Microalgae source of natural bioactive m

Microalgae – source of natural bioactive molecules as functional ingredients Luisa Gouveia1, Ana Evangelista Marques1, Joa˜o M Sousa1, Patrı´cia Moura2 and Narcisa M Bandarra2 Laborato´rio Nacional de Energia e Geologia, Unidade de Bioenergia, Estrada Pac¸o Lumiar, 1649-038 Lisboa, Portugal Tel ỵ351217127210 Fax: ỵ351217127195 E-mail luisa.gouveia@ineti.pt IPIMAR – INRB, Departamento de Inovac¸a˜o Tecnolo´gica e Valorizac¸a˜o dos Produtos da Pesca, Av Brasılia, 1449-005 Lisboa, Portugal Abstract Microalgae can provide an untapped number of important bioactive molecules (functional ingredients), and their incorporation in traditional foods (e.g breakfast cereals, spreads, breads, cookies, brownies, energy bars, mayonnaises, gelled desserts, pastas, emulsions, ice creams, and beverages), largely consumed on a daily basis, improves an individual’s state of wellbeing, reduces the risk of disease, and lowers health care costs Keywords: microalgae, functional ingredients, bioactive molecules Introduction Microalgae and other microaquatic plants are fascinating life forms They have evolved over time to not only survive but also to actually thrive in the earth’s harshest environments They this by naturally producing a remarkable array of protective and nourishing compounds – fatty acids, unique carotenoids, flavonoids, algal plant sterols, vitamins, phospholipids, non-digestible oligosacchară tles and Pire 2001) ides and nutrient-rich oils (O Microalgae appear to be more photosynthetically efficient than terrestrial plants (Pirt 1986), with higher biomass productivities, faster growth rates, and the highest CO2 fixation and O2 production rates when compared with higher plants Microalgae require a liquid medium for growth, which is easily achievable, and they can be cultivated in variable climates and on non-arable land, including marginal areas unsuitable for agricultural purposes (e.g desert and seashore environments) and in non-potable water or even as a waste treatment tool They use far less water than traditional crops and not displace food crop cultures, thereby reducing environmental impacts such as soil desertification and deforestation Their production is Food Science and Technology Bulletin: Functional Foods (2) 21–37 DOI: 10.1616/1476-2137.15884 Accepted March 2010 ISSN 1476-2137 # IFIS Publishing 2010 All Rights Reserved not seasonal, they can be harvested daily (Chisti 2007, 2008; Rodolfi et al 2009), they not need pesticides or herbicides, and they not produce contaminants Biomass production systems can be easily adapted to various levels of operational and technological skills, and microalgal cultures can be induced to produce high concentrations of bioactive compounds These systems can therefore potentially act as efficient producers of an untapped source of beneficial compounds We are entering into an extraordinary era for functional ingredients and for the functional foods industry Knowledge arising from epidemiological studies and often substantiated by preclinical and clinical studies demonstrates the relationship between diet and health Therefore, there are now widespread requirements with regards to functional foods varying from producers to consumers, regulatory agencies and health professionals The increased interest in functional foods, functional ingredients, nutraceuticals (Figure 1) and other natural health products has been recognised to promote good health, improve the state of wellbeing, decrease risk of disease and lower health care costs (Shahidi 2008) by maintaining a good quality of life As microalgae are a potential food source, they could possibly contribute to avoid malnuă tles and Pire 2001; Mazo trition in Third World countries (O et al 2004) Some functional foods markets are more active, with soft drinks, dairy, confectionery, bakery 22 Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al Figure Nutraceuticals (Wildman 2007) products, spreads and breakfast cereals accounting for the majority of all new products (Riaz 1999; Sloan 2000, 2001) It is clear that the organic and functional food markets are some of the strongest and most sustainable healthdriven markets in the world (Sloan 2002) Algae have been used as natural foods for a very long time, and today there is a significant amount of microalgaebased pills and tablets used as nutritional supplements Out of the thousands of microalgae species that are believed to exist (Chaumont 1993; Radmer and Parker 1994), a few thousand strains are kept in collections, while a few hundred have been investigated for their physicochemical content; only a handful have been exploited by the food and nutrition industry for human consumption and use as animal feed additives (Olaizola 2003) Of these few algae, Chlorella vulgaris and Spirulina pacifica are the main algae used in human nutrition, mainly as food supplements in the health food market (Moore 2001) These unicellular algae have been shown not only to improve the immune system (Merchant 2001) but also to decrease the symptoms associated with gastric ulcers (Hasuda and Mito 1966), constipation (Saito et al 1966), anaemia (Sonoda 1972), hypertension (Miyakoshi et al 1980), neurosis (Sonoda and Okuda 1978), fibromyalgia (Merchant and Andre 2001), as well as preventing tumours (Tanaka et al 1984; Konishi et al 1985) Spirulina naturally produces antioxidants (such as carotenoids and xanthophylls), as well as antimicrobial compounds, and has various possible health benefits such as alleviation of hyperlipidaemia and hypertension, protection against renal failure, increased growth of intestinal Lacto- bacillus and reduction of serum glucose levels (Vilchez et al 1997; Liand et al 2004) Chlorella seems to not only contribute to the prevention of atherosclerosis and hypercholesterolaemia due to its glycolipid and phospholipid composition, but also contributes to the prevention of tumours through its glycoprotein, peptide