- Email: [email protected]
Microalgae as a Novel Source of Antioxidants for Nutritional Applications
C H A P T E R
17 Microalgae as a Novel Source of Antioxidants for Nutritional Applications Koen Goiris1, Koenraad Muylaert2, Luc De Cooman1 1
KU Leuven Technology Campus Ghent, Laboratory of Enzyme, Fermentation and Brewing Technology, Ghent, Belgium; 2KU Leuven Kulak, Research Unit Aquatic Biology, Kortrijk, Belgium
1. OXIDATIVE STABILITY OF FOOD SYSTEMS
Termination:
1.1 Lipid Oxidation Along with microbial stability, oxidative stability is a determining factor in the overall shelf life of foodstuffs. In most cases, lipid oxidation determines the oxidative stability of foods, although other food components are also subject to oxidative transformations. Lipid oxidation is crucial for food stability as highly flavor-active aldehydes are formed, resulting in flavor deterioration. The most important mechanisms leading to lipid oxidation are spontaneous oxidation of lipids (auto-oxidation), light-induced oxidation (photo-oxidation), and lipoxygenase-mediated oxidation. The different stages in the process of auto-oxidation, which is a free-radical reaction, are given in Reactions (1)e(9): Initiation: LH þ X, /L,
(1)
Propagation: L, þ O2 /LO2 , ,
(2) ,
LO2 þ LH/LOOH þ L
Handbook of Marine Microalgae http://dx.doi.org/10.1016/B978-0-12-800776-1.00017-0
LO2 , þ LO2 , /LOOL þ O2
(4)
LO2 , þ L, /LOOL
(5)
,
,
L þ L /LL
(6)
Alternative Initiation: LOOH/LO, þ HO, ,
2 LOOH/LO þ LO2
,
Mnþ þ LOOH/LO, þ HO þ Mðnþ1Þþ
(7) (8) (9)
In the first step in Reaction (1), the lipid radical L is generated through the intervention of an initiator molecule that induces the abstraction of an a-methylenic hydrogen atom from the lipid LH. Once the free radical L is generated, Reactions (2) and (3) lead to a chain reaction; the lipid peroxide radical, which is formed in the fast reaction (2), attacks lipid molecules with formation of lipid hydroperoxides (3) and another lipid radical L . The function of chain-breaking antioxidants (ArOH) is the reaction with lipid peroxide, which halts the chain reaction, as given in Reaction (10):
(3)
269
LO2 , þ ArOH/LOOH þ ArO,
(10)
© 2015 Elsevier Inc. All rights reserved.
270
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
Effective antioxidants must react slowly with the substrate LH but rapidly with the LO2 and, secondly, the formed ArO radical must be relatively stable. The weaker the OH bond in ArOH, the more effective the antioxidant will be in donating an H-atom. Well-known chainbreaking antioxidants that inhibit lipid peroxidation are tocopherols and phenolics (Wright et al., 2001). There are two main pathways by which nonenzymatic antioxidants can deactivate radicals and prevent oxidative damage, namely hydrogen atom transfer (HAT) and single electron transfer (SET), which often occur in parallel. The dominant mechanism is predetermined by the properties of the antioxidant as well as the reaction environmentdthat is, solubility of the antioxidant and solvent used (Prior et al., 2005). The general reaction of the HAT mechanism, with AH being the hydrogen-donating antioxidant, is given by Reaction (11):
AH þ X, /A, þ XH
(11)
The second mechanism by which an antioxidant can deactivate radicals is single electron transfer, given by Reactions (12)e(14): X, þ AH/X þ AH,þ H2 O
AH,þ # A, þ H3 Oþ
þ
X þ H3 O /XH þ H2 O
(12) (13) (14)
Considering Reaction (13), it is obvious that the overall electron transfer mechanism is pH dependent and the reactivity of the antioxidant increases at higher pH values.
1.2 Commercial Antioxidants: Synthetic and Natural Alternatives To reduce the oxidative adulteration of food or bulk oils, many synthetic and natural antioxidants are added during food processing. Synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated
hydroxyanisol (BHA), and t-butyl hydroquinone, are restricted in their applications and levels (<0.02% of lipid content) because their toxicological safety has been debated (Namiki, 1990). Therefore, they are replaced with natural antioxidants where possible. Examples of natural antioxidants are tocopherols, polyphenols, and carotenoids. Well-known sources of natural sources of food-grade antioxidants are rosemary (rosmarinic acid), tea (catechins), and grape (flavonoids).
2. ANTIOXIDANT ACTIVITY IN MICROALGAE 2.1 Reactive Oxygen Species: Formation and Physiological Role The most important pro-oxidants in biological systems are reactive oxygen species (ROS), which are formed in varying physiological processes. In microalgae, ROS are continuously produced in chloroplasts, mitochondria, and peroxisomes (Figure 1). To avoid damage to cell components, production and scavenging of ROS must be strictly balanced; hence, antioxidant protective mechanisms must be in place. 2.1.1 Reactive Oxygen Species In its ground state, molecular oxygen or triplet oxygen can be considered a biradical because it contains two unpaired electrons in parallel spin. Ground-state oxygen can be converted to much more reactive ROS forms by energy transfer or by electron transfer reactions, leading to radical ROS and nonradical ROS (Figure 2). 2.1.2 Formation Sites of Reactive Oxygen Species In photosynthetic organisms, including microalgae, ROS are continuously produced as byproducts from various metabolic pathways (Apel and Hirt, 2004). Under light stress, excited
2. ANTIOXIDANT ACTIVITY IN MICROALGAE
FIGURE 1
271
Cellular pathways of reactive oxygen species (ROS) in microalgae. Based on Cirulis et al. (2013) and Laloi et al.
(2006).
FIGURE 2
Generation of different ROS by energy transfer (production of singlet oxygen) or sequential univalent reduction of ground-state triplet oxygen. Based on Apel and Hirt (2004).
272
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
triplet chlorophyll from the photosystem II reaction center in the chloroplasts may transfer its excitation energy onto triplet ground-state oxygen, yielding the highly reactive singlet oxygen. A second ROS-generating mechanism originates in the electron transfer system. When the lightdriven electron transport exceeds consumption of electrons needed for CO2 fixation or NADP supply is limited, molecular oxygen can be reduced by photosystem I to superoxide, which is rapidly converted to hydrogen peroxide by superoxide dismutase. Furthermore, in the peroxisomes, recycling of glycolate from photorespiration and fatty acid oxidation are both processes that produce hydrogen peroxide, which in its turn is mitigated by catalase. A third endogenous ROS source is the generation of superoxide by ubiquinone in the electron transport chain during oxidative phosphorylation in the mitochondria. However, mitochondrial ROS production is much lower than the production in chloroplasts. A last source of superoxide is the activity of NADPH oxidase in the plasma membrane.
2.2 ROS Detoxification in Microalgae To counteract the detrimental effects of ROS, all living organisms have several defensive systems at their disposal, both enzymatic and nonenzymatic. In this section, the most important ROS-associated enzymes and antioxidants found in microalgae are discussed. 2.2.1 Enzymatic Antioxidant Protection Superoxide dismutase (EC 1.15.1.1) consists of a mixture of metalloproteins differentiated by their metal cofactor. The three isoforms common to plants (CuZn-SOD, Fe-SOD, and Mn-SOD) are also present in microalgae (Janknegt et al., 2009). Superoxide dismutase catalyzes the neutralization of superoxide radicals with the formation of hydrogen peroxide and oxygen. Catalase (EC 1.11.1.6) catalyzes the conversion of hydrogen peroxide to water and oxygen. The catalase enzyme is sensitive to light, which
may affect the ability of photoautotrophic organisms to tolerate oxidative stress when exposed to high light irradiance. Another group of enzymes that catalyze reduction of hydrogen peroxide to water are peroxidases. They differ from catalase in their requirement of an electron donor that subsequently becomes oxidized. Ascorbate peroxidase (EC 1.11.1.11), present in the stroma and thylakoids of chloroplasts, has a significantly lower Km value for hydrogen peroxide than catalase and uses vitamin C as a specific electron donor (Asada and Akahashi, 1987). Glutathione peroxidase (EC 1.11.1.9) requires glutathione for the removal of hydrogen peroxide. Several isoforms have been detected in the microalgae Chlamydomonas reinhardtii (Dayer et al., 2008) and Chlorella sp. (Wang and Xu, 2012). 2.2.2 Nonenzymatic Antioxidant Protection L-Ascorbic acid or vitamin C, abundant in photosynthetic organisms, can reduce many ROS. Vitamin C is present in both cytosol and chloroplast where it takes part in the ascorbatee glutathione cycle to remove hydrogen peroxide (Mallick and Mohn, 2000). Next to its vital role in the elimination of hydrogen peroxide, vitamin C also scavenges superoxide, hydroxyl radicals, and lipid hydroperoxides (Lesser, 2006). In the chloroplast, vitamin C plays a crucial role in the regeneration of membrane-bound carotenoids and tocopherols, thereby protecting the photosynthetic apparatus (Mallick and Mohn, 2000). High levels of vitamin C have been reported in Chlorella sp. (Running et al., 2002), Dunaliella sp. (Barbosa et al., 2005), Chaetoceros calcitrans, and Skeletonema costatum (Brown et al., 1998, 1999). Production of vitamin C is stimulated by high light exposure (Barbosa et al., 2005), allelochemicals such as ethyl 2-methyl acetoacetate (Yang et al., 2011), or ultraviolet stress (Abd El-baky et al., 2004). Glutathione (GSH), a tripeptide (Glu-Cys-Gly) found in animals and photosynthetic organisms, acts as an antioxidant in many ways including
2. ANTIOXIDANT ACTIVITY IN MICROALGAE
reaction with superoxide, singlet oxygen, and hydroxyl radicals. Glutathione also acts as a chainbreaker of free-radical reactions and is crucial in the regeneration of ascorbate (Lesser, 2006). As a substrate for glutathione peroxidase, it donates the electrons necessary for decomposition of hydrogen peroxide (Kohen and Nyska, 2002). Tocopherols are located in the lipid bilayers of cell membranes. The most widespread homologs in nature are four tocopherols and four tocotrienols: a, b, g, and d -tocopherol as well as a, b, g, and d -tocotrienol (Colombo, 2010). Tocopherols and tocotrienols have the same basic chemical structure characterized by a long chain attached at the 2-position of a chromane ring. However, tocotrienols contain three conjugated double bonds in the aliphatic side chain instead of the saturated C16 side chain found in tocopherols (Figure 3). The most active antioxidant form is a-tocopherol, which is only synthesized in the chloroplasts of photosynthetic organisms. a-Tocopherol acts as an antioxidant through its ability to quench both singlet oxygen and (lipid) peroxides (Lesser, 2006; Mallick and Mohn, 2000). Although a-tocopherol is located in the membranes and the hydrophilic vitamin C is located in the liquid
FIGURE 3
273
phase, vitamin C is able to reduce the tocopheroxyl radical, thereby recycling the active form of tocopherol in the chloroplast (Buettner, 1993; Niki, 1991). a-Tocopherol is produced in high amounts by Dunaliella tertiolecta and Tetraselmis suecica. Carballo-Cardenas et al. (2003) demonstrated that production of a-tocopherol is highly variable throughout the growth cycle and that nutrient composition can be used to control its production in both species. In Dunaliella salina, a-tocopherol production is stimulated by UV-stress, nitrogen limitation, and salt stress (Abd El-baky et al., 2004). Another study showed that decreasing N-concentrations in the growth medium leads to increased a-tocopherol accumulation in Nannochloropsis oculata, but growth rate is reduced under these conditions (Durmaz, 2007). The lipophilic carotenoids are produced de novo by photoautotrophs. In photosynthetic organisms, carotenoids are present in the pigmenteprotein complexes of the thylakoid membranes of chloroplasts, where they fulfill a dual function (Cogdell et al., 1994). Some carotenoids (especially ketocarotenoids such as fucoxanthin, Figure 4) act as accessory light-harvesting pigments by
Chemical structures of tocopherols and tocotrienols.
