Antioxidant Compounds from Microalgae: A Review

1The demand for natural products isolated from microalgae has increased over the last decade and has drawn attention from the food, cosmetic and nutraceutical industries. Microalgae are eukaryotic unicellular cells that combine several advantages for the development of biotechnological applications: high biodiversity, photosynthetic yield, growth, productivity and a metabolic plasticity that can be orientated using culture conditions. Some of these metabolites are molecules of interest such as pigments (e.g., carotenoids), polyunsaturated fatty acids (PUFAs, e.g., the omega-3 or -6 fatty acids), polysaccharides, vitamins and sterols which can be introduced as dietary supplements in human nutrition and animal feed e.g.,. In addition, most of them are bioactive molecules with anti-inflammatory, antibacterial, anti-UV, antifungal, anticancer, and/or antioxidant activities which may bring added value to cosmetics, nutraceuticals or food products e.g.,.

2The demand for natural antioxidants as an alternative to synthetic antioxidants has increased. Indeed, many synthetic antioxidants (e.g., butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT)) are considered to have a carcinogenic and/or toxic effect on animal models. Although, most natural antioxidants currently available on the market are derived from terrestrial plants, microalgae are being more and more considered as a potential source of natural antioxidant compounds by the food industry and by the cosmetic and nutraceutical industries.

3Regarding the wide diversity of antioxidant compounds and mode of action combined with the diversity of ROS, this review first covers a global presentation of antioxidant molecules with their role and mode of action, to finally summarize and evaluate common and recent assays used to assess antioxidant activity of microalgae. The aim of this review is to improve our ability to choose the right assay to assess microalgae antioxidant activity regarding the antioxidant molecules studied. It also emphasizes and discusses the potential use of microalgae by the food industry for their antioxidant activity.

4An antioxidant is defined as “a substance that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate”. Antioxidant molecules produced by microalgae are used to protect the cell against reactive oxygen species (ROS) produced in response to biotic or abiotic stressors. Indeed, irradiance, UV, temperature, pH, metals, and nutrient can directly influence the production of antioxidant molecules in response to their availability, either through an excess or a limitation.

5Antioxidants used for ROS detoxification have enzymatic and nonenzymatic origins with intracellular or extracellular mode of action (e.g., singlet O2 quencher, radical scavenger, electron donor, hydrogen donor, peroxide decomposer, enzyme inhibitor, gene expression regulation, synergist, and metal-chelating agents).

6In microalgae, ROS are produced by electron transport chains in chloroplasts and mitochondria, by the activity of some enzymes such as peroxidases and oxidases and also by the activity of some photosensitizers such as the chlorophyll. The reactive oxygen species are therefore essentially generated in the chloroplasts and mitochondria but also in the peroxisomes. More generally, ROS refer to O2 derivatives that are more reactive than O2 itself. This includes free radicals that contain at least one unpaired electron, as well as nonradical molecules. Briefly, the activation of O2, in its stable state triplet oxygen (3O2), takes place (i) either by a transfer of energy large enough to reverse the spin of one of the electrons, which leads to the formation of singlet oxygen (1O2), or (ii) by an electron transfer that leads to the sequential reduction of 3O2 to superoxide radical (O2−•), hydrogen peroxide (H2O2) and hydroxyl radical (OH•).