and nucleotide contents (Borowitzka 1995) However, the most important substance in Chlorella seems to be b-1,3-glucan, which is an active immunostimulator, a free-radical scavenger and a reducer of blood lipids (Iwamoto 2004) Microalgae, however, are being seen as an untapped source of beneficial compounds that can be converted into sustainable functional ingredients The incorporation of microalgae into traditional foods (e.g breakfast cereals, spreads, breads, cookies, brownies, energy bars, mayonnaises, gelled desserts, pastas and beverages), which are largely consumed on a daily basis, may be beneficial whilst avoiding the potential problems associated with changing food habits and therefore solving possible resistance to the introduction of different products into one’s diet This strategy has many advantages from both the economic and the health hazard points of view and has proved to be efficient in the long term, whilst not presenting the drawbacks of traditional therapeutic actions based on medicines which generally have a short-term impact Bioactive molecules Microalgae are veritable miniature biochemical factories, providing a great diversity of primary and secondary metabolites, which they can biosynthesise, metabolise, Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al 23 Figure Chemical structures of chlorophyll-a and -b accumulate and secrete Microalgae, therefore, have potential applications in the food, pharmaceutical and cosmetic industries (Yamaguchi 1997) Like any other organisms, microalgae respond to changes in the external environment with changes in their intracellular environment Therefore, the manipulation of cultivation parameters (presence or absence of certain nutrients, temperature, light intensity, photoperiod and the microalgal growth phase; Otero et al 1997) stimulates the biosynthesis of compounds ranging from drugs to enzymes and natural antioxidants, some of high commercial value (Henriques et al 1998; Plaza 2008) The following sections give a description of the most important bioactive molecules of microalgae with food, pharmaceutical, cosmetic and trade interest 2.1 Pigments Only a small proportion of living matter is responsible for the beautiful gamut of colours commonly observed by simply looking around us However, pigment functionality goes beyond the aesthetic, and some colours are involved in processes essential for life on Earth: photosynthesis, protection and reproduction, amongst others To perform this range of functions, a large variety of compounds represented in nature (e.g chlorophylls, flavonoids, anthocyanins, carotenoids, betalains and quinones) are required One of the most obvious characteristics of microalgae is their colour Apart from chlorophylls, as the primary photosynthetic pigment, microalgae also form various accessory or secondary pigments such as phycobiliproteins and a wide range of carotenoids (carotenes and xanthophylls) 2.1.1 Chlorophylls All algae contain one or more types of chlorophyll: chlorophyll-a is the primary photosynthetic pigment in all algae (Figure 2) and is the only chlorophyll in cyanobacteria (blue-green algae) and rhodophyta Chlorophyta and euglenophyta algae contain chlorophyll-a and chlorophyll-b; chlorophylls -c, -d and -e can be found in several marine algae and freshwater diatoms Chlorophyll amounts are usually about 0.5–1.5% of dry weight (Baker and Gunther 2004; Gouveia et al 2008b) Chlorophylls have been traditionally used in medicine, and recent epidemiological studies provide evidence linking chlorophyll consumption to a decreased risk of colorectal cancer (Balder et al 2006) 2.1.2 Carotenoids Carotenoids are naturally occurring pigments that are responsible for the different colours of fruits, vegetables and other plants (Ben-Amotz and Fishler 1998) Carotenoids are usually yellow to red, isoprenoid polyene pigments derived from lycopene (Figure 3) They are synthesised de novo by photosynthetic organisms and some other microorganisms (Borowitzka 1988) In animals, carotenoids ingested in their diet are accumulated and metabolised by the organism, and are present in meat, eggs, fish skin (trout, salmon), the carapace of Crustacea (shrimp, lobster, Antarctic krill, crayfish), subcutaneous fat, skin, liver, integuments and in the feathers of birds (e.g poultry; Breithaupt 2007; Gouveia et al 2008b) Microalgae are also recognised as being an excellent source of natural food colourings and nutraceuticals and it is expected that they will surpass synthetic equivalents as well as other natural sources due to their sustainability of production and their renewable nature (Dufosse´ et al 2005) Carotenoids are already approved for use in foods such as the food colourings on labels such as E1161d (Blanchet et al 2007; Silaste et al 2007) Some microalgae can undergo a carotenogenesis process, in response to various environmental and cultural stresses (e.g light, 24 Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al Figure Chemical structures of some carotenoids: (a) lycopene, (b) b-carotene, (c) astaxanthin, (d) lutein, and (e) canthaxanthin temperature, salts, nutrients), where the alga stops growing and dramatically changes its carotenoid metabolism by accumulating secondary carotenoids as an adaptation to severe environments (Gouveia et al 1996b; Bhosale 2004) In algae, carotenoids seem to function primarily as photoprotective agents and as accessory light-harvesting pigments, thereby protecting the photosynthetic apparatus against solar radiation and related effects (Ben-Amotz et al 1987) They also play a role in phototropism and phototaxis (Borowitzka 1988) The function of carotenoids as antioxidants in plants shows interesting parallels with their potential role as antioxidants in foods and humans (Van den Berg et al 2000) The consumption of a diet rich in carotenoids has been recognised to reduce the risk of cardiovascular disease (Neuman et al 1999); to lower the prevalence