274
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
transferring light energy of wavelengths that cannot be captured by chlorophylls (Takaichi, 2011). Next, carotenoids also have a protective function by dissipating excess energy and by quenching ROS that are produced during photosynthesis (Goss and Jakob, 2010). The xanthophyll cycle, that is, the cyclical interconversion of violaxanthin, antheraxanthin, and zeaxanthin in chlorophytes, provides zeaxanthin needed for dissipation of excess energy from excited chlorophylls in photosynthetic organisms. In diatoms and dinoflagellates, an alternative xanthophyll cycle exists where diadinoxanthin is converted into diatoxanthin (diatoms) or dinoxanthin (dinoflagellates). Another mode of photoprotection by carotenoids is the quenching of excited triplet-state chlorophyll and singlet oxygen by b-carotene. The main mechanism in carotenoid photoprotection against singlet oxygen functions through electronic energy transfer as given by reaction (15) (Edge et al., 1997); however,
FIGURE 4
chemical quenching with formation of carotenoid epoxides also occurs (Liebler, 1993). 1
O2 þ CAR/3 O2 þ 3 CAR
(15)
During physical quenching (reaction (15)), the carotenoid triplet state is produced through energy transfer. This excited state can return to the ground state by dissipating energy as heat or by translocation over the conjugated double bond system. Therefore, the ability to quench singlet oxygen increases with longer chain lengths of the conjugated system (Edge et al., 1997). Next to the ability of carotenoids to quench singlet oxygen, they can also react with free radicals. Carotenoids can react with peroxyl radicals and are involved in recycling of phenoxyl radicals and tocopheroxyl radicals, which are formed upon reaction of phenolic antioxidants and tocopherols with peroxyl and alkoxyl radicals (Burke et al., 2001; Burton, 1989; Edge et al., 1997). However, unlike quenching of singlet oxygen, which mainly leads to
Chemical structures of some carotenoids occurring in microalgae.
2. ANTIOXIDANT ACTIVITY IN MICROALGAE
energy dissipation as heat, the reactions of carotenoids (or any antioxidant) with free radicals will lead to electron transfer or addition reactions. Three reaction mechanisms describe the reaction of free radicals with carotenoids, that is, electron transfer, hydrogen atom transfer, and radical addition to the carotenoid (Martínez and Barbosa, 2008; Martínez et al., 2008). In order to scavenge free radicals, carotenoids can either donate or accept unpaired electrons. Usually, antioxidant molecules become oxidized by donating electrons to the free radical. However, carotenoids can also quench free radicals by accepting an unpaired electron, rendering it harmless by translocation over the conjugated side chain. In a comparative study, it was observed that the apolar lycopene as well as the xanthophylls were the most effective carotenoids in reducing ferric ions (FRAP assay) (M€ uller et al., 2011). In the same study, it was further demonstrated that the group of carotenes (lycopene, a- and b-carotene) were more efficient quenchers of the ABTS þ radical than most of the xanthophylls and that keto-carotenoids were most efficient in scavenging peroxyl radicals, due to their more pronounced conjugated double bond systems. In microalgae, a distinction is usually made between primary and secondary carotenoids. Whereas primary carotenoids are structural and functional components of the photosynthetic apparatus, and thus essential for survival, secondary carotenoids are produced at high levels when cells are exposed to specific environmental stimuli (Jin et al., 2003). At present, carotenoids (both primary and secondary) are the most commercialized products from microalgae. One such carotenoid that can be sourced from microalgae is lutein. Lutein is also found in the human retina where it acts both as an optical filter and as an antioxidant to protect long-chain polyunsaturated fatty acids and is therefore important for our eye’s health (Rapp et al., 2000). In Scenedesmus, lutein production, as well as b-carotene production, can be stimulated
275
by increasing pH and temperature during cultivation (Guedes et al., 2011b). Further, Wei et al. (2008) showed that lutein content of heterotrophically grown Chlorella protothecoides increased in response to singlet oxygen, but was reduced when cells were exposed to hydroxyl radicals. Growth-limiting conditions, such as pH values of six or nine and a temperature of 33 C, were found to stimulate carotenogenesis in the chlorophyte Muriellopsis sp. (Del Campo et al., 2000), which is currently the commercial source of lutein. Lutein content in this species is the highest in early stationary phase. Another microalgal pigment, sold as antioxidant, is the secondary carotenoid astaxanthin, produced by Haematococcus pluvialis. Accumulation of astaxanthin occurs when H. pluvialis cells are exposed to stress, induced by a combination of high light, high salt levels and nitrogen deprivation (Boussiba, 2000; Wang et al., 2003). The last microalgal pigment that is currently produced commercially for its antioxidant properties is b-carotene, extracted from the halophile D. salina. Carotenogenesis in this microalga is induced by Fe2þ, as well as by UV-stress. Also in Chlorella vulgaris, an increase in carotenoid content is observed upon metal exposure (Mallick, 2004). A good overview of the optimum conditions for carotenoid production is given by Guedes et al. (2011a). A last group of antioxidant secondary metabolites are polyphenols, which are present, often at high levels, in virtually all plants (Pietta, 2000). Polyphenols comprise a structurally diverse group of components, including simple phenols, phenolic acids, flavonoids, tannins, and lignans. Polyphenols can inhibit lipid oxidation in different ways, that is, by directly scavenging HOCl, singlet oxygen, lipid peroxyl, superoxide and hydroxyl radicals, by metal chelation or by inhibiting lipoxygenase (Dugas et al., 2000; Rice-Evans et al., 1996; Salah et al., 1995). The general mechanism of radical scavenging by phenolics is given in Figure 5. In the first step, a hydrogen atom is donated to the radical, and an aroxyl radical is produced. This radical is
276
FIGURE 5
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
General radical scavenging scheme of phenolics. R represents superoxide anion, peroxyl, alkoxyl, or hydroxyl
radicals.
relatively stable through resonance and can interact with another radical yielding a quinone. This means that, in the general mechanism, one polyphenol molecule is able to quench two radical molecules. However, if high metal concentrations are present, the aroxyl radical can interact with oxygen, generating a quinone and a superoxide anion, rather than terminating the radical chain reactions. This mechanism is responsible for the undesired pro-oxidant effect of phenolics that can occur under specific conditions (Pietta, 2000). Next to their direct radical scavenging properties, polyphenols are able to chelate metals, hereby reducing the oxidative stress as transition metals are involved in ROS generation (Pietta, 2000). Although phenolics are well-studied antioxidant components in higher plants, the acknowledgment of their presence in microalgae is fairly recent. Li et al. (2007) and Hajimahmoodi et al. (2010) screened microalgae for polyphenol content and antioxidant activity and found large variations between samples. More recently, Goiris et al. (2012) indicated that the antioxidant potential of microalgae is not only determined by its carotenoid content but also other components, including phenolics, are important contributors to overall antioxidant activity. Next to the presence of simple phenols in microalgae (Klejdus et al., 2009; Onofrejov a et al., 2010), the presence of flavonoids in microalgae has been acknowledged, albeit at low levels (Goiris et al., 2014; Klejdus et al., 2010). Microalgae can further produce some remarkable polyphenolic antioxidant molecules such as marennine in the diatom Haslea ostrearia (Pouvreau et al., 2008), purpurogallin in the extremophile snow algae
Mesotaenium berggrenii (Remias et al., 2012) or even BHT, the well-known food additive (E321), which was found in the chlorophyte Botryococcus braunii and the cyanobacteria Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Oscillatoria sp. (Babu and Wu, 2008). In microalgae, little is known about the response of phenolic components to environmentally induced oxidative stress. Duval et al. (2000) examined the effects of UV-exposure on antioxidant properties of Chlamydomonas nivalis and observed an increase in phenolics upon exposure to UV-C light. Another study on potential effects of UV on phenolic content was performed with Scenedesmus quadricauda (Kovacik et al., 2010). This study found no significant changes in total phenolic content when cells were exposed to elevated levels of UV-A but noticed a 50% decrease in the flavonols quercetin and kaempherol. When the cells were exposed to UV-C, these flavonols were not found in the biomass, suggesting breakdown of these components by UV-C. On the other hand, benzoic acids increased upon UV-A exposure and cinnamic acid decreased when cells were exposed to UV-A or UV-C. Others found that production of BHT (Babu and Wu, 2008) was stimulated under high light irradiation in B. braunii, C. raciborskii, M. aeruginosa, and Oscillatoria sp. Other studies described the influence of metal stress on the phenolic content in microalgae. Ulloa et al. (2012) found that phenolic content was stimulated by strontium addition to cultures of T. suecica. Also in S. quadricauda (Kovacik et al., 2010) and Phaeodactylum tricornutum (Rico et al., 2013), phenolic content was higher when cells were exposed to metals. A better understanding on how polyphenol concentrations change in
3. POTENTIAL OF MICROALGAL ANTIOXIDANTS TO REDUCE LIPID OXIDATION IN FOODSTUFF
response to oxidative stress will clarify the role of polyphenols as antioxidants in microalgae and learn how to maximize production for use in the food, feed, or chemical industry.