7In plants and algae, singlet oxygen 1O2 is produced under high light by chloroplasts in the reaction center of the photosystem II (PSII) and to a lower extent in the antenna complex. In the antenna complex, triplet-excited chlorophyll (3Chl *) is formed from singlet-excited chlorophyll (1Chl *) by intersystem conversion. The chlorophyll in the triplet state has a longer lifespan than in the singlet state and can react with 3O2 to form the highly reactive 1O2. The singlet oxygen is responsible for extensive cell damage (e.g., protein, lipid and nucleic acid oxidation, chloroplasts and thylakoids membranes disruption and photoinhibition) around the production area. The reaction center of PSII is thus particularly threatened. The superoxide radical (O2−•) generation takes place in the chloroplast during photosynthesis, in the mitochondria during oxidative phosphorylation and in cell membranes through the activity of the NADPH oxidase. The superoxide radical is poorly reactive because it lacks the ability to modify macromolecules and is quickly transformed into hydrogen peroxide (H2O2). However, its protonated form is the precursor of much more reactive radicals. The hydrogen peroxide is formed by disproportionation of the O2−• a redox reaction that can be spontaneous or catalyzed by the superoxide dismutase (SOD). The hydrogen peroxide is also poorly reactive; however, it remains particularly toxic, as it can cross membranes, diffuse throughout the cell and oxidize sulfhydryl groups, causing the deactivation of essential enzymes. It can also react with DNA and more specifically with some transition metals (e.g., iron and copper) inducing the formation of highly reactive hydroxyl radicals by the Haber–Weiss reaction. The hydroxyl radical (OH•) is formed in the same cell compartments as the H2O2, i.e., in the stroma of the chloroplasts using the H2O2 generated by the photosystems, but needs the presence of reduced metal of transition. The hydroxyl radicals can induce lipid peroxidation, protein and nucleic acid denaturation. In addition, there are no enzymes that can detoxify these radicals; in excess, it might lead to cell death, and lipid peroxidation may also generate other very reactive free radicals (e.g., the perhydroxyl HO2•, alkyl radical, reactive aldehydes malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE)). Thus, the lipid-rich membranes and their functions are particularly affected by lipid peroxidation mainly through a decrease in membrane fluidity, an increase in their permeability and by enzyme, protein, ion channel and membrane receptor inactivation, which could lead to cell damage.

8Ascorbic acid or vitamin C (1) is one of the most abundant water-soluble antioxidants synthesized by plants (Figure 1). It is mainly present in the cytosol and chloroplasts where it can directly neutralize superoxide and hydroxyl radicals as well as singlet oxygen by electron transfer, in addition to its role in the detoxification of hydrogen peroxide during the ascorbate-glutathione cycle. Ascorbic acid is also involved in the protection of the photosynthetic apparatus through its participation in the regeneration of carotenoids of the xanthophyll cycle (cofactor of violaxanthin de-epoxidase) and α-tocopherol linked to membranes. It has been shown that ascorbate can also have a pro-oxidant action by the reduction of transition metals (Fe3+ to Fe2+ and Cu2+ to Cu+) which can reduce hydrogen peroxide to hydroxyl radical by the Fenton reaction.

9Glutathione (2) is a water-soluble tripeptide (l-γ-glutamyl-l-cysteinylglycine) present in all cellular compartments that play a crucial role in the antioxidant response (Figure 1). In addition to its role as a cofactor in the neutralization of hydrogen peroxide by glutathione peroxidase and in the regeneration of ascorbate in reduced form via the ascorbate-glutathione cycle, glutathione can directly deactivate superoxide and hydroxyl radicals as well as singlet oxygen. In addition, like ascorbate, glutathione participates in the regeneration of α-tocopherol in its reduced form.

10Tocopherols or vitamin E are fat-soluble molecules only synthesized by photosynthetic organisms and located in the lipid bilayers of membranes, mainly in those of chloroplasts. The name “vitamin E” groups together four natural forms of tocopherols (α-, β-, γ- and δ-) (3a–d) to which are added the four forms of tocotrienols (α-, β-, γ- and δ-) (4a–d) (Figure 1). Tocopherols and tocotrienols consist of a chromanol ring and a hydrophobic phytyl side chain, tocotrienols differing from tocopherols by the presence of three double bonds on the side chain.

11Tocopherols and tocotrienols have the capacity to neutralize lipid peroxyl radicals by giving a hydrogen atom from the hydroxyle group of the chromanol ring, thus making it possible to stop the chain reaction of lipid peroxidation. The reaction results in the formation of a hydroperoxide, which can be neutralized by the action of glutation peroxidase, and of a tocopheroxyl radical (for tocopherols) or tocotrienoxyl (for tocotrienols), which are less reactive. Tocopherols and tocotrienols can then be regenerated by the action of ascorbate and glutathione at the interface of the membrane and cytosol or by coenzyme Q (UQH2) in the membrane. Tocopherols can also deactivate singlet oxygen by two mechanisms: a physical quenching by charge transfer and a chemical reaction resulting in the formation of tocopherol quinone by irreversible opening of the chromanol ring.

