein from Scenedesmus sp. is compared, in which the DES composed of equimolar fenchyl alcohol and thymol (Fen-Thy) had the best performance, and the lutein extraction yield by Fen-Thy was even higher than ethyl acetate [39]. The ratio of hydrogen bond acceptors and donors of Fen-Thy has a significant effect on the lutein extraction yield. For example, when their ratio was below 1:1, the yield of lutein increased with the proportion of fenchyl alcohol. Additionally, Fen-Thy also enhanced the lutein stability under high temperature and light owing to hydrogen bond and Van der Waals interaction [39].

Terpene-based DESs were used to extract astaxanthin from crab shell residues by a straightforward solid-liquid extraction method. Astaxanthin yields were 3–657-fold higher than that at 6 h Soxhlet extraction from shrimp shells, mussels, and H. pluvialis when operating at 60°C for 2 h using the DES menthol: myristic acid (8:1) [36]. Three DESs composed of terpenes (thymol, menthol, and geraniol) and oleic acid were used for the extraction of astaxanthin from the microalga Haematococcus pluvialis [40]. All the DESs produced high astaxanthin recovery rates of about 60% for dried biomass, which were higher than the values of 40% with oleic acid alone. This showed that the extraction ability of oleic acid was effectively improved when using in the form of DES with one of the three terpenes, probably owing to a reduction of oleic acid viscosity or an increase in affinity for astaxanthin by π–π stacking interactions [40]. The DESs composed of thymol and oleic acid could increase astaxanthin stability after prolonged oxidative stress due to the antioxidant properties of thymol. This hind that some DESs can stabilize carotenoids, and acted as a storage system for sensitive molecules. The capacity of improving carotenoid extraction and stability combined with the intrinsic safety and edibility of the components of natural DESs makes the formulation of carotenoid-DES extract attractive to the food ingredients/additives industry.

3.3 DES recyclability from carotenoid extracts

The recovery of DESs and isolation of target compounds from the extract remain a challenge due to their low vapor pressure. The recyclability of DESs will bring benefits in reducing the costs of extraction processes for industrial scale-up. Noticeably, the recovery of carotenoids may not be indispensable for natural DESs because most of them are thought to be safe and can be directly used in food/pharmaceutical formulations because of their inherently benign character. For example, according to the standards set by the Flavor Extract Manufacturers’ Association (FEMA) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA), the composition of some natural DESs can be found as food additives or spices, thus these natural DES could be applied to food products. Recently, only a few studies focused on the recovery of DESs for carotenoid extractions, most did not consider recovery levels of the DESs and carotenoids.

Switchable DESs such as the DESs consisting of fatty acids were easily separated and recovered from the extract of carotenoids because the DES solvent polarity could be reversibly switched from hydrophobic to hydrophilic [50]. A switchable solvent system based on pH adjusting by a bio-friendly dilute amine solution (i.e., NH₄OH) and CO₂, has been developed, which mainly included: (1) switching hydrophobic to hydrophilic of DESs by adjusting pH using NH₄OH or CO₂ after extracting carotenoids; (2) after the polarity switching, the formed precipitate of carotenoids due to their low solubility in the hydrophilic media were separated by simple decantation or filtration; (3) recovering the DESs by pumping CO₂ or NH₄OH to cause a phase splitting and further by separating by centrifugation [37, 49, 51]. This switching recovery procedure was very efficient and required minimum intervention of external or complex lab equipment, which have been applied for carotenoid separation and DES recycling. For example, switchable DES solvents based on fatty acids or the combination of N, N-dimethyl cyclohexylamine, and n-butanol have been used in their hydrophobic form to extract and separate carotenoids, such as β-carotene from millet and pumpkin samples [37, 49]. The yield of β-carotene did not decrease, and over 91.0% of β-carotene from the millet could be directly recovered each time in five DES recycling processes [49]. Noticeably, it is also possible to extract some hydrophilic compounds by using the DES in a hydrophilic form after switching by adjusting pH. In this way, both polar and nonpolar target compounds could be successively extracted and separated, and DESs could also be reused. However, further studies of the development of available protocols for applying DESs in carotenoid extractions, with respect to their performance and recovery, the stability of carotenoids, production costs, and their potential effects on human health are still required.

4. Carotenoid applications

The market of carotenoids is expanding as a result of the increased use of carotenoids as food coloring and nutritional supplements due to their bioactive (i.e., antioxidant, anti-inflammatory) benefits and vitamins, as well as significant R&D efforts to accelerate the production of high-quality carotenoids. The carotenoid market shares are mainly divided into astaxanthin, capsanthin, lutein, β-carotene, lycopene, and others (Carotenoids Market, 2021–2031, Report Code: A04670). Especially, in 2021, astaxanthin accounted for the highest market share for carotenoid products in the market. The approval of the U.S. Food and Drug Administration for the application of astaxanthin as a color additive in fish and animal food applications boosts the carotenoids market demand. Furthermore, health benefits such as maintenance of skin glow, brain health improvement, and healthy vision, as well as growing awareness regarding natural ingredients drive customer preference toward algae-based astaxanthin products, thus supporting the growth of the global market. The companies identified in the global carotenoid market mainly include BASF SE (Germany), Koninklijke DSM N.V. (The Netherlands), FMC Corporation (USA), Chr. Hansen A/S (Denmark), Kemin Industries, Inc. (USA), Lycored Ltd. (Israel), and Cyanotech Corporation (USA). An increase in the competitiveness of business entities from China (i.e., NHU Co., Ltd) and India on the global market of carotenoids has also occurred.

