Algal Carotenoids: Chemistry, Sources, and Application

Recently, the isolation and identification of various biologically active secondary metabolites from algae have been of scientific interest, with particular attention paid to carotenoids, widely distributed in various photosynthetic organisms, including algal species. Carotenoids are among the most important natural pigments, with many health-promoting effects. Since the number of scientific studies on the presence and profile of carotenoids in algae has increased exponentially along with the interest in their potential commercial applications, this review aimed to provide an overview of the current knowledge (from 2015) on carotenoids detected in different algal species (12 microalgae, 21 green algae, 26 brown algae, and 43 red algae) to facilitate the comparison of the results of different studies. In addition to the presence, content, and identification of total and individual carotenoids in various algae, the method of their extraction and the main extraction parameters were also highlighted.


Introduction
Carotenoids are the most widely distributed lipid-soluble pigments in nature. They are common in various photosynthetic organisms (higher plants, fungi, algae, and bacteria), where they are primarily biosynthesized and are responsible for the various colors, hues, and shades [1]. They play several roles: during photosynthesis they act as accessory pigments for light collection, they are involved in preventing damage from excess light and serve as one of the most important cell antioxidants, they are important in the reproductive cycle as their color attracts pollinators, they are provitamins, etc. [2,3]. Carotenoids are a very complex and heterogeneous group of compounds whose greatest structural diversity is found in organisms of the marine environment. Some carotenoids are restricted exclusively to aquatic sources. Complex habitats and harsh conditions have led algae to produce a wide range of specific and bioactive compounds. Due to the diverse bioactivity of algae and their use in food, medicine, and cosmetics, they are currently being intensively studied for the production of carotenoid compounds [4][5][6][7].
Currently, commercially available carotenoids are usually produced by chemical synthesis, which involves high economic costs and has a negative impact on the environment. These facts have led to a significant increase in demand for natural sources of carotenoids, with a focus on marine organisms, especially algae. According to the Scopus database, the number of publications and citations related to carotenoids in algae has increased in the last decade, and it is still permanently increasing ( Figure 1).
In this review, articles published from 2015 (via keywo Science direct databases) on the presence, isolation, and pounds (total and individual) in various microalgal and mac  Carotenoids are able to absorb ultraviolet (UV) and light in the visible region of the spectrum because they have conjugated double bonds in their structure (chromophore). They usually have three absorption maxima in the visible region of the spectrum (between 430 and 480 nm), and only a few of them have maxima in the UV region (e.g., phytoene). Therefore, the differences in the spectral characteristics of individual carotenoids are often small but very important for their identification. Carotenoid absorption maximums depend on the nature of the carotenoid (polyenic extremity, presence of the C=O conjugated groups, and the cis-trans configuration of the molecule). It should also be noted that the used solvent has a great influence on the absorption maximum of the compound [38][39][40]. The total carotenoid content of isolates is usually determined by measuring the absorbance of the sample at a specific wavelength and calculating it from the absorbance values reported in the literature. In this method, the content of total carotenoids is usually expressed as equivalents of β-carotene [8,10,11,37,39,40].
The separation and quantification of carotenoids is usually performed by high-performance liquid chromatography (HPLC) using different stationary phases (octyl-C8, octadecyl-C18, and C30), with C18 being the most commonly used and C30 being more efficient in separating the geometric isomers. Their separation can be performed by normal and reversed phase HPLC, but the separation of carotenes by normal phase HPLC is usually not good [41]. The most common detectors are ultraviolet (UV), visible (Vis), and diode array detector (DAD). In these methods, carotenoids are identified based on their retention times, UV/visible spectral characteristics, and/or mass spectra and compared with data obtained for standard compounds tested under the same conditions. Since some carotenoids cannot be identified from their absorbance or spectrum alone, mass spectrometry (MS), MS/MS, electrospray ionization (ESI), time-of-flight (TOF) MS, etc., are often used [11,37,42].
The great diversity of carotenoid structures and their susceptibility to degradation make their identification and quantification very difficult. In addition, there is no reference method for their extraction and detection, they are often present at low concentrations and surrounded by numerous interfering substances, and commercial standards are often not available, further complicating their identification and quantification.

Algal Carotenoids
The carotenoid profile of algae is similar to that of higher plants in terms of their species, location, and distribution [1]. However, carotenoid composition varies qualitatively and quantitatively among different species, especially since the extraction method is not generally standardized. Various abiotic and biotic factors such as the growth stage, harvest location and period, depth, nutrient quantity and quality, temperature, salinity, light exposure, etc. also vary. The most important carotenoids from microalgae and macroalgae are astaxanthin, fucoxanthin, β-carotene, lutein, siphonaxanthin, zeaxanthin, violaxanthin, neoxanthin, and antheraxanthin [4,5,43,44]. Figure 2 shows the chemical structures of selected carotenoids that have been detected in algae.