of metabolic syndrome, adiposity and serum triglyceride concentrations in middle-aged and elderly men (Sluijs et al 2009); to diminish the prevalence of certain types of cancer, such as breast and lung cancer (Tavani et al 1997; De Stefani et al 1999, 2001), as well as atherosclerosis, cataracts and age-related macular degeneration; and to enhance immune resistance to viral, bacterial, fungal and parasitic infections (Cooper et al 1999; Stahl and Sies 2005; Tapiero et al 2004) The mechanisms by which carotenoids exert their health benefits are not yet well understood (Mathews-Roth 1991) Nevertheless, it was established that longer chromophores have better quenching of singlet oxygen in lipidic systems (Palozza and Krinsky 1992; Liebler 1993) Capsanthin and canthaxanthin have shown better antioxidant activity (AAC) than lutein and b-carotene, respectively It appears that activity depends on the number of double bonds, keto groups and cyclopentane rings present in the carotenoid structure (Kobayashi and Sakamoto 1999); carotenoids have been proposed as food additives to prevent degradation (Nielsen et al 1996) Moreover, when their AAC is evaluated on triglycerides, lutein, lycopene and b-carotene seem to act as pro-oxidants, but when evaluation is carried out in the presence of g-tocopherol, the phenomenon is reverted It is suggested that tocopherols protect carotenoids against radical auto-oxidation (Haila et al 1996) More than 600 known carotenoids have been reported in nature and about 50 have provitamin A activity; however, only very few carotenoids are used commercially, such as b-carotene and astaxanthin, and of lesser importance, lutein, zeaxanthin, lycopene and bixin which are used in animal feeds, pharmaceuticals, cosmetics and food colourings (Gouveia et al 2008b) The main carotenoids produced industrially by microalgae are b-carotene from Dunaliella salina and astaxanthin from Haematococcus pluvialis b-Carotene is both recognised as safe and as having positive health effects because of its provitamin A activity (Baker and Gunther 2004) It serves as an essential nutrient and is in high demand on the market as a natural food colouring agent, as an additive to cosmetics, and also as a health food (Raja et al 2007) b-Carotene is routinely used in soft drinks, cheeses and in butter or margarine Astaxanthin is a strong colouring agent and has many functions in animals such as having an impact on growth, reproduction and the immune system, as well as enhancing eye health, improving muscle strength and endurance, protecting the skin from premature ageing and reducing inflammation and UVA damage (Blomhoff et al 1992; Tsuchiya et al 1992; Beckett and Petkovich 1999) Astaxanthin is used in aquaculture in the following ways (Margalith 1999; Lorenz and Cysewski 2000):         Antioxidant Hormone precursor Immune enhancer Enhancer of provitamin A activity In reproduction In growth In maturation In photoprotection Astaxanthin has been shown to be a more potent antioxidant than other carotenoids as well as vitamin E (500 times more; Miki et al 1982; Terao 1989) Some reports Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al have also supported the assumption that daily ingestion of astaxanthin may protect body tissues from oxidative damage, and this might be a practical and beneficial strategy in health management (Dufosse´ et al 2005) In general, astaxanthin is used as a dietary supplement for its potential health benefits (Stewart et al 2008) 2.1.3 Phycobiliproteins Besides chlorophyll and carotenoid lipophilic pigments, cyanobacteria (blue-green algae), rhodophyta (red algae) and cryptomonad algae contain phycobiliproteins, deepcoloured water-soluble fluorescent pigments, which are major components of a complex assemblage of photosynthetic light-harvesting antenna pigments – the phycobilisomes (Glazer 1994) This group of pigments possesses a large spectrum of applications, such as being used as highly sensitive fluorescence markers in clinical diagnosis and for labelling antibodies; they are also used in multi-colour immunofluorescence or fluorescence-activated cell-sorter analysis (Becker 1994; Sekar and Chandramohan 2007) This compound has also shown several pharmacological properties, including antioxidant, anti-inflammatory, neuroprotective and hepatoprotective effects (Bhat and Madyastha 2000; Romay et al 2003; Benedetti et al 2004) Phycobiliproteins are formed by a protein backbone covalently linked to tetrapyrrole chromophoric prosthetic groups, named phycobilins (Figure 4) The main natural resources of phycobiliproteins are the cyanobacterium Spirulina (Arthrospira) for phycocyanin (blue) and the rhodophyte Porphyridium for phycoerythrin (red) Phycocyanin is currently used in Japan and China as a bright blue natural colour in food products such as chewing gums, candies, dairy products, jellies, ice creams, soft drinks (e.g Pepsi1 blue) and also in cosmetics such as lipsticks, eyeliners and eye shadows (Sekar and Chandramohan 2007) 2.2 Fatty acids a-Linolenic acid (ALA, 18:3 o3) is the essential fatty acid precursor of the o3 series synthesised in plant organisms Figure Chemical structure of a phycocyanobilin attached by thioether linkage to the apoprotein 25 using the o12- and o15-desaturases (Calder 2004; Moyad 2005) However, ALA cannot be synthesised by animals due to the lack of these desaturase enzymes (Nakamura and Nara 2003), and therefore its essential importance for mammals was recognised (Burr and Burr 1930) In fact, the long-chain o3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA; 20:5 o3) and docosahexaenoic acid (DHA; 22:6 o3) are very important from a nutritional point of view and can be mainly found in marine fish products (Figure 5) However, it is important to take into account that both fatty acids are mainly produced by unicellular marine microalgae that are consumed by other marine species (Moyad 2005) and accumulated through the trophic chain