2.3 Other Antioxidants Next to the antioxidant components commonly found in other plants, some microalgae produce specific types of antioxidants such as the phycobilin proteins phycocyanin in cyanobacteria (Benedetti et al., 2004; Huang et al., 2007; Thangam et al., 2013; Yoshikawa and Belay, 2008) and phycoerythrin in rhodophytes and cyanobacteria (Huang et al., 2007). Especially the cyanobacteria Arthrospira platensis is known to produce high amounts of phycocyanin (Oliveira et al., 2009). Also dimethylsulfide/ dimethylsulfoxide (Sunda et al., 2002) and sulfated polysaccharides (Tannin-Spitz et al., 2005) contribute to the antioxidant pool of microalgae.
3. POTENTIAL OF MICROALGAL ANTIOXIDANTS TO REDUCE LIPID OXIDATION IN FOODSTUFF 3.1 Current Knowledge and Applications Over the last decade, increasing evidence has been gathered on the potential of microalgal extracts for retarding lipid oxidation. For instance, Ranga Rao et al. (2006) studied the effect of crude acetone extracts of B. braunii on lipid peroxidation in model systems. The relatively high degree of inhibition of lipid peroxidation, in comparison with the synthetic antioxidant BHT, was ascribed to carotenoids and polyphenols in the extracts. Tannin-Spitz et al. (2005) studied the effect of sulfated polysaccharides from Porphyridium cruentum on lipid oxidation and found inhibition rates of up to 80% at a concentration of 10 mg mL1 Benedetti et al. (2004) reported the use of a phycocyanin-rich extract from the cyanophyte
277
Aphanizomenon flos-aquae to reduce cupperinduced oxidation of plasma lipids and found that at a concentration of 1 mM phycocyanin, oxidation of blood lipids was reduced by a factor three. Lee et al. (2010) assessed the antioxidant properties of the microalgae Halochlorococcum porphyrae and Oltamannsiellopsis unicellularis using both solvent extracts and enzymatic digests. Lipid peroxidation was strongly inhibited by all methanolic extracts, as well as the ethyl acetate fraction of H. porphyrae and the chloroform fraction of O. unicellularis which inhibited lipid peroxidation similar to a-tocopherol. In addition, some enzymatic digests exhibited effects similar to the synthetic antioxidant BHT. Several studies demonstrated the efficacy of ethanolic extracts of Chlorella sp. on lipid peroxidation and measured similar degrees of inhibition compared to BHT (Choochote et al., 2014; Rodriguez-Garcia and Guil-Guerrero, 2008). Another study by Natrah et al. (2007) screened 14 samples of Malaysian indigenous microalgae and identified six species, that is, S. quadricauda, C. vulgaris, N. oculata, Tetraselmis tetrathele and especially Isochrysis galbana and C. calcitrans, of which crude methanolic extracts inhibited the oxidation of linoleic acid to the same extent as the commercial antioxidants BHA and BHT. Recently, the use of whole biomass of C. vulgaris and H. pluvialis has been shown to retard lipid oxidation in bulk oils (Lee et al., 2013) as well as in food emulsions (Gouveia et al., 2005). Although all studies mentioned above describe the activity against lipid oxidation in view of radical scavenging activity of the extracts, earlier work by Matsukawa et al. (1997) looked at the potential of microalgal extracts for inhibition of two important oxidative enzymes that are involved in oxidation of lipids and proteins, that is, lipoxygenase and tyrosinase, respectively. In this study, it was indicated that ethanol extracts contained efficient inhibitors of the lipoxygenase activity, whereas methanol extracts showed the highest tyrosinase inhibition, compared to ethanol and aqueous extracts.
278
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
3.2 Future Perspectives Although the efficacy of microalgal antioxidants toward lipid oxidation has been proven, little data are available on how these novel antioxidant formulations perform compared to commercially available antioxidant products, both from a cost-perspective and from the antioxidant action in real food systems. Further, the active principles still need to be further characterized to allow standardization and commercialization. Thirdly, growth conditions should be optimized for maximal productivity of antioxidant components. Finally, a big hurdle that has to be taken before incorporating microalgal products in foodstuffs are the legislative constrictions concerning novel foods. Since only a few species are currently allowed for human consumption, many microalgae with high antioxidant potential still await approval before they can be used in foodstuff.
References Abd El-baky, H.H., El-Baz, F.K., El-baroty, G.S., 2004. Production of antioxidant by the green alga Dunaliella salina. Int. J. Agri. Biol. 6 (1), 49e57. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Ann. Rev. Plant Biol. 55, 373e399. Asada, K., Akahashi, M., 1987. Production and scavenging of active oxygen in photosynthesis. In: Kyle, D.J., Osmond, C.B., Arntzen, C.J. (Eds.), Photoinhibition. Elsevier, Amsterdam, The Netherlands, pp. 228e287. Babu, B., Wu, J.-T., 2008. Production of natural butylated hydroxytoluene as an antioxidant by freshwater phytoplankton 1. J. Phycol. 44 (6), 1447e1454. Barbosa, M.J., Zijffers, J.W., Nisworo, A., Vaes, W., van Schoonhoven, J., Wijffels, R.H., 2005. Optimization of biomass, vitamins, and carotenoid yield on light energy in a flat-panel reactor using the A-stat technique. Biotech. Bioeng. 89 (2), 233e242. Benedetti, S., Benvenuti, F., Pagliarani, S., Francogli, S., Scoglio, S., Canestrari, F., 2004. Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenon flos-aquae. Life Sci. 75 (19), 2353e2362. Boussiba, S., 2000. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Phys. Plant. 108, 111e117.
Brown, M.R., Mular, M., Miller, I., Farmer, C., Trenerry, C., 1999. The vitamin content of microalgae used in aquaculture. J. App. Phycol. 11, 247e255. Brown, M.R., Skabo, S., Wilkinson, B., 1998. The enrichment and retention of ascorbic acid in rotifers fed microalgal diets. Aquacult. Nutr. 4, 151e156. Buettner, G.R., 1993. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300, 535e543. Burke, M., Edge, R., Land, E.J., Mcgarvey, D.J., Truscott, T.G., 2001. One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments. FEBS Lett. 500, 132e136. Burton, G.W., 1989. Antioxidant action of carotenoids. J. Nutr. 119, 109e111. Carballo-Cardenas, E.C., Minh Tuan, P., Janssen, M., Wijffels, R.H., 2003. Vitamin E (a-tocopherol) production by the marine microalgae Dunaliella tertiolecta and Tetraselmis suecica in batch cultivation. Biomol. Eng. 20 (4e6), 139e147. Choochote, W., Suklampoo, L., Ochaikul, D., 2014. Evaluation of antioxidant capacities of green microalgae. J. Appl. Phycol. 26 (1), 43e48. Cirulis, J.T., Scott, J.A., Ross, G.M., 2013. Management of oxidative stress by microalgae. Can. J. Physiol. Pharmacol. 91 (1), 15e21. Cogdell, R.J., Gillbro, T., Andersson, P.O., Liu, R.S.H., Asato, A.E., 1994. Carotenoids as accessory light-harvesting pigments. Pure Appl. Chem. 66 (5), 1041e1046. Colombo, M.L., 2010. An update on vitamin E, tocopherol and tocotrienol-perspectives. Molecules 15 (4), 2103e2113. Dayer, R., Fischer, B.B., Eggen, R.I.L., Lemaire, S.D., 2008. The peroxiredoxin and glutathione peroxidase families in Chlamydomonas reinhardtii. Genetics 179 (1), 41e57. Del Campo, J.a, Moreno, J., Rodríguez, H., Vargas, M.a, Rivas, J., Guerrero, M.G., 2000. Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta). J. Biotech. 76 (1), 51e59. Dugas, A.J., Casta~ neda-Acosta, J., Bonin, G.C., Price, K.L., Fischer, N.H., Winston, G.W., 2000. Evaluation of the total peroxyl radical-scavenging capacity of flavonoids: structure-activity relationships. J. Nat. Prod. 63 (3), 327e331. Durmaz, Y., 2007. Vitamin E (a-tocopherol) production by the marine microalgae Nannochloropsis oculata (Eustigmatophyceae) in nitrogen limitation. Aquaculture 272 (1e4), 717e722. Duval, B., Shetty, K., Thomas, W.H., 2000. Phenolic compounds and antioxidant properties in the snow alga Chlamydomonas nivalis after exposure to UV light. J. Appl. Phycol. 11, 559e566. Edge, R., McGarvey, D.J., Truscott, T.G., 1997. The carotenoids as anti-oxidantsda review. J. Photochem. Photobiol. B 41 (3), 189e200.