4.1 Carotenoid as coloring agents

Carotenoids are mainly used as colorants, one of the groups of food additives by legislations (Regulation (EC) No. 1333/2008; (China) GB2760-2014; (Canada) NOM/ADM-0099) of different countries, in which natural coloring agents have been gaining popularity in the food sector. Carotenoids present a range of colors among intense yellow (diverse xanthophylls), orange (β-carotene), and red (lycopene). According to the standards for food additives, the dosage of common carotenoids such as lycopene, β-carotene, and lutein is different according to different food types and legislations. For example, the maximum consumption of lycopene in the standard (China) GB2760-2014 changed between 0.015 and 0.39 g/kg according to different food types, however for the standards (EC) No. 1333/2008 and (Canada) NOM/ADM-0099, the values are 5–500 mg/kg, and 3–100 mg/kg, respectively. In the European Union, all food additives are subject to a safety evaluation by the European Food Safety Authority before they are permitted for use in the European Union by the European Commission. The European Food Safety Authority has made recommendations defining safe daily intakes of carotenoids. For example, as an additive the acceptable daily intake (ADI) for lycopene is 0.5 mg/kg body weight per day, and the value for lutein from Tagetes erecta is 1 mg/kg body weight per day. In addition, natural astaxanthin from Haematococcus pluvialis is used as a nutritional supplement with a daily recommended consumption of 12 mg per day by the U.S. Food and Drug Administration. Noticeably, Haematococcus pluvialis and krill oil rich in astaxanthin, as well as lutein ester from Marigold Chrysanthemum have been used as new food raw materials in China, and their maximum consumptions are 0.8 g, 3 g, and 12 mg per day, respectively. Although Haematococcus pluvialis and lutein ester can be used in general food without any limitation, there are clear restrictions on their use in infant food.

4.2 Carotenoid as diet supplements

The development of the market of carotenoid-containing products results from various economic, demographic, and sociocultural factors. The senior citizen segment constitutes a major portion of the population in the developed world and is the largest user of preventive and protective medication. Health problems faced by the older population such as vision damage is often associated with insufficient intake of vitamins. Balanced consumption can be observed in contemporary consumer behaviors. These are anticipated to boost the demand for carotenoid diet supplements, thereby driving the growth of the carotenoid industry. Commercial nutraceutical carotenoid-based supplements including Trunature astaxanthin, NATURE’S BOUNTY lutein, Jamieson lycopene, NOW natural beta-carotene, and NATURGIN Fucoxanthin, in the form of capsules (or soft gel) or tablets are currently available (Table 2). The contents of carotenoids in these carotenoid-containing products range from 1 to 100 mg/capsule (piece). The functional claim of these carotenoid-containing products mainly focuses on skin health, anti-aging, treating eye disorders, preventing heart disease, cancer (prostate), and obesity due to high antioxidant properties. Especially, the products rich in β-carotene act as diet supplements of vitamin A; astaxanthin plays a positive role in anti-aging and skin care; lutein and zeaxanthin are used to prevent ocular oxidative stress, supporting eye and retina health; lycopene helps to protect the prostate, and treat prostate disease; fucoxanthin is helpful for prevention and treatment of obesity and improving liver health.

Table 2.

Examples of carotenoid-containing products.

5. Conclusion

Z-isomerization of carotenoids (i.e., lycopene and astaxanthin) can promote their extraction and bioavailability. Z-isomers of carotenoids can be applied in various health products with broad development prospects owing to its physiological activities (i.e., antioxidant, and anti-inflammatory) and high bioavailability. Thermal Z-isomerization of carotenoids using natural catalysts especially sulfur-containing compounds, as well as isothiocyanate-functionalized silica was promising for the food industry. Carotenoids used in the food sector as colorants or diet supplements are mostly extracted from various processing by-products. Traditional extraction of carotenoids using organic solvents requires an extensive downstream operation for the purification of the target carotenoids. Some natural DESs as green/safety solvents can be directly included in the final product as food additives or spices, which makes that there are two options for the use of carotenoids-DES extracts as dietary supplements in the food industry: (1) including the obtained extracts directly in food products without the elimination of DESs; (2) developing a process to isolate the target carotenoids and recycle the DES solvents. Noticeably, before carotenoids-DES extracts are directly used in food products, DESs should be guaranteed to be enough safety. In conclusion, in order to promote the development and application of carotenoids as colorants or diet supplements, food processing technologies (i.e., Z-isomerization using natural catalyst, extraction by natural DESs) need to be developed to improve the content of Z-carotenoids in processed food and promote the industrial application. However, the expansion of the worldwide market for carotenoids is hampered by strict regulatory and approval standards. Relevant standards (i.e., GB2760-2014; (EC) No. 1333/2008) of carotenoids, especially Z-carotenoids, need to be improved.

Acknowledgments

This work was supported by the grants from National Natural Science Foundation of China (32302056); Zhejiang Provincial Natural Science Foundation (Q23C200037); China Postdoctoral Science Foundation (2022M712847).

Conflict of interest

The authors declare no conflict of interest.