Carotenoids in Microalgae
The main carotenoids produced by marine microalgae are β-carotene, astaxanthin, lutein, fucoxanthin, zeaxanthin, echinenone, and violaxanthin [45], but various environmental factors such as temperature, light, salinity, and nutrient content affect their production [46]. The cell walls of microalgae are an obstacle to the extraction of carotenoids. Therefore, to increase extraction yield, cell disruption (by mechanical or nonmechanical methods) is required, which is often combined with other methods to weaken and/or  Table 1 provides an overview of the studies on the extraction of carotenoids from microalgae. As can be seen, various microalgae species have been studied, and the presence of different carotenoids has been reported, such as fucoxanthin, lutein, neoxanthin, violaxanthin, α-carotene, β-carotene, etc.
Astaxanthin is an orange-red-colored xanthophyll with hydroxyl-and keto-groups [52][53][54]. Among algal species, Haematococcus pluvialis has the highest ability to biosynthesize astaxanthin and the greatest potential for its accumulation [5], while the microalga Dunaliella salina has been recognized as an important industrial source of β-carotene, a carotene with two beta rings at both ends of the molecule (β-ionone ring substitutions), especially due to its wide geographic distribution, ease of cultivation, and ability to exist under extreme environmental conditions. Its major advantage is that it serves as a precursor of vitamin A [5,46,55]. As for the solvents used, Table 1 shows that alcoholic solvents and acetone are the most commonly used.  UV/Vis-ultraviolet-visible; HPLC-high-performance liquid chromatography; LC-liquid chromatography; DAD-diode array detector; MS-mass spectrometry; QTOF-quadrupole time-of-flight; MetOH-methanol; ACE-acetone; EtOH-ethanol; Chl-chloroform; DCM-dichloromethane; DME-dimethyl ether; EtOAc-ethyl acetate; Hex-hexane; Hep-heptane; SFE-supercritical fluid extraction; CSE-conventional solvent extraction; PLE-pressurized liquid extraction; UAME-ultrasound-assisted microextraction; UAE-ultrasound-assisted extraction; PEF-pulsed electric field; MAE-microwave-assisted extraction; MFAE-magnetic-field-assisted extraction; RT-room temperature; fw-fresh weight. From the reported results, the content of total carotenoids varied in the different studies and the highest content was found in D. salina (up to 25 mg/g) in the study by Tirado and Calvo [47]. The authors used supercritical fluid extraction as a green method for the extraction of carotenoids, while supercritical CO 2 with EtOH/MetOH was used as the extraction solvent. Zhao et al. [21] reported an extremely high concentration of astaxanthin in H. pluvialis (111.2 mg/g), Ishika et al. [51] reported a similarly high concentration of fucoxanthin in Chaetoceros muelleri (2.92 mg/g), and Gayathri et al. [26] reported similar results for lutein in Chlorella salina (2.76 mg/g). Other studies also reported the presence of other carotenoids in microalgae samples: β-carotene, adonixanthin, antheraxanthin, neoxanthin, and echinenone in H. pluvialis [59] and neoxanthin, violaxanthin, α-carotene, and β-carotene in Desmodesmus sp. F51 [58].

Carotenoids in Green Algae (Chlorophyta)
The most abundant carotenoids in green algae are β-carotene, lutein, violaxanthin, and zeaxanthin, which are more widely distributed in green algal species than in higher plants [1]. As can be seen from the studies reported in Table 2, conventional extraction methods are generally used for the isolation and extraction of carotenoids from green algae, while only two studies [62,63] used extraction methods with ultrasound. Again, acetone is the most commonly used extraction solvent.

Carotenoids in Brown Algae (Phaeophyta)
Brown algae contain more xanthophylls than carotenes, and this prevalence is responsible for their coloration and activity [89]. The major pigments in brown algae are fucoxanthin, β-carotene, and violaxanthin [1]. Fucoxanthin is the major carotenoid in brown algae. It is an allelic carotenoid, a 5,6-monoepoxide that has nine conjugated double bonds and oxygen-containing functional groups (hydroxyl, epoxy, carbonyl, and carboxyl groups). This unique structure distinguishes it from other carotenoids [5,90,91]. Its content varies greatly depending on location, season, and other factors (pH and salinity). For example, fucoxanthin concentration has been found to increase in winter in response to sunlight limitation [90,91].
Fucoxanthin and the other carotenoids were isolated from different species of brown algae using different extraction and detection methods that resulted in different concentrations. Again, the most commonly used method to isolate carotenoids was CSE, followed by UAE [62,[92][93][94], but the use of other methods has also been reported [94][95][96]. In addition, hexane and alcoholic solvents were used in most cases ( Table 3).