REFERENCES
Goiris, K., Muylaert, K., Fraeye, I., Foubert, I., De Brabanter, J., De Cooman, L., 2012. Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. J. App. Phycol. 24 (6), 1477e1486. Goiris, K., Muylaert, K., Voorspoels, S., Noten, B., De Paepe, D., Baart, G.J.E., E Baart, G.J., 2014. Detection of flavonoids in microalgae from different evolutionary lineages. J. Phycol. 50 (3), 483e492. Goss, R., Jakob, T., 2010. Regulation and function of xanthophyll cycle-dependent photoprotection in algae. Photosynth. Res. 106 (1e2), 103e122. Gouveia, L., Raymundo, A., Batista, A.P., Sousa, I., Empis, J., 2005. Chlorella vulgaris and Haematococcus pluvialis biomass as colouring and antioxidant in food emulsions. Eur. Food Res. Technol. 222 (3e4), 362e367. Guedes, A.C., Amaro, H.M., Malcata, F.X., 2011a. Microalgae as sources of carotenoids. Mar. Drugs 9 (4), 625e644. Guedes, A.C., Amaro, H.M., Pereira, R.D., Malcata, F.X., 2011b. Effects of temperature and pH on growth and antioxidant content of the microalga Scenedesmus obliquus. Biotech. Progr. 27 (5), 1218e1224. Hajimahmoodi, M., Faramarzi, M.A., Mohammadi, N., Soltani, N., Oveisi, M.R., Nafissi-Varcheh, N., 2010. Evaluation of antioxidant properties and total phenolic contents of some strains of microalgae. J. Appl. Phycol. 22 (1), 43e50. Huang, Z., Guo, B.J., Wong, R.N.S., Jiang, Y., 2007. Characterization and antioxidant activity of selenium-containing phycocyanin isolated from Spirulina platensis. Food Chem. 100 (3), 1137e1143. Janknegt, P., De Graaff, C.M., Van De Poll, W., Visser, R., Rijstenbil, J., Buma, A., 2009. Short-term antioxidative responses of 15 microalgae exposed to excessive irradiance including ultraviolet radiation. Eur. J. Phycol. 44 (4), 525e539. Jin, E., Polle, J.E.W., Lee, H.K.U.M., Hyun, S.M.I.N., Chang, M.A.N., 2003. Xanthophylls in microalgae: from biosynthesis to biotechnological mass production and application. J. Microbiol. Biotech. 13, 165e174. Klejdus, B., Kopecký, J., Benesova, L., Vacek, J., 2009. Solidphase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species. J. Chrom. A 1216 (5), 763e771. Klejdus, B., Lojkov a, L., Plaza, M., Sn oblova, M., Sterbova, D., 2010. Hyphenated technique for the extraction and determination of isoflavones in algae: ultrasound-assisted supercritical fluid extraction followed by fast chromatography with tandem mass spectrometry. J. Chrom. A 1217 (51), 7956e7965. Kohen, R., Nyska, A., 2002. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol. Pathol. 30 (6), 620e650.
279
Kovacik, J., Klejdus, B., Backor, M., 2010. Physiological responses of Scenedesmus quadricauda (Chlorophyceae) to UV-A and UV-C light. Photochem. Photobiol. 86, 612e616. Kovacik, J., Klejdus, B., Hedbavny, J., Backor, M., 2010. Effect of copper and salicylic acid on phenolic metabolites and free amino acids in Scenedesmus quadricauda (Chlorophyceae). Plant Sci. 178 (3), 307e311. Laloi, C., Przybyla, D., Apel, K., 2006. A genetic approach towards elucidating the biological activity of different reactive oxygen species in Arabidopsis thaliana. J. Exp. Bot. 57 (8), 1719e1724. Lee, S.-H., Lee, J.-B., Lee, K.-W., Jeon, Y.-J., 2010. Antioxidant properties of tidal pool microalgae, Halochlorococcum porphyrae and Oltamannsiellopsis unicellularis from Jeju Island, Korea. Algae 25 (1), 45e56. Lee, Y.-L., Chuang, Y.-C., Su, H.-M., Wu, F.-S., 2013. Freezedried microalgae of Nannochloropsis oculata improve soybean oil’s oxidative stability. Appl. Microbiol. Biotech. 97 (22), 9675e9683. Lesser, M.P., 2006. Oxidative stress in marine environments: biochemistry and physiological ecology. Ann. Rev. Physiol. 68 (3), 253e278. Li, H., Cheng, K., Wong, C., Fan, K., Chen, F., Jiang, Y., 2007. Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. Food Chem. 102 (3), 771e776. Liebler, D.C., 1993. Antioxidant reactions of carotenoids. Ann. N. Y. Acad. Sci. 691 (1), 20e31. Mallick, N., 2004. Copper-induced oxidative stress in the chlorophycean microalga Chlorella vulgaris: response of the antioxidant system. J. Plant Physiol. 161 (5), 591e597. Mallick, N., Mohn, F.H., 2000. Reactive oxygen species: response of algal cells. J. Plant Physiol. 157, 183e193. Martínez, A., Barbosa, A., 2008. Antiradical power of carotenoids and vitamin E: testing the hydrogen atom transfer mechanism. J. Phys. Chem. B 112 (51), 16945e16951. Martínez, A., Rodríguez-Girones, M.a, Barbosa, A., Costas, M., 2008. Donator acceptor map for carotenoids, melatonin and vitamins. J. Phys. Chem. A 112 (38), 9037e9042. Matsukawa, R., Dubinsky, Z., Masaki, K., Takeuchi, T., Karube, I., 1997. Enzymatic screening of microalgae as a potential source of natural antioxidants. Appl. Biochem. Biotech. 66 (3), 239e247. M€ uller, L., Fr€ ohlich, K., B€ ohm, V., 2011. Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (aTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chem. 129 (1), 139e148. Namiki, M., 1990. Antioxidants/antimutagens in food. Crit. Rev. Food Sci. Nutr. 29 (4), 273e300. Natrah, F.M.I., Yusoff, F.M., Shariff, M., Abas, F., Mariana, N.S., 2007. Screening of Malaysian indigenous microalgae for antioxidant properties and nutritional value. J. App. Phycol. 19 (6), 711e718.
280
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
Niki, E., 1991. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am. J. Clin. Nutr. 54, 1119Se1124S. Oliveira, E.G., Rosa, G.S., Moraes, M.A., Pinto, L.A.A., 2009. Characterization of thin layer drying of Spirulina platensis utilizing perpendicular air flow. Bioresour. Technol. 100 (3), 1297e1303. Onofrejov a, L., Vasíckova, J., Klejdus, B., Stratil, P., Misurcov a, L., Kracmar, S., Vacek, J., 2010. Bioactive phenols in algae: the application of pressurized-liquid and solid-phase extraction techniques. J. Pharm. Biomed. Anal. 51 (2), 464e470. Pietta, P.G., 2000. Flavonoids as antioxidants. J. Nat. Prod. 63 (7), 1035e1042. Pouvreau, J.-B., Morançais, M., Taran, F., Rosa, P., Dufosse, L., Guerard, F., Pondaven, P., 2008. Antioxidant and free radical scavenging properties of marennine, a blue-green polyphenolic pigment from the diatom Haslea ostrearia (Gaillon/ Bory) Simonsen responsible for the natural greening of cultured oysters. J. Agric. Food Chem. 56 (15), 6278e6286. Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53 (10), 4290e4302. Ranga Rao, A., Sarada, R., Baskaran, V., Ravishankar, G.A., 2006. Antioxidant activity of Botryococcus braunii extract elucidated in vitro models. J. Agric. Food Chem. 54 (13), 4593e4599. Rapp, L.M., Maple, S.S., Choi, J.H., 2000. Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina. Invest. Ophthalmol. Vis. Sci. 41 (5), 1200e1209. Remias, D., Schwaiger, S., Aigner, S., Leya, T., Stuppner, H., L€ utz, C., 2012. Characterization of an UV- and VISabsorbing, purpurogallin-derived secondary pigment new to algae and highly abundant in Mesotaenium berggrenii (Zygnematophyceae, Chlorophyta), an extremophyte living on glaciers. FEMS Microbiol. Ecol. 79 (3), 638e648. Rice-Evans, C., Miller, N.J., Paganga, G., 1996. Structureactivity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20 (7), 933e956. Rico, M., L opez, A., Santana-Casiano, J.M., Gonzalez, A.G., Gonz alez-D avila, M., 2013. Variability of the phenolic profile in Phaeodactylum tricornutum diatom growing under copper and iron stress. Limnol. Oceanogr. 58 (1), 144e152. Rodriguez-Garcia, I., Guil-Guerrero, J.L., 2008. Evaluation of the antioxidant activity of three microalgal species for use as dietary supplements and in the preservation of foods. Food Chem. 108 (3), 1023e1026. Running, J.A., Severson, D.K., Schneider, K.J., 2002. Extracellular production of L-ascorbic acid by Chlorella protothecoides, Prototheca species, and mutants of P. moriformis during aerobic culturing at low pH. J. Ind. Microbiol. Biotechnol. 29 (2), 93e98.
Salah, N., Miller, N.J., Paganga, G., Tijburg, L., Bolwell, G.P., Rice-Evans, C., 1995. Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch. Biochem. Biophys. 322 (2), 339e346. Sunda, W., Kieber, D.J., Kiene, R.P., Huntsman, S., 2002. An antioxidant function for DMSP and DMS in marine algae. Nature 418, 317e320. Takaichi, S., 2011. Carotenoids in algae: distributions, biosyntheses and functions. Mar. Drugs 9 (6), 1101e1118. Tannin-Spitz, T., Bergman, M., Van-Moppes, D., Grossman, S., Arad, S.(M.), 2005. Antioxidant activity of the polysaccharide of the red microalga Porphyridium sp. J. Appl. Phycol. 17 (3), 215e222. Thangam, R., Suresh, V., Princy, W.a., Rajkumar, M., SenthilKumar, N., Gunasekaran, P., Kannan, S., 2013. C-Phycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest. Food Chem. 140 (1e2), 262e272. ~ez, M.J., Ulloa, G., Otero, A., Sanchez, M., Sineiro, J., N un Fabregas, J., 2012. Effect of Mg, Si, and Sr on growth and antioxidant activity of the marine microalga Tetraselmis suecica. J. App. Phycol. 24 (5), 1229e1236. Wang, B., Zarka, A., Trebst, A., Boussiba, S., 2003. Astaxanthin accumulation in Haematococcus pluvialis (chlorophyceae) as an active photoprotective process under high irradiance. J. Phycol. 39, 1116e1124. Wang, X., Xu, X., 2012. Molecular cloning and functional analyses of glutathione peroxidase homologous genes from Chlorella sp. NJ-18. Gene 501 (1), 17e23. Wei, D., Chen, F., Chen, G., Zhang, X., Liu, L., Zhang, H., 2008. Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress. Sci. China, Ser. C: Life Sci. 51 (12), 1088e1093. Wright, J.S., Johnson, E.R., DiLabio, G.A., 2001. Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 123 (6), 1173e1183. Yang, C.-Y., Liu, S.-J., Zhou, S.-W., Wu, H.-F., Yu, J.-B., Xia, C.-H., 2011. Allelochemical ethyl 2-methyl acetoacetate (EMA) induces oxidative damage and antioxidant responses in Phaeodactylum tricornutum. Pestic. Biochem. Physiol. 100 (1), 93e103. Yoshikawa, N., Belay, A., 2008. Single-laboratory validation of a method for the determination of c-phycocyanin and allophycocyanin in Spirulina (Arthrospira) supplements and raw materials by spectrophotometry. J. AOAC Int. 91 (3), 524e529.