12Phenolic compounds are a large family of molecules: more than 8000 phenolic structures have been described to date in the plant kingdom. These molecules contain at least one aromatic ring carrying one or more hydroxyl groups (Figure 1). The main families of compounds are phenolic acids, tocopherols described above, flavonoids and tannins as well as stilbenes and lignans. Phenolics are an important class of antioxidants in higher plants and macroalgae but have only recently been studied in microalgae. However, the total content of phenolic compounds has been shown to contribute to the antioxidant activity of microalgae extracts. The main molecules identified to date in microalgae are phloroglucinol (5) and phenolic acids derived from hydroxybenzoic acid (6) and hydroxycinnamic acid (7). Several studies have also shown the presence of weak concentrations of flavonoids e.g.,. All of these molecules are found in higher plants where their concentration is generally higher than in microalgae.

13Phenolic acids can neutralize ROS primarily by hydrogen atom transfer. The antioxidant activity of the different molecules is directly linked to their chemical structure such as the number of hydroxyl groups or their position on the aromatic cycle. The reaction results in the formation of a phenoxyl radical which is stabilized by the delocalization of the single electron around the aromatic ring (resonance stabilization). Phenolic acids also have the ability to inactivate radicals by monoelectronic transfer, and some can chelate the transition metals involved in the Fenton reaction thus preventing the formation of the highly reactive hydroxyl radical.

14Among the pigments, we can also mention marennine, a blue-green pigment produced by Haslea ostrearia, which shows particularly interesting anti-free radical and antioxidant properties.

15Carotenoids are the most common pigments in nature, and more than 750 molecules have been described in algae, higher plants, bacteria and fungi (Figure 2). They are fat-soluble molecules belonging to the terpenoids family containing a central chain with a system of conjugated double bonds, which can carry cyclic end groups. Carotenoids are separated into two groups: carotenes which contain only carbon and hydrogen atoms, and xanthophylls which contain at least one oxygen atom (hydroxyl, epoxy, ketone functions, for example).

16Carotenoids are mainly present in the pigment-protein complexes of the thylakoid membrane, but certain species of microalgae can also accumulate carotenoids (β-carotene (8) and astaxanthin (9)) in lipid globules located in the stroma of the chloroplast or in the cytoplasm. Some carotenoids are only found in specific classes of algae and so be used as chemotaxonomic markers.

17The role of carotenoids is on the one hand to transfer light energy to chlorophylls and on the other hand to protect the photosynthetic system by deactivating ROS and preventing their formation. The first photoprotection mechanism involves xanthophylls associated with the antennal complexes of the PSII which allows the dissipation of an excess of light energy without damage, according to a series of reactions called the “xanthophyll cycle”. In excess light, violaxanthin (10) is converted to antheraxanthin (11) and then to zeaxanthin (12) by de-epoxidation provided by violaxanthin de-epoxidase, which uses ascorbate as cofactor. This enzyme, bound to thylakoids in the lumen, is activated by an acidic pH, an excess of proton in the lumen signaling that the light energy absorbed exceeds the capacity of the electron transport chain. The de-epoxidation of violaxanthin to zeaxanthin is a very rapid phenomenon on the order of a few minutes and reversible at low light intensity or in darkness by the action of zeaxanthin epoxidase. Zeaxanthin, unlike violaxanthin, can deactivate 1Chl* by dissipating its energy by heat. This nonphotochemical quenching (NPQ) mechanism decreases the lifespan of 1Chl* and therefore prevents the formation of 3Chl* and then singlet oxygen in the PSII. In addition, by dissipating the excess energy, the possibilities of reducing O2 to the superoxide radical O2−• in the PSI are minimized (less electron leakage in the transport chain). The violaxanthin cycle takes place primarily in chlorophytes. There is an alternative xanthophyll cycle, with similar photoprotective functions, in certain classes of microalgae (heterokonts, haptophytes, euglenophytes and dinophyceae) for which diadinoxanthin (13) is converted to diatoxanthin (14).

18At high light intensity, the probability of 3Chl* formation is high despite the action of the xanthophyll cycle. In antenna complexes, carotenoids are located near chlorophylls and can thus quickly neutralize 3Chl* by triplet–triplet transfers before they react with 3O2 to form 1O2. Carotenoids can also directly deactivate singlet oxygen if it is formed. This ability to deactivate 1O2 is particularly important in the reaction center of PSII where there are no carotenoids in close proximity to the special pair of chlorophylls which can change to the triplet state and then react with the 3O2 without that the reaction is not neutralized beforehand by the carotenoids. Carotenoids therefore deactivate the 1O2 that is formed in the reaction center, thus protecting the photosynthetic system from oxidative damage. The deactivation of 3Chl* and of 1O2 results in the formation of triplet carotenoids (3CAR*) which de-excite without damage by dissipating the excess energy absorbed in the form of heat and can again intervene in a deactivation cycle.