Carotenoids in Red Algae (Rhodophyta)
The dominant carotenoids in red algae are zeaxanthin, lutein, and αand β-carotene, but in contrast to the α-:β-carotene ratio typical of higher plants, the α-carotene content is much higher in red algae [1]. A review of recent studies identifying carotenoids in red algae is presented in Table 4. Red algal carotenoids were mostly extracted by conventional extraction with solvents (alcohols, acetone, hexane, ethyl acetate, or water), while only some studies used ultrasound to assist the extraction .

Potential Applications of Algal Carotenoids
The most commercially interesting carotenoids include astaxanthin, β-carotene, lutein, and zeaxanthin, which are widely distributed in algae, making these organisms an important source of natural carotenoids. Fucoxanthin, abundant in brown algae, is considered a therapeutic and nutritional ingredient with a unique chemical structure that enables its reactions in many physiological functions and ensures its strong biological properties. Algal carotenoids (fucoxanthin and others) derived from Indian brown algae (Padina tetrastromatica) have been studied against oxidative stress in rats [115]. It was found that lipid oxidation induced by retinol deficiency (plasma and liver) was reduced by the supplementation of fucoxanthin (plasma 7-85% and liver 24-72%) versus β-carotene (plasma-51-76% and liver 33-65%) by enhancing the activity of catalase and glutathione transferase enzymes. Similarly, Jang et al. [116] reported the ability of fucoxanthin from Laminaria japonica to impart hepatoprotective effects under oxidative stress, suggesting its inclusion in the formulation of nutraceuticals. For other algae-derived carotenoids, protective properties such as cardioprotective, hepatoprotective, photoprotective, renal protective, and various health-promoting and beneficial properties such as antioxidant, anti-obesity, antitumor, antidiabetic, anti-inflammatory, and hepatoprotective have been confirmed in the literature [29,45,91,101,[115][116][117][118][119][120][121][122][123]. Due to these properties, algae-derived carotenoids have been investigated for various applications. Most commonly, they are used as dietary supplements and food colorants, for the production of functional/nutraceutical foods and animal feeds, for the formulation of food packaging, in health care, and in cosmetics [65,[124][125][126]. The main challenges for their industrial application are the extraction method, reported variations in yield, and their unstable nature [124,[126][127][128]. Based on the proposed concerns, several studies have been conducted and reported on the ability of carotenoid-rich algae as a blend, coating, film, and additive to various food matrices, which have been carefully presented in previous reports [124,[129][130][131].
Taking carotenoids from marine algae as natural antioxidants has been shown to be effective in reducing obesity and weight gain. The molecular mechanism of obesity has been linked to inflammation and oxidative stress, which lead to the development of other metabolic diseases (e.g., type 2 diabetes, hypertension, and liver disease). Antioxidants from marine algae have been suggested as potential replacements for conventional treatments such as surgery or drugs. Carotenoids from algae have been associated with the regulation of key factors in adipogenesis, glucose levels, and fatty acid metabolism [119]. In addition, algal carotenoids can be used for the nutrient fortification of foods. They have been incorporated as powders or oils into various food matrices such as pasta, bread, cookies, vegetable soup, and yogurt [132][133][134]. The formulation and dosage for each product still need to be optimized as high doses of some carotenoids or algae are considered unacceptable by consumers, mainly because of their color and flavor properties. However, in the right concentration, they could provide nutrient fortification and antioxidant activity and even prolong the shelf life of foods. On the contrary, there are studies confirming that a pigment such as fucoxanthin can be successfully used as a colorant to improve the appearance of and provide bioactivity to foods and beverages [90].
Algal carotenoids showed potential in food packaging film formulations. Films formulated with algal extract (Fucus vesiculosus) exhibited lower lipid oxidation in chicken breast samples, which was attributed to higher carotene content [135]. Similarly, Sáez et al. [136] reported the preservative action of carotenoid-rich water extracts from algae on rainbow trout fillets. The application inhibited the growth of total viable counts and lipid oxidation and helped to preserve the quality of fillets by improving their water holding capacity [136]. Recently, Pereira et al. [137] summarized studies and meta-analyses on the health-related properties and effects of marine-derived carotenoids.
Algae-derived carotenoids have also been added to feed to improve the color of fish (salmon and trout), crustaceans, and eggs [138]. These organisms represent a conventional food source for humans, and diets rich in carotenoids have been associated with health benefits. The carotenoid profile is largely dependent on feed composition [139]. Traditionally, carotenoids have been used extensively as colorants in foods; however, with the development of natural sources for the extraction of carotenoids and their extensive assimilability to synthetic forms, they found further applications in the feed industry [138]. The application of algal pigment extracts rich in carotenoids has shown the ability to enhance immunity to Vibrio infections and promote weight gain in shrimp production [140]. Another study by Abdel-Rahim et al. [141] concluded that dietary supplementation of algae apart from the findings of Aftab Uddin et al. [140] had imparted cold tolerance. In the case of beef feed supplemented with algae, it lowered the carbon footprint by lowering the methane production in vitro without loss of efficiency, which supported its use in feeds [142].
Pigments from marine algae are also used as active ingredients in cosmetics, where their addition delays skin aging and protects against UV radiation, which leads to the formation of reactive oxygen species. The formation of these components can damage DNA and lead to hyperpigmentation, premature aging, sunburn, and skin cancer [143]. The presence of carotenoids in skin tissue occurs through two mechanisms: diffusion from the body, e.g., adipose tissue and plasma, and/or secretion by sebaceous glands and reabsorption. In addition, the content and profile of carotenoids reflect those present in plasma, the most important being lutein, β-carotene, lycopene, zeaxanthin, β-cryptoxanthin, and colorless pigments (phytoene and phytofluene). The presence of carotenoids on the skin surface is associated with the reduction of oxidation and inflammation, leading to other effects such as inhibition of metalloproteases, inhibition of UVA-induced expression of heme oxygenase 1, prevention of mitochondrial DNA mutations, and photoimmunomodulation. In a study by Grether-Beck et al. [144], oral supplementation of lutein and lycopene was shown to result in photoprotection. The effect was at the molecular level by inhibiting UVA1 and UVA/B-induced gene expression. Astaxanthin was found to decrease the expression of matrix metalloproteinases that degrade collagen and elastin. It has also been associated with improvement in sebum oil levels, wrinkling, elasticity, and hydration [145].
Brown algae have also been used as fertilizers with improved effects on plants. Nurjannah et al. [146] used a fermented brown algae extract (Sargassum sp.) on Zea mays L. to test the effect on corn growth. Parameters such as the height, stem circumference, cob length, and diameter improved after spraying with algal extracts compared with the control and urea-phosphate-potassium fertilizer application. Baroud et al. [147] tested the effects of brown algal extracts (C. gibraltarica, F. spiralis, and Bifurcaria bifurcate) on the germination, growth, and biochemical profile of tomatoes. Improvements in the germination rate and seedling biomass were observed. Compared with the control, the biochemical composition was higher in terms of protein content (21.59 mg/g to 54.64 mg/g), pigments (0.38 mg/g to 0.61 mg/g), and polysaccharide content (21.04 mg/g to 56.38 mg/g). Brown algae were also used to improve the quality of postharvest products. Extracts of brown algae Sargassum crassifolium, S. cristaefolium, S. aquifolium, and Turbinaria murayana were used as sprays for tomatoes. More fruits (15 fruits/plant) were reported when algae were added to the urea than in the control (9 fruits/plant). In addition to harvest and storage, tomatoes sprayed with brown algae had better texture after 7 days of storage at room temperature [148].