17 Microalgae as a Novel Source of Antioxidants for Nutritional Applications Koen Goiris1, Koenraad Muylaert2, Luc De Cooman1 1
KU Leuven Technology Campus Ghent, Laboratory of Enzyme, Fermentation and Brewing Technology, Ghent, Belgium; 2KU Leuven Kulak, Research Unit Aquatic Biology, Kortrijk, Belgium
1. OXIDATIVE STABILITY OF FOOD SYSTEMS
Termination:
1.1 Lipid Oxidation Along with microbial stability, oxidative stability is a determining factor in the overall shelf life of foodstuffs. In most cases, lipid oxidation determines the oxidative stability of foods, although other food components are also subject to oxidative transformations. Lipid oxidation is crucial for food stability as highly flavor-active aldehydes are formed, resulting in flavor deterioration. The most important mechanisms leading to lipid oxidation are spontaneous oxidation of lipids (auto-oxidation), light-induced oxidation (photo-oxidation), and lipoxygenase-mediated oxidation. The different stages in the process of auto-oxidation, which is a free-radical reaction, are given in Reactions (1)e(9): Initiation: LH þ X, /L,
(1)
Propagation: L, þ O2 /LO2 , ,
(2) ,
LO2 þ LH/LOOH þ L
Handbook of Marine Microalgae http://dx.doi.org/10.1016/B978-0-12-800776-1.00017-0
LO2 , þ LO2 , /LOOL þ O2
(4)
LO2 , þ L, /LOOL
(5)
,
,
L þ L /LL
(6)
Alternative Initiation: LOOH/LO, þ HO, ,
2 LOOH/LO þ LO2
,
Mnþ þ LOOH/LO, þ HO þ Mðnþ1Þþ
(7) (8) (9)
In the first step in Reaction (1), the lipid radical L is generated through the intervention of an initiator molecule that induces the abstraction of an a-methylenic hydrogen atom from the lipid LH. Once the free radical L is generated, Reactions (2) and (3) lead to a chain reaction; the lipid peroxide radical, which is formed in the fast reaction (2), attacks lipid molecules with formation of lipid hydroperoxides (3) and another lipid radical L . The function of chain-breaking antioxidants (ArOH) is the reaction with lipid peroxide, which halts the chain reaction, as given in Reaction (10):
(3)
269
LO2 , þ ArOH/LOOH þ ArO,
(10)
© 2015 Elsevier Inc. All rights reserved.
270
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
Effective antioxidants must react slowly with the substrate LH but rapidly with the LO2 and, secondly, the formed ArO radical must be relatively stable. The weaker the OH bond in ArOH, the more effective the antioxidant will be in donating an H-atom. Well-known chainbreaking antioxidants that inhibit lipid peroxidation are tocopherols and phenolics (Wright et al., 2001). There are two main pathways by which nonenzymatic antioxidants can deactivate radicals and prevent oxidative damage, namely hydrogen atom transfer (HAT) and single electron transfer (SET), which often occur in parallel. The dominant mechanism is predetermined by the properties of the antioxidant as well as the reaction environmentdthat is, solubility of the antioxidant and solvent used (Prior et al., 2005). The general reaction of the HAT mechanism, with AH being the hydrogen-donating antioxidant, is given by Reaction (11):
AH þ X, /A, þ XH
(11)
The second mechanism by which an antioxidant can deactivate radicals is single electron transfer, given by Reactions (12)e(14): X, þ AH/X þ AH,þ H2 O
AH,þ # A, þ H3 Oþ
þ
X þ H3 O /XH þ H2 O
(12) (13) (14)
Considering Reaction (13), it is obvious that the overall electron transfer mechanism is pH dependent and the reactivity of the antioxidant increases at higher pH values.
1.2 Commercial Antioxidants: Synthetic and Natural Alternatives To reduce the oxidative adulteration of food or bulk oils, many synthetic and natural antioxidants are added during food processing. Synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated
hydroxyanisol (BHA), and t-butyl hydroquinone, are restricted in their applications and levels (<0.02% of lipid content) because their toxicological safety has been debated (Namiki, 1990). Therefore, they are replaced with natural antioxidants where possible. Examples of natural antioxidants are tocopherols, polyphenols, and carotenoids. Well-known sources of natural sources of food-grade antioxidants are rosemary (rosmarinic acid), tea (catechins), and grape (flavonoids).
2. ANTIOXIDANT ACTIVITY IN MICROALGAE 2.1 Reactive Oxygen Species: Formation and Physiological Role The most important pro-oxidants in biological systems are reactive oxygen species (ROS), which are formed in varying physiological processes. In microalgae, ROS are continuously produced in chloroplasts, mitochondria, and peroxisomes (Figure 1). To avoid damage to cell components, production and scavenging of ROS must be strictly balanced; hence, antioxidant protective mechanisms must be in place. 2.1.1 Reactive Oxygen Species In its ground state, molecular oxygen or triplet oxygen can be considered a biradical because it contains two unpaired electrons in parallel spin. Ground-state oxygen can be converted to much more reactive ROS forms by energy transfer or by electron transfer reactions, leading to radical ROS and nonradical ROS (Figure 2). 2.1.2 Formation Sites of Reactive Oxygen Species In photosynthetic organisms, including microalgae, ROS are continuously produced as byproducts from various metabolic pathways (Apel and Hirt, 2004). Under light stress, excited
2. ANTIOXIDANT ACTIVITY IN MICROALGAE
FIGURE 1
271
Cellular pathways of reactive oxygen species (ROS) in microalgae. Based on Cirulis et al. (2013) and Laloi et al.
(2006).
FIGURE 2
Generation of different ROS by energy transfer (production of singlet oxygen) or sequential univalent reduction of ground-state triplet oxygen. Based on Apel and Hirt (2004).
272
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
triplet chlorophyll from the photosystem II reaction center in the chloroplasts may transfer its excitation energy onto triplet ground-state oxygen, yielding the highly reactive singlet oxygen. A second ROS-generating mechanism originates in the electron transfer system. When the lightdriven electron transport exceeds consumption of electrons needed for CO2 fixation or NADP supply is limited, molecular oxygen can be reduced by photosystem I to superoxide, which is rapidly converted to hydrogen peroxide by superoxide dismutase. Furthermore, in the peroxisomes, recycling of glycolate from photorespiration and fatty acid oxidation are both processes that produce hydrogen peroxide, which in its turn is mitigated by catalase. A third endogenous ROS source is the generation of superoxide by ubiquinone in the electron transport chain during oxidative phosphorylation in the mitochondria. However, mitochondrial ROS production is much lower than the production in chloroplasts. A last source of superoxide is the activity of NADPH oxidase in the plasma membrane.
2.2 ROS Detoxification in Microalgae To counteract the detrimental effects of ROS, all living organisms have several defensive systems at their disposal, both enzymatic and nonenzymatic. In this section, the most important ROS-associated enzymes and antioxidants found in microalgae are discussed. 2.2.1 Enzymatic Antioxidant Protection Superoxide dismutase (EC 1.15.1.1) consists of a mixture of metalloproteins differentiated by their metal cofactor. The three isoforms common to plants (CuZn-SOD, Fe-SOD, and Mn-SOD) are also present in microalgae (Janknegt et al., 2009). Superoxide dismutase catalyzes the neutralization of superoxide radicals with the formation of hydrogen peroxide and oxygen. Catalase (EC 1.11.1.6) catalyzes the conversion of hydrogen peroxide to water and oxygen. The catalase enzyme is sensitive to light, which
may affect the ability of photoautotrophic organisms to tolerate oxidative stress when exposed to high light irradiance. Another group of enzymes that catalyze reduction of hydrogen peroxide to water are peroxidases. They differ from catalase in their requirement of an electron donor that subsequently becomes oxidized. Ascorbate peroxidase (EC 1.11.1.11), present in the stroma and thylakoids of chloroplasts, has a significantly lower Km value for hydrogen peroxide than catalase and uses vitamin C as a specific electron donor (Asada and Akahashi, 1987). Glutathione peroxidase (EC 1.11.1.9) requires glutathione for the removal of hydrogen peroxide. Several isoforms have been detected in the microalgae Chlamydomonas reinhardtii (Dayer et al., 2008) and Chlorella sp. (Wang and Xu, 2012). 2.2.2 Nonenzymatic Antioxidant Protection L-Ascorbic acid or vitamin C, abundant in photosynthetic organisms, can reduce many ROS. Vitamin C is present in both cytosol and chloroplast where it takes part in the ascorbatee glutathione cycle to remove hydrogen peroxide (Mallick and Mohn, 2000). Next to its vital role in the elimination of hydrogen peroxide, vitamin C also scavenges superoxide, hydroxyl radicals, and lipid hydroperoxides (Lesser, 2006). In the chloroplast, vitamin C plays a crucial role in the regeneration of membrane-bound carotenoids and tocopherols, thereby protecting the photosynthetic apparatus (Mallick and Mohn, 2000). High levels of vitamin C have been reported in Chlorella sp. (Running et al., 2002), Dunaliella sp. (Barbosa et al., 2005), Chaetoceros calcitrans, and Skeletonema costatum (Brown et al., 1998, 1999). Production of vitamin C is stimulated by high light exposure (Barbosa et al., 2005), allelochemicals such as ethyl 2-methyl acetoacetate (Yang et al., 2011), or ultraviolet stress (Abd El-baky et al., 2004). Glutathione (GSH), a tripeptide (Glu-Cys-Gly) found in animals and photosynthetic organisms, acts as an antioxidant in many ways including
2. ANTIOXIDANT ACTIVITY IN MICROALGAE
reaction with superoxide, singlet oxygen, and hydroxyl radicals. Glutathione also acts as a chainbreaker of free-radical reactions and is crucial in the regeneration of ascorbate (Lesser, 2006). As a substrate for glutathione peroxidase, it donates the electrons necessary for decomposition of hydrogen peroxide (Kohen and Nyska, 2002). Tocopherols are located in the lipid bilayers of cell membranes. The most widespread homologs in nature are four tocopherols and four tocotrienols: a, b, g, and d -tocopherol as well as a, b, g, and d -tocotrienol (Colombo, 2010). Tocopherols and tocotrienols have the same basic chemical structure characterized by a long chain attached at the 2-position of a chromane ring. However, tocotrienols contain three conjugated double bonds in the aliphatic side chain instead of the saturated C16 side chain found in tocopherols (Figure 3). The most active antioxidant form is a-tocopherol, which is only synthesized in the chloroplasts of photosynthetic organisms. a-Tocopherol acts as an antioxidant through its ability to quench both singlet oxygen and (lipid) peroxides (Lesser, 2006; Mallick and Mohn, 2000). Although a-tocopherol is located in the membranes and the hydrophilic vitamin C is located in the liquid
FIGURE 3
273
phase, vitamin C is able to reduce the tocopheroxyl radical, thereby recycling the active form of tocopherol in the chloroplast (Buettner, 1993; Niki, 1991). a-Tocopherol is produced in high amounts by Dunaliella tertiolecta and Tetraselmis suecica. Carballo-Cardenas et al. (2003) demonstrated that production of a-tocopherol is highly variable throughout the growth cycle and that nutrient composition can be used to control its production in both species. In Dunaliella salina, a-tocopherol production is stimulated by UV-stress, nitrogen limitation, and salt stress (Abd El-baky et al., 2004). Another study showed that decreasing N-concentrations in the growth medium leads to increased a-tocopherol accumulation in Nannochloropsis oculata, but growth rate is reduced under these conditions (Durmaz, 2007). The lipophilic carotenoids are produced de novo by photoautotrophs. In photosynthetic organisms, carotenoids are present in the pigmenteprotein complexes of the thylakoid membranes of chloroplasts, where they fulfill a dual function (Cogdell et al., 1994). Some carotenoids (especially ketocarotenoids such as fucoxanthin, Figure 4) act as accessory light-harvesting pigments by
Chemical structures of tocopherols and tocotrienols.