The fatty acids in microalgae are biosynthesised through the addition of acetate (C-2) units; almost all are straight chain and with an even number of carbon atoms, predominantly between C-12 and C-22 (Cohen 1986) The main saturated fatty acids present in these structures are acids with 12, 14, 16 and 18 carbon atoms A wide variety of unsaturated fatty acids are found in algae, with chains between 16 and 22 carbon atoms and double bonds in cisconfiguration The marine microalgae generally contain the higher level fatty acids C-20 and C-22, which are highly unsaturated when compared with those from freshwater species (Behrens and Kyle 1996) The biosynthesis of o3 PUFA occurs in plant cells, and these fatty acids are often found in high concentrations in marine algae (Reitan et al 1994) Moreover, in algae, o3 and o6 fatty acids with 18 carbon atoms are important components of photosynthetically active thylakoid membranes in chloroplasts, where these fatty acids are synthesised (Ahlgren et al 1992) The metabolism of fatty acids in microalgae is not very different from the metabolism in higher plants and mammals (Behrens and Kyle 1996) Haptophyceae is one of the classes of marine microalgae commonly used in mariculture These algae contain a wide variety of fatty acids with chains between C-16 and C-22 (Sukenik and Wahnon 1991; Behrens and Kyle 1996) One of the microalgae used as a nutritional source of o3 PUFA is Isochrysis galbana (Bandarra et al 2003; Durmaz et al 2008a) The nutritional value of this microalga is related to its biochemical composition, specifically the level of EPA and DHA Many marine animals have a limitation in the synthesis of EPA and DHA, and this deficiency can be remedied by incorporating enriched o3 PUFA microalgae in the diet of these species Certain animals, such as oyster and shrimp, may not have an absolute need for EPA and DHA, but their growth rates and survival increase substantially when these fatty acids are included in their diet (Volkman et al 1989) Besides the importance in aquaculture, the interest in o3 and o-PUFA from microalgae has increased mainly for pharmaceutical and biotechnological applications 26 Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al Figure Chemical structure of o3 PUFA of high pharmaceutical and nutritional value (Otero et al 1997) From a medical point of view, DHA and EPA have various positive effects on human metabolism such as reducing the levels of cholesterol, triglycerides, low-density lipoprotein and very low-density lipoprotein-2 and preventing rheumatoid arthritis, stroke, Alzheimer’s disease, psoriasis and certain types of cancer They also have anti-inflammatory properties and are essential for pre- and post-natal growth of the retina and brain (Calder 2004; Chapkin et al 2007) Some microalgae synthesise fatty acids which are of particular interest, such as g-linolenic acid (18:3 o6; Arthrospira spp.), arachidonic acid (20:4 o6; Porphyridium spp.), EPA (Nannochloropsis spp., Phaeodactylum spp., Nitzschia spp., Isochrysis spp and Diacronema spp.) and DHA (Crypthecodinium spp., Schizochytrium spp., Ulkenia spp.; Chini Zittelli et al 1999; Bandarra et al 2003; Donato et al 2003; Molina Grima et al 2003; Spolaore et al 2006) The high content of DHA found in some algae is currently used to enrich infant formulas to produce supplements with characteristics similar to breast milk Consequently, the marketing of DHA from microalgae demonstrates the value of these organisms as a source of fatty acids (Behrens and Kyle 1996) In fact, the microalga Schizochytrium, which contain 50–85% lipids and more than 20% DHA (w/w), has been used for the production of functional foods Crypthecodinium spp and Ulkenia spp are also good sources of DHA used in dairy products such as milk, yoghurt, cheese and ice cream (Barclay and van Elswyk 2001) It is estimated that healthy human adults are able to convert 18:3 o3 to EPA at a rate lower than 5%, and convert EPA to DHA at a rate below 0.05%; moreover, this is not possible in children and the elderly (Burdge and Calder 2005; Wang et al 2006a, 2006b) This statement confirms the importance of the inclusion of EPA and DHA in one’s daily diet, and it is recommended by the ISSFAL (2004) that a daily dose of 500 mg of EPA plus DHA should be taken by healthy subjects and g should be taken to prevent the incidence of secondary cardiovascular diseases 2.3 Proteins During the 1950s, the use of microalgae as sources of protein was of great interest because of the high protein content of various microalgae species (Soletto et al 2005) In addition, the amino acid pattern of almost all algae compares favourably with that of other food proteins As the cells are capable of synthesising all amino acids, they can provide those that are essential to humans and animals (Guil-Guerrero et al 2004) As with other bioactive compounds synthesised by microalgae, the amino acid composition of microalgae, especially free amino acids, varies greatly between species, as well as with growth conditions and growth phase (Borowitzka 1988; Gouveia et al 2008b) 2.4 Polyphenols Polyphenols are antioxidant compounds with health benefits reported to range from improved cardiovascular health to protection against certain cancers and Alzheimer’s disease (Ares et al 2009) However, fortifying foods with polyphenols is limited by the inherent bitter taste of these compounds, and food manufacturers are acutely aware of the need to make healthy products taste good The addition of other compounds such as sucrose, sucralose, polydextrose and milk can reduce the bitterness, astringency and characteristic flavour of polyphenol extracts (Ares et al 2009) 2.5 Polysaccharides Polysaccharides are widely used in the food industry primarily as gelling and thickening agents Many commercially used polysaccharides such as agar, alginates and carrageenans are extracted from macroalgae (e.g Laminaria, Gracilaria, Macrocystis; Borowitzka 