19Carotenoids are considered to be the most efficient molecules in deactivating 1O2 owing to their system of conjugated double bonds. Thus, the greater the number of conjugated double bonds is, the more effective the carotenoid will be. Carotenoids also have the ability to react with free radicals through three mechanisms: hydrogen atom transfer, monoelectronic transfer and adduct formation.

20The interactions between carotenoids and free radicals are complex. Indeed, many parameters are involved, such as the nature of the radical, the polarity of the reaction medium, the partial pressure of oxygen, the interactions with other antioxidants, such as ascorbate or tocopherols, and the concentration and structure of the carotenoid (number of conjugated double bonds, presence and types of oxygen functions, presence of end groups, cis- or trans-configuration, etc.). Carotenoids can, for example, react with a peroxyl radical (ROO•), which is added to the polyene chain of the carotenoid forming an adduct ROO-CAR• which can react with another peroxyl radical forming a nonradical product ROO-CAR-OOR, thus allowing one to break the reaction chain of lipid peroxidation. This phenomenon takes place at low partial pressure of oxygen; however, at higher partial pressure, the ROO-CAR• radical can react with 3O2 to form a ROO-CAR-OO• radical which acts as a pro-oxidant and could in this case contribute to the spread of lipid peroxidation.

21There are other more specific antioxidant molecules produced by certain microalgae:

22Mycosporins-like amino acids (MAA) form a family of thirty-five molecules. They are colorless, water-soluble molecules found in a wide variety of marine organisms. In microalgae, the most abundant MAAs are mycosporin-glycine (15), porphyra 334 (16), shinorin (17), asterina-330 (18), palythene (19) and palythine (20) (Figure 3). The main function of these molecules is UV protection, but some of them have also been shown to have antioxidant properties. In particular, they can inhibit lipid peroxidation and neutralize singlet oxygen and certain free radicals.

23Polysaccharides are polymers composed of osidic units linked to glycosidic bonds attached to the cell wall or released into the medium (exopolysaccharides). Several polysaccharides derived from microalgae have shown antioxidant activity against free radicals; however, this in vitro activity remains quite low.

24Phycobiliproteins are water-soluble pigments participating in the photosynthesis of certain groups of microalgae. They are composed of a protein and a chromophore called phycobilin particularly effective at absorbing red, orange, yellow and green light, which is not optimally absorbed by chlorophyll a. There are four different structures: phycoerythrobilin (21), phycourobilin (22), phycocyanobilin (23) and phycoviolobilin (24) (Figure 3). They can neutralize ROS and chelate or reduce ferrous ions.

25Many antioxidant assays have been developed with different types of reactions to highlight the wide variety of antioxidant molecules and ROS, which act with different mechanisms. It is important to note that there is no single ideal test, and it is necessary to use several tests with different mechanisms of action to evaluate the whole antioxidant capacity of an extract or molecule.

26The majority of the assays are based on the two main mechanisms of action of antioxidants (AH) to deactivate radicals (X•):

27Hydrogen atom transfer (or HAT):

28These reactions are generally fast; they are completed in seconds to minutes. The effectiveness of the antioxidant is determined by its ability to give a hydrogen atom (homolytic dissociation energy); therefore, the weaker the A-H bond, the more effective the antioxidant is.

29Single electron transfer (SET):

30These reactions are slower than hydrogen transfer reactions. The reaction is pH dependent, and the effectiveness of the antioxidant is mainly determined by its ionization potential. In general, the ionization potential decreases with increasing pH leading to an increase in the ability to donate an electron by deprotonation.