274
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
transferring light energy of wavelengths that cannot be captured by chlorophylls (Takaichi, 2011). Next, carotenoids also have a protective function by dissipating excess energy and by quenching ROS that are produced during photosynthesis (Goss and Jakob, 2010). The xanthophyll cycle, that is, the cyclical interconversion of violaxanthin, antheraxanthin, and zeaxanthin in chlorophytes, provides zeaxanthin needed for dissipation of excess energy from excited chlorophylls in photosynthetic organisms. In diatoms and dinoflagellates, an alternative xanthophyll cycle exists where diadinoxanthin is converted into diatoxanthin (diatoms) or dinoxanthin (dinoflagellates). Another mode of photoprotection by carotenoids is the quenching of excited triplet-state chlorophyll and singlet oxygen by b-carotene. The main mechanism in carotenoid photoprotection against singlet oxygen functions through electronic energy transfer as given by reaction (15) (Edge et al., 1997); however,
FIGURE 4
chemical quenching with formation of carotenoid epoxides also occurs (Liebler, 1993). 1
O2 þ CAR/3 O2 þ 3 CAR
(15)
During physical quenching (reaction (15)), the carotenoid triplet state is produced through energy transfer. This excited state can return to the ground state by dissipating energy as heat or by translocation over the conjugated double bond system. Therefore, the ability to quench singlet oxygen increases with longer chain lengths of the conjugated system (Edge et al., 1997). Next to the ability of carotenoids to quench singlet oxygen, they can also react with free radicals. Carotenoids can react with peroxyl radicals and are involved in recycling of phenoxyl radicals and tocopheroxyl radicals, which are formed upon reaction of phenolic antioxidants and tocopherols with peroxyl and alkoxyl radicals (Burke et al., 2001; Burton, 1989; Edge et al., 1997). However, unlike quenching of singlet oxygen, which mainly leads to
Chemical structures of some carotenoids occurring in microalgae.
2. ANTIOXIDANT ACTIVITY IN MICROALGAE
energy dissipation as heat, the reactions of carotenoids (or any antioxidant) with free radicals will lead to electron transfer or addition reactions. Three reaction mechanisms describe the reaction of free radicals with carotenoids, that is, electron transfer, hydrogen atom transfer, and radical addition to the carotenoid (Martínez and Barbosa, 2008; Martínez et al., 2008). In order to scavenge free radicals, carotenoids can either donate or accept unpaired electrons. Usually, antioxidant molecules become oxidized by donating electrons to the free radical. However, carotenoids can also quench free radicals by accepting an unpaired electron, rendering it harmless by translocation over the conjugated side chain. In a comparative study, it was observed that the apolar lycopene as well as the xanthophylls were the most effective carotenoids in reducing ferric ions (FRAP assay) (M€ uller et al., 2011). In the same study, it was further demonstrated that the group of carotenes (lycopene, a- and b-carotene) were more efficient quenchers of the ABTS þ radical than most of the xanthophylls and that keto-carotenoids were most efficient in scavenging peroxyl radicals, due to their more pronounced conjugated double bond systems. In microalgae, a distinction is usually made between primary and secondary carotenoids. Whereas primary carotenoids are structural and functional components of the photosynthetic apparatus, and thus essential for survival, secondary carotenoids are produced at high levels when cells are exposed to specific environmental stimuli (Jin et al., 2003). At present, carotenoids (both primary and secondary) are the most commercialized products from microalgae. One such carotenoid that can be sourced from microalgae is lutein. Lutein is also found in the human retina where it acts both as an optical filter and as an antioxidant to protect long-chain polyunsaturated fatty acids and is therefore important for our eye’s health (Rapp et al., 2000). In Scenedesmus, lutein production, as well as b-carotene production, can be stimulated
275
by increasing pH and temperature during cultivation (Guedes et al., 2011b). Further, Wei et al. (2008) showed that lutein content of heterotrophically grown Chlorella protothecoides increased in response to singlet oxygen, but was reduced when cells were exposed to hydroxyl radicals. Growth-limiting conditions, such as pH values of six or nine and a temperature of 33 C, were found to stimulate carotenogenesis in the chlorophyte Muriellopsis sp. (Del Campo et al., 2000), which is currently the commercial source of lutein. Lutein content in this species is the highest in early stationary phase. Another microalgal pigment, sold as antioxidant, is the secondary carotenoid astaxanthin, produced by Haematococcus pluvialis. Accumulation of astaxanthin occurs when H. pluvialis cells are exposed to stress, induced by a combination of high light, high salt levels and nitrogen deprivation (Boussiba, 2000; Wang et al., 2003). The last microalgal pigment that is currently produced commercially for its antioxidant properties is b-carotene, extracted from the halophile D. salina. Carotenogenesis in this microalga is induced by Fe2þ, as well as by UV-stress. Also in Chlorella vulgaris, an increase in carotenoid content is observed upon metal exposure (Mallick, 2004). A good overview of the optimum conditions for carotenoid production is given by Guedes et al. (2011a). A last group of antioxidant secondary metabolites are polyphenols, which are present, often at high levels, in virtually all plants (Pietta, 2000). Polyphenols comprise a structurally diverse group of components, including simple phenols, phenolic acids, flavonoids, tannins, and lignans. Polyphenols can inhibit lipid oxidation in different ways, that is, by directly scavenging HOCl, singlet oxygen, lipid peroxyl, superoxide and hydroxyl radicals, by metal chelation or by inhibiting lipoxygenase (Dugas et al., 2000; Rice-Evans et al., 1996; Salah et al., 1995). The general mechanism of radical scavenging by phenolics is given in Figure 5. In the first step, a hydrogen atom is donated to the radical, and an aroxyl radical is produced. This radical is
276
FIGURE 5
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
General radical scavenging scheme of phenolics. R represents superoxide anion, peroxyl, alkoxyl, or hydroxyl
radicals.
relatively stable through resonance and can interact with another radical yielding a quinone. This means that, in the general mechanism, one polyphenol molecule is able to quench two radical molecules. However, if high metal concentrations are present, the aroxyl radical can interact with oxygen, generating a quinone and a superoxide anion, rather than terminating the radical chain reactions. This mechanism is responsible for the undesired pro-oxidant effect of phenolics that can occur under specific conditions (Pietta, 2000). Next to their direct radical scavenging properties, polyphenols are able to chelate metals, hereby reducing the oxidative stress as transition metals are involved in ROS generation (Pietta, 2000). Although phenolics are well-studied antioxidant components in higher plants, the acknowledgment of their presence in microalgae is fairly recent. Li et al. (2007) and Hajimahmoodi et al. (2010) screened microalgae for polyphenol content and antioxidant activity and found large variations between samples. More recently, Goiris et al. (2012) indicated that the antioxidant potential of microalgae is not only determined by its carotenoid content but also other components, including phenolics, are important contributors to overall antioxidant activity. Next to the presence of simple phenols in microalgae (Klejdus et al., 2009; Onofrejov a et al., 2010), the presence of flavonoids in microalgae has been acknowledged, albeit at low levels (Goiris et al., 2014; Klejdus et al., 2010). Microalgae can further produce some remarkable polyphenolic antioxidant molecules such as marennine in the diatom Haslea ostrearia (Pouvreau et al., 2008), purpurogallin in the extremophile snow algae
Mesotaenium berggrenii (Remias et al., 2012) or even BHT, the well-known food additive (E321), which was found in the chlorophyte Botryococcus braunii and the cyanobacteria Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Oscillatoria sp. (Babu and Wu, 2008). In microalgae, little is known about the response of phenolic components to environmentally induced oxidative stress. Duval et al. (2000) examined the effects of UV-exposure on antioxidant properties of Chlamydomonas nivalis and observed an increase in phenolics upon exposure to UV-C light. Another study on potential effects of UV on phenolic content was performed with Scenedesmus quadricauda (Kovacik et al., 2010). This study found no significant changes in total phenolic content when cells were exposed to elevated levels of UV-A but noticed a 50% decrease in the flavonols quercetin and kaempherol. When the cells were exposed to UV-C, these flavonols were not found in the biomass, suggesting breakdown of these components by UV-C. On the other hand, benzoic acids increased upon UV-A exposure and cinnamic acid decreased when cells were exposed to UV-A or UV-C. Others found that production of BHT (Babu and Wu, 2008) was stimulated under high light irradiation in B. braunii, C. raciborskii, M. aeruginosa, and Oscillatoria sp. Other studies described the influence of metal stress on the phenolic content in microalgae. Ulloa et al. (2012) found that phenolic content was stimulated by strontium addition to cultures of T. suecica. Also in S. quadricauda (Kovacik et al., 2010) and Phaeodactylum tricornutum (Rico et al., 2013), phenolic content was higher when cells were exposed to metals. A better understanding on how polyphenol concentrations change in
3. POTENTIAL OF MICROALGAL ANTIOXIDANTS TO REDUCE LIPID OXIDATION IN FOODSTUFF
response to oxidative stress will clarify the role of polyphenols as antioxidants in microalgae and learn how to maximize production for use in the food, feed, or chemical industry.