1988) Nevertheless, most microalgae produce polysaccharides and some of them could have industrial and commercial applications, considering the fast growth rates and the possibility to control the environmental conditions regulating their growth Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al The most promising microalga for commercial purposes is the unicellular red alga Porphyridium cruentum, which produces a sulphated galactan exopolysaccharide (EPS) that can replace carrageenans in many applications (Spolaore et al 2006) Another example is Chlamydomonas mexicana, which releases up to 25% of its total organic production as extracellular polysaccharides, which have found applications as soil conditioners in the United States (Borowitzka 1988) Polysaccharides account for the bulk of microalgal carbohydrates and are often divided into three groups: reserve glucans, extracellular polysaccharides and cell-wall polysaccharides (Granum 2002) Branched starches and b-1,3glucans are the most common reserve glucans in microalgae Red microalgae were shown to produce different structures of starch, as well as amylose-, amylopectin- and glycogen-type polyglucans (Shimonaga et al 2007) A particular structure of starch which is more highly branched than amylopectin, named floridean starch, constitutes a major carbohydrate storage product of some unicellular red algae, e.g Galdieria sulphuraria and Cyanidioschyzon merolae (Barbier et al 2005) Because of the nature of the glycosidic linkages of these polysaccharides, they are commonly linked with bioethanol production by saccharification and subsequent or simultaneous fermentation (Ogaki et al 2009) b-1,3-Glucans, also called chrysolaminaran, are frequently found in diatoms These are polymers of b-(1?3)-linked glucopyranoside units with a degree of polymerisation of 20–60 and a variable degree of branching in the b-(1?6) and b-(1?2) positions (Figure 6; Granum 2002; Størseth et al 2005) Chrysolaminaran constitutes both an important storage product for photosynthetically fixed carbon and a major respiratory substrate of marine diatoms, and it can make up to 20– 30% of the cell dry weight (Sheehan 1998) b-(1?3)-DGlucans display immunostimulant activity, which is considered to be closely associated with the b-(1?6)branches (Størseth et al 2005) Microalgae can secrete extracellular polymeric substances, which can be formed as capsular material that closely surrounds the producing microbial cell or as looseslime matrices that are released more widely into the surrounding environment A large proportion (40–95%) of this polymeric material is EPS, which plays an important role in cellular attachment and adhesion to surfaces, as well as formation of a protective layer, and prevention of cellular desiccation (Nichols et al 2009) The constituent monosaccharides of EPS are very diverse and may vary with the growth phase (Allard and Tazi 1993; Giroldo et al 2005; Sua´rez et al 2005; Mishra and Jha 2009) One example of these EPS is the soluble cell-wall polysaccharide complex, which encapsulates the cells of Porphyridium spp It is composed of about 10 different sugars, of which the major ones are xylose, glucose and Figure Chemical structure of b-1,3-glucan branches in C-2 and C-6 (Granum 2002) 27 with galactose (Geresh et al 2002), glucuronic acid and halfester sulphate groups, and it dissolves continuously into the medium (Arad 1988; Geresh et al 2009) The sulphated heteropolymers produced by Porphyridium spp are considered to have relevant biological activity, exhibiting anti-retroviral, bio-lubricant, anti-inflammatory, hypocholesterolaemic and anti-cell proliferation activities (Dvir et al 2000; Talyshinsky et al 2002; Ginzberg et al 2008) There are indications that the sulphate group is the chemically and biologically active moiety of the Porphyridium spp polysaccharide (Keidan et al 2006) Another interesting example of microalgae polymeric substances comes from the aqueous extract of Chlorella pyrenoidosa because of its immunostimulatory properties (Sua´rez et al 2005) It is commercially available as RespondinTM, and its activity is thought to be due to its polysaccharide content It was found to contain structurally unique polysaccharides of alternate D-galactopyranose and L-arabinofuranose units (Sua´rez et al 2005; Figure 7) From the same extract, cyclic b-(1?2)-D-glucans were also isolated (Sua´rez et al 2008) In prokaryotes, these cyclic compounds are thought to be involved in osmoregulation The fraction of b-(1?2)-D-glucans isolated from C pyrenoidosa comprises a mixture of cyclic and linear b-(1?2)-D-glucans The ring sizes of cyclic and linear b-(1?2)-D-glucans range from 18 to 35 and 20 glucose units, respectively These relatively low-molecularweight glucans also showed some immunostimulatory activity 2.6 Vitamins and minerals Microalgal biomass represents a valuable source of nearly all essential vitamins (e.g A, B1, B2, B6, B12, C, E, nicotinate, biotin, folic acid and pantothenic acid) and a balanced 28 Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al Figure Cyclic b-(1?2)-D-glucans isolated from Chlorella pyrenoidosa (Sua´rez et al 2008) mineral content (e.g Na, K, Ca, Mg, Fe, Zn and trace minerals; Becker 2004) The high levels of vitamin B12 and iron in some microalgae, such as Spirulina, makes them particularly suitable as nutritional supplements for vegetarian individuals The vitamin content of an alga depends on the genotype, the stage in the growth cycle, the nutritional status of the alga and the light intensity (photosynthetic rate) The vitamin content is therefore amenable to manipulation by varying the culture conditions as well as by strain selection or genetic engineering However, vitamin cell content fluctuates with environmental factors, harvesting treatment and biomass drying methods (Borowitzka 1988; Brown et al 1999) 2.7 Tocopherols Tocopherols (vitamin E) have a saturated side chain, with the main difference among