31Other methods can also be used to evaluate the capacity of antioxidants to chelate transition metals or to inhibit the lipid peroxidation chain reaction. The most commonly used methods to evaluate the antioxidant activity of microalgae are presented in Table 1, and the most relevant results to assess antioxidant activity of microalgae extracts by in vitro chemical methods are presented in Table 2. Some cell-based antioxidant activity assays are presented, although few results using microalgae are found in the literature (Table 3). In addition, there does not seem to be any specific assays to evaluate antioxidant activity of microalgae on an animal model. Indeed, in most cases, microalgae are administrated to animals by food with a defined period and dosage; the testing animals are then sacrificed, and common in vitro chemical antioxidant activity assays (TBARS mostly) are used on animal tissues or blood by comparing with animals that did not consume microalgae (Table 4).

32The studies included in this part have been selected using Scopus and Google Scholar databases, using the terms “microalgae” in combination with “antioxidant”, “antioxidant activity”, “antioxidant capacity” or “antioxidant properties” as keywords. The research was limited to publication with an impact factor higher than 0.5, published until 2020. The studies have been selected based on these criteria: studies using in vitro (Table 2) or in cellular assays (Table 3) reporting the antioxidant activity of crude extract of eukaryotic microalgae. Studies focusing in the antioxidant activity of specific purified metabolite(s), or antioxidant enzyme activity have not been considered.

33In addition, we have included a nonexhaustive selection of studies evaluating the antioxidant activity of microalgae in different animal models (Table 4).

34The main publications evaluating the antioxidant activity of crude microalgal extracts by in vitro chemical tests are presented in Table 2. In these studies, more than two hundred strains of microalgae were evaluated. The most studied genera are Chlorella (29 strains), Scenedesmus and Tetraselmis (14 strains) (Supplementary Materials Figure S1).

35The most commonly used assays to evaluate the antioxidant activity of microalgae are the DPPH (36 studies out of 52 referenced), ABTS (20 studies) and FCA assays (13 studies) (Supplementary Materials Figure S2). Overall, the results are very heterogeneous depending on the species of microalgae studied and the tests used to measure antioxidant activity. The protocols of the assays vary from one study to another, notably in terms of the extraction method, the solvents used, the reaction time and the concentrations tested. In addition, the results are expressed in different ways, making it difficult to compare the results. For example, for the DPPH assay, the results are expressed in percentage of inhibition for a given concentration (at different concentrations according to the studies), in IC50, in equivalent trolox (per unit of weight of extract or per unit of dry weight) or in equivalent ascorbic acid. Finally, the use of a reference product as a point of comparison is not systematic, and the choice of the reference product is not always relevant according to the assay used.

36Nevertheless, several studies highlight the potential of microalgae as a source of antioxidants:

37Chloromonas sp. and Botryidiopsidaceae sp. (ethanolic extracts) show a strong ability to neutralize DPPH radicals (IC50 of 0.97 and 1.53 µg mL−1, respectively) and ABTS (IC50 of 0.95 µg mL−1 and 1.79 µg mL−1) similar to vitamin C. The ABTS assay also revealed interesting activities of Scenedesmus obliquus (IC50 of 41 µg mL−1,), Haematococcus pluvialis (activity up to 1974 µmol TE g−1 extract for supercritical H2O extraction,) and Dunaliella salina (activity up to 1118 µmol TE g−1 extract with hexane extraction,). Interesting results are also obtained with the DPPH assay for Galdieria sulphuraria, Ettlia carotinosa, Neochloris texensis, Chlorella minutissima, Chlorella vulgaris, Schizochytrium limacinum, Stichococcus bacillaris and Crypthecodinium cohnii with inhibition percentages between 89% and 95% with aqueous or methanolic extracts at concentrations of 250 µg mL−1.

38Natrah et al. showed that Chaetoceros calcitrans, Scenedesmus quadricauta, Isochrysis galbana, Chlorella vulgaris, Nannochloropsis oculata, and Tetraselmis tetrahele had a strong ability to inhibit lipid peroxidation with inhibition percentages ranging from 88% to 98% for methanolic extracts at 80 µg mL−1 with the TBARS assay and between 88.4 and 97% for extracts at 200 µg mL−1 with the FTC assay (Ferric ThioCyanate assay, indirect measurement of the quantity of hydroperoxides formed during the first stages of lipid oxidation). The ability of the genera Tetraselmis to inhibit lipid peroxidation is confirmed by Coulombier et al. who have obtained an IC50 up to 3,4 µg mL−1 with a methanol-dichloromethane extract. Euglena tuba also seems to be an interesting species for its ability to inhibit lipid peroxidation (IC50 with TBARS assay = 42 µg mL−1) and to neutralize the superoxide radical (IC50 = 5.2 µg mL−1,). Some species show good ability to neutralize superoxide radical such as Chaetoceros sp. (1029 µmol TE g−1 dichloromethane extract), Nannochloropsis sp. (3224 µmol TE g−1 methanol extract), Chlorella stigmatophora and Phaeodactylum tricornutum (IC50 of 48.37 and 68.61 µg mL−1 with aqueous extracts,). Chloroform and methanol extracts of Chaetoceros sp. also show interesting results with the FRAP assay (610 and 492.50 µmol TE g−1,). Good results are also obtained with the TAC assay with IC50 below 100 µg mL−1 for methanolic extracts of Chlorella vulgaris and Chlamydomonas reinhardtii.