2.3 Other Antioxidants Next to the antioxidant components commonly found in other plants, some microalgae produce specific types of antioxidants such as the phycobilin proteins phycocyanin in cyanobacteria (Benedetti et al., 2004; Huang et al., 2007; Thangam et al., 2013; Yoshikawa and Belay, 2008) and phycoerythrin in rhodophytes and cyanobacteria (Huang et al., 2007). Especially the cyanobacteria Arthrospira platensis is known to produce high amounts of phycocyanin (Oliveira et al., 2009). Also dimethylsulfide/ dimethylsulfoxide (Sunda et al., 2002) and sulfated polysaccharides (Tannin-Spitz et al., 2005) contribute to the antioxidant pool of microalgae.
3. POTENTIAL OF MICROALGAL ANTIOXIDANTS TO REDUCE LIPID OXIDATION IN FOODSTUFF 3.1 Current Knowledge and Applications Over the last decade, increasing evidence has been gathered on the potential of microalgal extracts for retarding lipid oxidation. For instance, Ranga Rao et al. (2006) studied the effect of crude acetone extracts of B. braunii on lipid peroxidation in model systems. The relatively high degree of inhibition of lipid peroxidation, in comparison with the synthetic antioxidant BHT, was ascribed to carotenoids and polyphenols in the extracts. Tannin-Spitz et al. (2005) studied the effect of sulfated polysaccharides from Porphyridium cruentum on lipid oxidation and found inhibition rates of up to 80% at a concentration of 10 mg mL1 Benedetti et al. (2004) reported the use of a phycocyanin-rich extract from the cyanophyte
277
Aphanizomenon flos-aquae to reduce cupperinduced oxidation of plasma lipids and found that at a concentration of 1 mM phycocyanin, oxidation of blood lipids was reduced by a factor three. Lee et al. (2010) assessed the antioxidant properties of the microalgae Halochlorococcum porphyrae and Oltamannsiellopsis unicellularis using both solvent extracts and enzymatic digests. Lipid peroxidation was strongly inhibited by all methanolic extracts, as well as the ethyl acetate fraction of H. porphyrae and the chloroform fraction of O. unicellularis which inhibited lipid peroxidation similar to a-tocopherol. In addition, some enzymatic digests exhibited effects similar to the synthetic antioxidant BHT. Several studies demonstrated the efficacy of ethanolic extracts of Chlorella sp. on lipid peroxidation and measured similar degrees of inhibition compared to BHT (Choochote et al., 2014; Rodriguez-Garcia and Guil-Guerrero, 2008). Another study by Natrah et al. (2007) screened 14 samples of Malaysian indigenous microalgae and identified six species, that is, S. quadricauda, C. vulgaris, N. oculata, Tetraselmis tetrathele and especially Isochrysis galbana and C. calcitrans, of which crude methanolic extracts inhibited the oxidation of linoleic acid to the same extent as the commercial antioxidants BHA and BHT. Recently, the use of whole biomass of C. vulgaris and H. pluvialis has been shown to retard lipid oxidation in bulk oils (Lee et al., 2013) as well as in food emulsions (Gouveia et al., 2005). Although all studies mentioned above describe the activity against lipid oxidation in view of radical scavenging activity of the extracts, earlier work by Matsukawa et al. (1997) looked at the potential of microalgal extracts for inhibition of two important oxidative enzymes that are involved in oxidation of lipids and proteins, that is, lipoxygenase and tyrosinase, respectively. In this study, it was indicated that ethanol extracts contained efficient inhibitors of the lipoxygenase activity, whereas methanol extracts showed the highest tyrosinase inhibition, compared to ethanol and aqueous extracts.
278
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
3.2 Future Perspectives Although the efficacy of microalgal antioxidants toward lipid oxidation has been proven, little data are available on how these novel antioxidant formulations perform compared to commercially available antioxidant products, both from a cost-perspective and from the antioxidant action in real food systems. Further, the active principles still need to be further characterized to allow standardization and commercialization. Thirdly, growth conditions should be optimized for maximal productivity of antioxidant components. Finally, a big hurdle that has to be taken before incorporating microalgal products in foodstuffs are the legislative constrictions concerning novel foods. Since only a few species are currently allowed for human consumption, many microalgae with high antioxidant potential still await approval before they can be used in foodstuff.
References Abd El-baky, H.H., El-Baz, F.K., El-baroty, G.S., 2004. Production of antioxidant by the green alga Dunaliella salina. Int. J. Agri. Biol. 6 (1), 49e57. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Ann. Rev. Plant Biol. 55, 373e399. Asada, K., Akahashi, M., 1987. Production and scavenging of active oxygen in photosynthesis. In: Kyle, D.J., Osmond, C.B., Arntzen, C.J. (Eds.), Photoinhibition. Elsevier, Amsterdam, The Netherlands, pp. 228e287. Babu, B., Wu, J.-T., 2008. Production of natural butylated hydroxytoluene as an antioxidant by freshwater phytoplankton 1. J. Phycol. 44 (6), 1447e1454. Barbosa, M.J., Zijffers, J.W., Nisworo, A., Vaes, W., van Schoonhoven, J., Wijffels, R.H., 2005. Optimization of biomass, vitamins, and carotenoid yield on light energy in a flat-panel reactor using the A-stat technique. Biotech. Bioeng. 89 (2), 233e242. Benedetti, S., Benvenuti, F., Pagliarani, S., Francogli, S., Scoglio, S., Canestrari, F., 2004. Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenon flos-aquae. Life Sci. 75 (19), 2353e2362. Boussiba, S., 2000. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Phys. Plant. 108, 111e117.
Brown, M.R., Mular, M., Miller, I., Farmer, C., Trenerry, C., 1999. The vitamin content of microalgae used in aquaculture. J. App. Phycol. 11, 247e255. Brown, M.R., Skabo, S., Wilkinson, B., 1998. The enrichment and retention of ascorbic acid in rotifers fed microalgal diets. Aquacult. Nutr. 4, 151e156. Buettner, G.R., 1993. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch. Biochem. Biophys. 300, 535e543. Burke, M., Edge, R., Land, E.J., Mcgarvey, D.J., Truscott, T.G., 2001. One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments. FEBS Lett. 500, 132e136. Burton, G.W., 1989. Antioxidant action of carotenoids. J. Nutr. 119, 109e111. Carballo-Cardenas, E.C., Minh Tuan, P., Janssen, M., Wijffels, R.H., 2003. Vitamin E (a-tocopherol) production by the marine microalgae Dunaliella tertiolecta and Tetraselmis suecica in batch cultivation. Biomol. Eng. 20 (4e6), 139e147. Choochote, W., Suklampoo, L., Ochaikul, D., 2014. Evaluation of antioxidant capacities of green microalgae. J. Appl. Phycol. 26 (1), 43e48. Cirulis, J.T., Scott, J.A., Ross, G.M., 2013. Management of oxidative stress by microalgae. Can. J. Physiol. Pharmacol. 91 (1), 15e21. Cogdell, R.J., Gillbro, T., Andersson, P.O., Liu, R.S.H., Asato, A.E., 1994. Carotenoids as accessory light-harvesting pigments. Pure Appl. Chem. 66 (5), 1041e1046. Colombo, M.L., 2010. An update on vitamin E, tocopherol and tocotrienol-perspectives. Molecules 15 (4), 2103e2113. Dayer, R., Fischer, B.B., Eggen, R.I.L., Lemaire, S.D., 2008. The peroxiredoxin and glutathione peroxidase families in Chlamydomonas reinhardtii. Genetics 179 (1), 41e57. Del Campo, J.a, Moreno, J., Rodríguez, H., Vargas, M.a, Rivas, J., Guerrero, M.G., 2000. Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta). J. Biotech. 76 (1), 51e59. Dugas, A.J., Casta~ neda-Acosta, J., Bonin, G.C., Price, K.L., Fischer, N.H., Winston, G.W., 2000. Evaluation of the total peroxyl radical-scavenging capacity of flavonoids: structure-activity relationships. J. Nat. Prod. 63 (3), 327e331. Durmaz, Y., 2007. Vitamin E (a-tocopherol) production by the marine microalgae Nannochloropsis oculata (Eustigmatophyceae) in nitrogen limitation. Aquaculture 272 (1e4), 717e722. Duval, B., Shetty, K., Thomas, W.H., 2000. Phenolic compounds and antioxidant properties in the snow alga Chlamydomonas nivalis after exposure to UV light. J. Appl. Phycol. 11, 559e566. Edge, R., McGarvey, D.J., Truscott, T.G., 1997. The carotenoids as anti-oxidantsda review. J. Photochem. Photobiol. B 41 (3), 189e200.
REFERENCES
Goiris, K., Muylaert, K., Fraeye, I., Foubert, I., De Brabanter, J., De Cooman, L., 2012. Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. J. App. Phycol. 24 (6), 1477e1486. Goiris, K., Muylaert, K., Voorspoels, S., Noten, B., De Paepe, D., Baart, G.J.E., E Baart, G.J., 2014. Detection of flavonoids in microalgae from different evolutionary lineages. J. Phycol. 50 (3), 483e492. Goss, R., Jakob, T., 2010. Regulation and function of xanthophyll cycle-dependent photoprotection in algae. Photosynth. Res. 106 (1e2), 103e122. Gouveia, L., Raymundo, A., Batista, A.P., Sousa, I., Empis, J., 2005. Chlorella vulgaris and Haematococcus pluvialis biomass as colouring and antioxidant in food emulsions. Eur. Food Res. Technol. 222 (3e4), 362e367. Guedes, A.C., Amaro, H.M., Malcata, F.X., 2011a. Microalgae as sources of carotenoids. Mar. Drugs 9 (4), 625e644. Guedes, A.C., Amaro, H.M., Pereira, R.D., Malcata, F.X., 2011b. Effects of temperature and pH on growth and antioxidant content of the microalga Scenedesmus obliquus. Biotech. Progr. 27 (5), 1218e1224. Hajimahmoodi, M., Faramarzi, M.A., Mohammadi, N., Soltani, N., Oveisi, M.R., Nafissi-Varcheh, N., 2010. Evaluation of antioxidant properties and total phenolic contents of some strains of microalgae. J. Appl. Phycol. 22 (1), 43e50. Huang, Z., Guo, B.J., Wong, R.N.S., Jiang, Y., 2007. Characterization and antioxidant activity of selenium-containing phycocyanin isolated from Spirulina platensis. Food Chem. 100 (3), 1137e1143. Janknegt, P., De Graaff, C.M., Van De Poll, W., Visser, R., Rijstenbil, J., Buma, A., 2009. Short-term antioxidative responses of 15 microalgae exposed to excessive irradiance including ultraviolet radiation. Eur. J. Phycol. 44 (4), 525e539. Jin, E., Polle, J.E.W., Lee, H.K.U.M., Hyun, S.M.I.N., Chang, M.A.N., 2003. Xanthophylls in microalgae: from biosynthesis to biotechnological mass production and application. J. Microbiol. Biotech. 13, 165e174. Klejdus, B., Kopecký, J., Benesova, L., Vacek, J., 2009. Solidphase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species. J. Chrom. A 1216 (5), 763e771. Klejdus, B., Lojkov a, L., Plaza, M., Sn oblova, M., Sterbova, D., 2010. Hyphenated technique for the extraction and determination of isoflavones in algae: ultrasound-assisted supercritical fluid extraction followed by fast chromatography with tandem mass spectrometry. J. Chrom. A 1217 (51), 7956e7965. Kohen, R., Nyska, A., 2002. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicol. Pathol. 30 (6), 620e650.