them being the number and position of methyl groups in the aromatic ring The corresponding tocotrienols (a-, b-, g- and d-) are characterised by an unsaturated side chain (Figure 8) To establish the relationship between structure and biological activity of tocopherols and tocotrienols, it is necessary to consider two structural characteristics: the presence of hydroxyl and methyl groups in the aromatic fraction of the molecule, and the aliphatic side chain at C-2 The unsaturation of the side chain of tocotrienols causes a decrease in the molecular activity (Kasparek 1980; Eitenmiller 1997) Tocopherols are lipid-soluble antioxidants synthesised only by photosynthetic organisms (Go´mez-Coronado et al 2004) The activ- Figure Structure of tocopherols and tocotrienols ity of this vitamin in microalgae and other organisms of vegetable origin is mainly due to a-tocopherol that represents about 80% of total tocopherols and tocotrienols (Chen et al 1998) The significance of vitamin E has been subsequently proven as a radical chain-breaking antioxidant because of its well-documented ability to protect the integrity of tissues, and it also plays an important role in life processes (Huo et al 1997; Vismasa et al 2003) Vitamin E has always been considered as an essential constituent because of its recognised AAC and ability to protect DNA, protein amino acids and lipids (PUFAs) from oxidative damage by neutralising free radicals, which can initiate chain reactions (Kasparek 1980; Huo et al 1997; Brown et al 1999) In fact, tocopherols are a class of dietary antioxidants (Clark 1996), with a-tocopherol currently being widely used in food as a natural lipophylic antioxidant (Chen et al 1998) The role of these antioxidants is to interrupt the process of lipid oxidation by donating a hydrogen atom of a peroxide radical, stopping the chain reaction in which it destroys the lipid molecules Often the tocopherols are preferred to synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylhydroxytoluene (BHT) primarily because of their unquestionable safety at the level of toxicity and their low volatility which gives them great stability at high temperatures Another reason for using these molecules is their high solubility in fats and oils (Clark 1996) Animal cells are unable to synthesise vitamin E and must obtain it from plant sources (Vismasa et al 2003) Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al a-Tocopherol is the most abundant form that has the highest AAC in vivo and also has an important function in electron transport reactions and cell membrane stabilisation related to membrane permeability and fluidity (Carballo-Ca´rdenas et al 2003) From the physiological point of view, the tocopherol appears to be unique in meeting all the specifications of biologically active lipid antioxidants (Kasparek 1980), presenting a synergistic effect with the phospholipids (Bandarra et al 1999), which is an important component of membranes Thus, optimal concentrations of tocopherols may be affected by the quality and quantity of phospholipids and other minor compounds (Jung and Min 1990) According to Eitenmiller (1997), vitamin E undergoes large losses as a result of its AAC Such oxidative losses are accelerated by light, temperature, alkaline pH and various metals, particularly iron and copper The variability of vitamin E in microalgae is inherent to the species and environmental conditions (i.e light and temperature; Huo et al 1997) In microalgae, a-tocopherols are located in chloroplasts, whereas g- and d-tocopherols are found in extrachloroplastic fractions of the cell According to Draper (1980), the partitioning of different isomers of vitamin E suggests several possibilities:    With the exception of a-tocopherol, all the other tocopherol isomers could not cross the chloroplast membrane All tocopherols can be synthesised in a specific site of the cell and subsequently be dispersed to different locations of the cell (which would imply that these isomers could have different functions) Tocopherols can be synthesised in different cell locations Moreover, tocopherols have a widespread occurrence in nature, being present in both photosynthetic (e.g leaves) and non-photosynthetic (e.g seedlings) tissues of higher plants and algae In green microalgae, Isochrysis galbana and Diacronema vlkianum, an increase in a-tocopherol levels during the growing phases was reported by Bandarra et al (2003), Donato et al (2003) and Durmaz et al (2008a, 2008b) These authors also pointed out that these levels of a-tocopherols were higher in these microalgae when compared with conventional foods known to be rich in vitamin E However, the microalga Euglena has the highest tocopherol content among the several genera of yeast, moulds and algae tested (Kusmic et al 1999) The benefits of vitamin E on human health have been the subject of several studies that confirm its beneficial effect in preventing degenerative disorders such as cardiovascular diseases, arteriosclerosis, certain cancers and light-induced pathologies of skin and eyes (Clark 1996; 29 Eitenmiller 1997; Go´mez-Coronado et al 2004) For this reason, it has been suggested that its use as a supplement or nutraceutical could have a positive impact on health (Carballo-Ca´rdenas et al 2003; Vismasa et al 2003) Furthermore, increased intake of vitamin E contributes to the enhancement of the immune response It is noteworthy that vitamin E is a protective agent and not a therapeutic agent Deficiency has been associated with problems in digestion, absorption and fat transportation (Clark 1996) The recommended daily dose of a-tocopherol is 15 mg, which increases when the diet is rich in unsaturated fatty acids A normal supply results in a concentration of 0.7–1.6 mg/100 mL in blood plasma; however, a poor level would be when the concentration of atocopherol levels is below 0.4 mg/100 mL 2.8 Other antioxidants Antioxidants are now thought to possibly prevent the