Future Directions
The bioactivity of algal carotenoids and the increasing awareness of their potential health-promoting properties make them attractive for application in various industries and fields, including nutraceuticals, food, feed, pharmaceuticals, and cosmetics [53]. The global market for carotenoids is expected to grow from an estimated USD 1.5 billion in 2019 to USD 2.0 billion in 2026 [91]. Algae production is still concentrated in Asian countries, with China dominating with a total production of over 56% of global aquaculture [149]. Microalgae are already widely used for the commercial production of carotenoids, and their use is rapidly increasing in various sectors due to their fast growth rate, resource sustainability, significantly higher production of carotenoids compared with macroalgae or terrestrial plants, and ability to quickly adapt to new or changing growth conditions [20]. Macroalgae also produce more biomass than higher plants (they grow more than ten times faster) and can be grown in both fresh and marine water, and their cultivation is carried out without the use of pesticides and/or antibiotics, etc. [149], which in turn highlights their industrial potential for isolating valuable constituents. Therefore, future research in this field, especially to develop new technologies to improve the efficiency of algae extraction, is needed.
Accordingly, the production of carotenoids from algae has a bright future, but there are still some major challenges that need to be overcome, mainly related to the cost of algae production, optimization of harvesting and extraction of key compounds, and stability and storage of the isolated products. Also, the current issues of global warming and sea level rise are negatively impacting algal biomass production and quality, leading to losses and degradation of beneficial target components. The numerous current investigations in this scientific field are an indication that this industry is growing exponentially and will certainly lead to more competitive processes and final products with a wide range of applications.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.