279
Kovacik, J., Klejdus, B., Backor, M., 2010. Physiological responses of Scenedesmus quadricauda (Chlorophyceae) to UV-A and UV-C light. Photochem. Photobiol. 86, 612e616. Kovacik, J., Klejdus, B., Hedbavny, J., Backor, M., 2010. Effect of copper and salicylic acid on phenolic metabolites and free amino acids in Scenedesmus quadricauda (Chlorophyceae). Plant Sci. 178 (3), 307e311. Laloi, C., Przybyla, D., Apel, K., 2006. A genetic approach towards elucidating the biological activity of different reactive oxygen species in Arabidopsis thaliana. J. Exp. Bot. 57 (8), 1719e1724. Lee, S.-H., Lee, J.-B., Lee, K.-W., Jeon, Y.-J., 2010. Antioxidant properties of tidal pool microalgae, Halochlorococcum porphyrae and Oltamannsiellopsis unicellularis from Jeju Island, Korea. Algae 25 (1), 45e56. Lee, Y.-L., Chuang, Y.-C., Su, H.-M., Wu, F.-S., 2013. Freezedried microalgae of Nannochloropsis oculata improve soybean oil’s oxidative stability. Appl. Microbiol. Biotech. 97 (22), 9675e9683. Lesser, M.P., 2006. Oxidative stress in marine environments: biochemistry and physiological ecology. Ann. Rev. Physiol. 68 (3), 253e278. Li, H., Cheng, K., Wong, C., Fan, K., Chen, F., Jiang, Y., 2007. Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. Food Chem. 102 (3), 771e776. Liebler, D.C., 1993. Antioxidant reactions of carotenoids. Ann. N. Y. Acad. Sci. 691 (1), 20e31. Mallick, N., 2004. Copper-induced oxidative stress in the chlorophycean microalga Chlorella vulgaris: response of the antioxidant system. J. Plant Physiol. 161 (5), 591e597. Mallick, N., Mohn, F.H., 2000. Reactive oxygen species: response of algal cells. J. Plant Physiol. 157, 183e193. Martínez, A., Barbosa, A., 2008. Antiradical power of carotenoids and vitamin E: testing the hydrogen atom transfer mechanism. J. Phys. Chem. B 112 (51), 16945e16951. Martínez, A., Rodríguez-Girones, M.a, Barbosa, A., Costas, M., 2008. Donator acceptor map for carotenoids, melatonin and vitamins. J. Phys. Chem. A 112 (38), 9037e9042. Matsukawa, R., Dubinsky, Z., Masaki, K., Takeuchi, T., Karube, I., 1997. Enzymatic screening of microalgae as a potential source of natural antioxidants. Appl. Biochem. Biotech. 66 (3), 239e247. M€ uller, L., Fr€ ohlich, K., B€ ohm, V., 2011. Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (aTEAC), DPPH assay and peroxyl radical scavenging assay. Food Chem. 129 (1), 139e148. Namiki, M., 1990. Antioxidants/antimutagens in food. Crit. Rev. Food Sci. Nutr. 29 (4), 273e300. Natrah, F.M.I., Yusoff, F.M., Shariff, M., Abas, F., Mariana, N.S., 2007. Screening of Malaysian indigenous microalgae for antioxidant properties and nutritional value. J. App. Phycol. 19 (6), 711e718.
280
17. MICROALGAE AS A NOVEL SOURCE OF ANTIOXIDANTS FOR NUTRITIONAL APPLICATIONS
Niki, E., 1991. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am. J. Clin. Nutr. 54, 1119Se1124S. Oliveira, E.G., Rosa, G.S., Moraes, M.A., Pinto, L.A.A., 2009. Characterization of thin layer drying of Spirulina platensis utilizing perpendicular air flow. Bioresour. Technol. 100 (3), 1297e1303. Onofrejov a, L., Vasíckova, J., Klejdus, B., Stratil, P., Misurcov a, L., Kracmar, S., Vacek, J., 2010. Bioactive phenols in algae: the application of pressurized-liquid and solid-phase extraction techniques. J. Pharm. Biomed. Anal. 51 (2), 464e470. Pietta, P.G., 2000. Flavonoids as antioxidants. J. Nat. Prod. 63 (7), 1035e1042. Pouvreau, J.-B., Morançais, M., Taran, F., Rosa, P., Dufosse, L., Guerard, F., Pondaven, P., 2008. Antioxidant and free radical scavenging properties of marennine, a blue-green polyphenolic pigment from the diatom Haslea ostrearia (Gaillon/ Bory) Simonsen responsible for the natural greening of cultured oysters. J. Agric. Food Chem. 56 (15), 6278e6286. Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53 (10), 4290e4302. Ranga Rao, A., Sarada, R., Baskaran, V., Ravishankar, G.A., 2006. Antioxidant activity of Botryococcus braunii extract elucidated in vitro models. J. Agric. Food Chem. 54 (13), 4593e4599. Rapp, L.M., Maple, S.S., Choi, J.H., 2000. Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina. Invest. Ophthalmol. Vis. Sci. 41 (5), 1200e1209. Remias, D., Schwaiger, S., Aigner, S., Leya, T., Stuppner, H., L€ utz, C., 2012. Characterization of an UV- and VISabsorbing, purpurogallin-derived secondary pigment new to algae and highly abundant in Mesotaenium berggrenii (Zygnematophyceae, Chlorophyta), an extremophyte living on glaciers. FEMS Microbiol. Ecol. 79 (3), 638e648. Rice-Evans, C., Miller, N.J., Paganga, G., 1996. Structureactivity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20 (7), 933e956. Rico, M., L opez, A., Santana-Casiano, J.M., Gonzalez, A.G., Gonz alez-D avila, M., 2013. Variability of the phenolic profile in Phaeodactylum tricornutum diatom growing under copper and iron stress. Limnol. Oceanogr. 58 (1), 144e152. Rodriguez-Garcia, I., Guil-Guerrero, J.L., 2008. Evaluation of the antioxidant activity of three microalgal species for use as dietary supplements and in the preservation of foods. Food Chem. 108 (3), 1023e1026. Running, J.A., Severson, D.K., Schneider, K.J., 2002. Extracellular production of L-ascorbic acid by Chlorella protothecoides, Prototheca species, and mutants of P. moriformis during aerobic culturing at low pH. J. Ind. Microbiol. Biotechnol. 29 (2), 93e98.
Salah, N., Miller, N.J., Paganga, G., Tijburg, L., Bolwell, G.P., Rice-Evans, C., 1995. Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch. Biochem. Biophys. 322 (2), 339e346. Sunda, W., Kieber, D.J., Kiene, R.P., Huntsman, S., 2002. An antioxidant function for DMSP and DMS in marine algae. Nature 418, 317e320. Takaichi, S., 2011. Carotenoids in algae: distributions, biosyntheses and functions. Mar. Drugs 9 (6), 1101e1118. Tannin-Spitz, T., Bergman, M., Van-Moppes, D., Grossman, S., Arad, S.(M.), 2005. Antioxidant activity of the polysaccharide of the red microalga Porphyridium sp. J. Appl. Phycol. 17 (3), 215e222. Thangam, R., Suresh, V., Princy, W.a., Rajkumar, M., SenthilKumar, N., Gunasekaran, P., Kannan, S., 2013. C-Phycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest. Food Chem. 140 (1e2), 262e272. ~ez, M.J., Ulloa, G., Otero, A., Sanchez, M., Sineiro, J., N un Fabregas, J., 2012. Effect of Mg, Si, and Sr on growth and antioxidant activity of the marine microalga Tetraselmis suecica. J. App. Phycol. 24 (5), 1229e1236. Wang, B., Zarka, A., Trebst, A., Boussiba, S., 2003. Astaxanthin accumulation in Haematococcus pluvialis (chlorophyceae) as an active photoprotective process under high irradiance. J. Phycol. 39, 1116e1124. Wang, X., Xu, X., 2012. Molecular cloning and functional analyses of glutathione peroxidase homologous genes from Chlorella sp. NJ-18. Gene 501 (1), 17e23. Wei, D., Chen, F., Chen, G., Zhang, X., Liu, L., Zhang, H., 2008. Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress. Sci. China, Ser. C: Life Sci. 51 (12), 1088e1093. Wright, J.S., Johnson, E.R., DiLabio, G.A., 2001. Predicting the activity of phenolic antioxidants: theoretical method, analysis of substituent effects, and application to major families of antioxidants. J. Am. Chem. Soc. 123 (6), 1173e1183. Yang, C.-Y., Liu, S.-J., Zhou, S.-W., Wu, H.-F., Yu, J.-B., Xia, C.-H., 2011. Allelochemical ethyl 2-methyl acetoacetate (EMA) induces oxidative damage and antioxidant responses in Phaeodactylum tricornutum. Pestic. Biochem. Physiol. 100 (1), 93e103. Yoshikawa, N., Belay, A., 2008. Single-laboratory validation of a method for the determination of c-phycocyanin and allophycocyanin in Spirulina (Arthrospira) supplements and raw materials by spectrophotometry. J. AOAC Int. 91 (3), 524e529.