incidence of many diseases such as cancers, cardiovascular and age-related diseases, by protecting cells against oxidative damage Microalgae are photoautotrophic organisms that are exposed to high oxygen and radical stresses and consequently have developed several efficient protective systems against reactive oxygen species and free radicals (Pulz and Gross 2004; Gouveia et al 2008b) Natrah et al (2007) reported a stronger AAC exhibited by methanolic microalgal crude extracts (e.g from Isochrysis galbana, Chlorella vulgaris, Nannochloropsis oculata, Tetraselmis tetrathele, Chaetoceros calcitrans) when compared with a-tocopherol Rodriguez-Garcia and GuilGuerrero (2008) reported that the yields for C vulgaris, Phaeodactylum tricornutum and Porphyridium cruentum were in the range of 24–30%, with the highest yield from C vulgaris Measures of AAC showed that, above a concentration of 1.5 mg/mL, extracts from both C vulgaris and P tricornutum significantly exceeded BHA and BHT Indeed, at a concentration of 2.5 mg/mL, the extracts from C vulgaris had five times the AAC when compared with the synthetic alternatives (Rodriguez-Garcia and Guil-Guerrero 2008) In fact, AAC values of C vulgaris seem to be among the highest AAC reported to date in the literature for any biological extract or pure compound tested (Rodriguez-Garcia and Guil-Guerrero 2008) Since use of the synthetic antioxidants BHT and BHA has been called into question in terms of their safe use (they are believed to be carcinogenic and tumourigenic if given in high doses; Schildermann et al 1995; Aruoma 2003), there is increasing interest in using microalgae as a natural source of antioxidants, both for cosmetic and functional food use In particular, C vulgaris has potential important applications in the food and dietary supplement industries 30 Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al Figure Basic structure of sterols 2.9 Sterols Sterols are a group of triterpenoids, a family of natural products derived from biosynthetic squalene, which adopts a cyclic structure In higher plants and algae, the first compound to be formed in the cyclisation of squalene, the first product in the cyclic biosynthetic pathway, is cycloartenol (Goodwin 1980) These biomolecules are an important family of lipids and are found in most eukaryotic cells, mainly in the plasma membrane (the barrier between the intracellular and extracellular media; Voet and Voet 1990) The basic structure of sterols is composed of four rings: A, B, C and D (Figure 9) The first three are arranged in a chair-shaped conformation, while ring D usually adopts a planar conformation While the rings B and C, and C and D are connected in a trans-conformation, the rings A and B can connect in a trans- or cis-conformation A characteristic of sterols is the presence of a hydroxyl group on C-3 of ring A (Belitz and Grosh 1999) From an analytical point of view, the sterols represent one of the most important groups in the unsaponifiable fraction (Lognay et al 1989) These complex molecules are different in animals and plants, showing differences in their biosynthesis Animal cells and fungi usually contain one major sterol, i.e cholesterol and ergosterol, respectively More than 100 types of phytosterols have been reported in plant species, but the most abundant sterol in plants are sitosterol, campesterol and stigmasterol (Nabil and Cosson 1996; Fernandes and Cabral 2007; Figure 10) The lipid fraction of plants contains 0.15–0.90% of sterols, with sitosterol being the major component (Belitz and Grosh 1999) Sterols in plants exist in the form of free alcohols of fatty acid esters, steryl glycosides and acylated steryl glycosides (Fernandes and Cabral 2007) Not much is known regarding the importance of sterols in plants other than their importance as structural components of cell membranes It is believed that variations in the structure of these molecules have implications for cellular plasticity and permeability of the membrane (Volkman et al 1981; Ibrahim et al 1990; Ponomarenko Figure 10 Common examples of phytosterols et al 2004) A recent work pointed out that those small differences in sterol structure can give rise to significant differences in membrane properties The ability of sterols to modify membrane properties in different ways points to the mutual interplay between sterols and membrane mechanical properties and its potential relevance for evolution (Hodzic et al 2008) The importance of sterols in microalgae is of increasing interest since:    The presence of these natural products in microalgae determines their food value These compounds are useful biomarkers for identifying sources of organic matter in sediments They are not only essential components of biomembranes but also have functions in cell proliferation and signal transduction of microalgae and other eukaryotic organisms, modulating the activity of membrane-bound enzymes (Ponomarenko et al 2004) In contrast with higher plants, microalgae contain a larger diversity of different sterols Microalgae contain different types of sterols that are characteristic of each species (Easa et al 1994; Barret et al 1995) Moreover, studies on bivalve production have suggested that the type and quantity of sterols present in dietary microalgae were directly related to the bivalve Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al 31 Table Major microalgae commercialised for human nutrition (Adapted from Pulz and Gross 2004; Spolaore et al 2006; Hallmann 2007) Microalga Spirulina (Arthrospira) Chlorella Dunaliella salina Aphanizomenon flos-aquae Major producers Products Hainan Simai Pharmacy Co (China) Earthrise Nutritionals (California, USA) Cyanotech Corp (Hawaii, USA) Powders, extracts Myanmar Spirulina factory (Myanmar) Taiwan Chlorella Manufacturing Co (Taiwan) Kloătze (Germany) Cognis Nutrition and Health (Australia) Blue Green Foods (USA) Vision (USA) growth rate (Wikfors et al 1991) Hence, studies on sterol