39The antioxidant activity of the genus Chlorella has been demonstrated by several authors with different antioxidant assays. In addition to the results obtained with the DPPH, TBARS, FTC, TAC, and superoxide radical neutralization assays presented above, Aremu et al. obtained IC50 up to 25 µg mL−1 with the β-carotene bleaching assay for Chlorella minutissima and Chlorella sp. and Plaza et al. showed activities up to 1008 µmol TE g−1 of Chlorella vulgaris extract with the ORAC assay.

40Overall, few links are made between these antioxidant activities and the metabolites involved. Still, correlations have been shown with carotenoid content, phenolic compound content including flavonoids and gallic acid and vitamin E content.

41Despite cellular assays potentially giving more biological relevant information, as they take into account the bioavailability and metabolism of the tested compounds, we found only four studies using cellular assays to determine antioxidant activity of microalgae extract (Table 3). Those studies use different antioxidant cellular assays and different cell models (mouse fibroblast, macrophage or lymphoma cells and human liver cancer cell line).

42Chloroform, methanol, acetone and 70% ethanol extracts of Chaetoceros calcitrans showed high nitric oxide scavenging activity in mouse macrophage with IC50 values of 3.46, 3.83, 15.35 and 17.94 µg mL−1, respectively, that is closed to reference compounds (IC50 of 4.7 and 6.1 µg mL−1 for quercetin and curcumin,). This strong inhibitory activity of nitric oxide was attributed to the carotenoid content of Chaetoceros calcitrans (fucoxanthin, astaxanthin, violaxanthin, zeaxanthin, canthaxanthin and lutein). Karawita et al. showed that Pediastrum duplex extract has a good protective effect against DNA damage induced by hydrogen peroxyde exposure (Comet assay). Indeed, a decrease of 80% of DNA damage on mouse lymphoma cells was measured with Pediastrum extract at 100 µg mL−1 compared to control with no microalgae extract. Good antioxidant activity was also measured with CAA (cellular antioxidant activity) and CLPAA (cellular lipid peroxidation antioxidant activity) assays on human liver cancer cell line with Ostreopsis ovata and Alexandrium minutum; however, both species extracts showed toxicity in cytotoxicity assay.

43Similarly to cellular assays, the evaluation of the antioxidant activity of microalgae extracts by in vivo experimentations are limited compared to in vitro assays (Table 4). Those studies used different antioxidant in vitro assays couple with other physiological measurement, such as antioxidant enzyme activity, on various animal models (e.g., shrimps, chicken, catfish, rats, turbots or trouts, Table 4) to assess the effect of microalgae. The microalgae (Schizochytrium sp., Chlorella vulgaris, Amphora coffeaformis, Schizochytrium limacinum, Acutodesmus obliquus, Nannochloropsis spp., Tetraselmis chuii and Botryococcus braunii) were mostly included in the animal feed as dry microalgae with a percentage of inclusion mainly going from 1–10% or as a molecule equivalent of given antioxidant compounds. The results are variable depending on species from no effect of the microalgae tested to a decrease in oxidative stress measurements such as the malondialdehyde or hydrogen peroxide content or a decrease in DNA damage. In most cases, it seems that the inclusion of microalgae directly in the fed has a positive effect on the animal physiology, which is promising regarding further used of microalgae in the food industry either in human or animal nutrition as functional ingredients. It also raises the question of the bioavailability of an antioxidant compound in the algal matrices and thus of the digestibility of the microalgae tested.