compositions of unstudied microalgae, including new and rare species, may be of great value The first in-depth study on algal sterol classes started in 1930 by Heilbron, Phipers and Wright (Goldberg et al 1982) More recently, research has shown that some lipid constituents of microalgae, with particular emphasis on sterols and fatty acids, may be useful as biomarkers to support taxonomic classification (Volkman et al 1997; Bouzidi et al 2008) Contrary to the profile of fatty acids, which may vary in some unicellular algae due to changes in growth conditions, the composition of sterols is unaffected by such factors and is therefore increasingly used in taxonomic studies (Billard et al 1990; Patterson et al 1994; Volkman et al 1997) The major sterols in red algae are C-27 compounds; cholesterol occurs in substantial amounts and is generally the primary sterol Desmosterol and 22E dehydrocholesterol are also present in high concentrations and may even be the major sterol in the Gigartinales class (Bouzidi et al 2008) On the other hand, in brown algae, the dominant sterol is fucosterol, and cholesterol is present only in low amounts, such as for Bifurcaria bifurcate, Cladostephus hirsitus, Dictyota dichotoma and Cystoseira sedoides In green algae, there is no single major sterol, and the dominant sterol seems to vary within orders and families (Bouzidi et al 2008) Moreover, in green marine microalgae such as Isochrysis galbana and Diacronema vlkianum, sitosterol was the main sterol identified (Bandarra et al 2003; Donato et al 2003; Durmaz et al 2008a, 2008b) Sitosterol, administered orally as a suspension, was used as a drug to treat hypercholesterolaemia in the United World production (tonnes/year) 3000 Tablets, powders, extracts Tablets, powders, beverages, extracts Tablets, chips, pasta and liquid extract Tablets, powders, nectar, noodles 2000 Powders Powders and b-carotene 1200 Capsules, crystals Powder, capsules, crystals 500 States (Clark 1996) Sterols are not absorbed by the body but inhibit the absorption of cholesterol This feature is primarily due to the presence of double bonds in the stigmasterol and alkyl group in campesterol, stigmasterol and b-sitosterol (Ibrahim et al 1990; Gurr 1996) Sitostanol is a product that is obtained by chemical reduction of sitosterol and seems to have a superior effect in the reduction of blood cholesterol levels (Gurr 1996) On the other hand, it has been found that many polyhydroxysterols from marine organisms have anti-cancer, cytotoxic and other biological activities (Cui et al 2000; Tang et al 2002; Han et al 2003; Volkman 2003) Since phytosterols reduce the level of cholesterol in the blood, they are valuable in the development of functional foods Microalgal biomass market The microalgal biomass market produces about 5000 tonnes/year of dry matter and generates a turnover of about US $1.25 Â 109 per year (Pulz and Gross 2004) All over the world, commercial production of microalgae for human nutrition is already a reality Numerous combinations of microalgae or mixtures with other health foods can be found in the market as nutritional supplements in the form of tablets, powders, capsules, pills and liquids (Table 1) They can also be incorporated into food products (e.g pastas, biscuits, breads, snack foods, candies, yoghurts and soft drinks), providing the health-promoting effects that are associated with microalgal biomass, probably related to a general immune-modulating effect (Belay 1993) The authors have used C vulgaris green and carotenogenic (Gouveia et al 1996b) in several food products 32 Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al for pigmentation purposes (egg yolks, rainbow trout, seabream, ornamental fishes, emulsions such as mayonnaises, biscuits, puddings and pastas; Gouveia et al 1996a, 1996b, 2002, 2003, 2006, 2007; Gouveia and Rema 2005; Gomes et al 2002; Batista et al 2007, 2008; Fradique et al 2008) Haematococcus pluvialis and Spirulina maxima were used for ornamental fish pigmentation (Gouveia et al 2003; Gouveia and Rema 2005) and food products such as puddings and pastas (Gouveia et al 2008a, 2008c; Fradique et al 2008) Isochrysis galbana and Diacronema vlkianum have been investigated as a potential source of PUFAs and have already been tested by the authors in puddings and pastas (Fradique et al 2008; Gouveia et al 2008a) Conclusions Nutritional issues highlight the relationship between diet and chronic diseases We are moving beyond preventing the most important diseases worldwide (such as 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secondary prevention studies: a systematic review American Journal of Clinical Nutrition 84: 5-17 Wang, Y., Han, F., Hu, B., Li, J and Yu, W 2006b In vivo prebiotic properties of alginate oligosaccharides prepared through enzymatic hydrolysis of alginate Nutrition Research 26: 597603 View publication stats 37 Wikfors, G H., Gladu, P K and Patterson, G W 1991 In search of the ideal algal diet for oysters: recent progress, with emphasis on sterol The Journal of Shellfish Research 10: 292 Wildman, R 2007 Handbook of nutraceuticals and functional foods, 2nd ed CRC Press, Boca Raton, FL, USA Yamaguchi, K 1997 Recent advances in microalgal bioscience in Japan, with special reference to utilization of biomass and metabolites: a review Journal of Applied Phycology 8: 487-502  ... 1992) The metabolism of fatty acids in microalgae is not very different from the metabolism in higher plants and mammals (Behrens and Kyle 1996) Haptophyceae is one of the classes of marine microalgae. .. dietary microalgae were directly related to the bivalve Microalgae – source of natural bioactive molecules as functional ingredients L Gouveia et al 31 Table Major microalgae commercialised for human... some of high commercial value (Henriques et al 1998; Plaza 2008) The following sections give a description of the most important bioactive molecules of microalgae with food, pharmaceutical, cosmetic