44In the food industry, antioxidants are used for human and animal nutrition as functional ingredients to provide nutritional benefits to a product (e.g., orange juice enriched with vitamin C), and as preservatives to extend the shelf life of foods and beverages to prevent their degradation by oxidation. The use of antioxidant ingredients in food products intended for humans is highly regulated by country-specific laws owing to their potential toxicity. In the European Union, there is a list of authorized antioxidant additives, some of which may be of natural origin such as vitamin C (E300-E304), vitamin E (E306-E309), guaiac resin (E314) and rosemary extract (E392). Certain carotenoids are also authorized as dyes but can have an antioxidant role such as β-carotene (E160a), lycopene (E160d), lutein (E161b), violaxanthin (E161e), zeaxanthin (E161h), canthaxanthin (E161g) or astaxanthin (E161j). For foods and ingredients that were not significantly consumed before 1997, such as most microalgae, the "Novel Food" regulation framework was to be applied in Europe. New microalgae on the market must obtain this authorization; however, to receive it, it has to be demonstrated that the product does not present any risk in terms of safety for human health as some microalgae are known to produce phytotoxins. In addition, and beyond the regulatory framework, to be of interest to the food industry, an antioxidant should not affect the color, smell and taste of the food and should be effective at low concentrations (0.001–0.01%), be easily usable, stable during processing and storage and be inexpensive. The use of microalgae may thus be regarded as promising additive for human food, livestock feed and shelf life; however, it greatly depends on the microalgae productivity and nutrient compositions in protein, carbohydrates, lipids, vitamins, and antioxidants, which also strongly depend on species, mode of cultivation and culture medium composition e.g.,. Currently, around 10 species of microalgae or microalgae extract are authorized for human consumption in Europe as a food or food ingredient.

45For livestock feed, antioxidant additives are subject to authorization before going on the market, an authorization that remains only valid ten years. On the other hand, raw materials are not subject to authorization, but a contribution of microalgae as an antioxidant in animal feed could only be considered as a raw material if it also provides proteins, minerals, fats, fibers, energy or carbohydrates. Microalgae presents growth rate and dietary value of interest (e.g., polyunsaturated fatty acids, vitamins, pigments, polysaccharides) for livestock feed or aquaculture feed either fish, live feed and shellfish applications (e.g., in Table 4). Indeed, in aquaculture, the polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (DHA) and docosahexanoic acid (DHA) are of nutritional importance in aquafeeds and are hitherto ensured by inclusion of fish oil in aquafeeds. However, this resource is limited, and microalgae offer an alternative to fish oil. In addition, microalgae are not only seen as a source of PUFAs but also as source of other metabolites of interest such as pigments, polysaccharides, vitamins (e.g., vitamin E and C) and sterols which are introduced as dietary supplements for dietetic and therapeutic purposes. In terms of applications, antioxidant molecules (asthaxanthin, lutein, β-carotene) carotenoids are produced by a wide variety of microalgae (see Table 2).

46Antioxidant molecules from microalgae are more and more considered as a potential source of natural antioxidant compounds by the food, the cosmetic and nutraceutical industries as they may bring benefits to their products.

47However, it is very crucial to assess properly the antioxidant activity of an algal extract owing to the wide diversity of antioxidant compounds and the mode of action combined with the diversity of ROS involved. This review highlights the lack of standardization between extractions procedures used to assess antioxidant activity from microalgae matrices, and more disturbingly, it highlights the inappropriateness between the assay used and the molecules studied. These often hamper the comparison between studies and bring the authors to false or incorrect interpretation of their results.

48Therefore, although all the assays have their merits and demerits, the appropriate selection of a given assay was to be made based on the mode of action of a studied molecule in front of the principle and mechanism of an assay, especially in vitro assays. In addition to the need of normalization of the extraction procedures and to the appropriate use of an assay, we conclude that it is crucial to combine many assays to assess microalgae full antioxidant activity.

49This review also highlights that microalgae are rich in antioxidant molecules with more or less potent activities, which can be used as an ingredient in food, cosmetic and nutraceutical industries. In addition, research publications are available on modern in vitro chemical methods, but application on cellular assays and in vivo experimentations are still lacking. There is a need to develop models to improve our ability to assess the activity of antioxidant molecule on these kinds of models to further improve industrial adaption and application.