Acessibilidade / Reportar erro

Metabolites of interest for food technology produced by microalgae from the Northeast Brazil1 1 Parte da Tese de Doutorado da primeira autora apresentada ao Programa de Pós-Graduação em Ciência e Tecnologia de Alimentos - Universidade Federal da Paraíba/UFPB

Metabólitos de interesse à tecnologia de alimentos produzidos por microalgas do Nordeste do Brasil

ABSTRACT

There is an increasing demand for bioprospection focusing on microalgae isolated from the northeastern region of Brazil with potential importance for food industries. To attend that need, we evaluated the characteristics of 12 regional species of microalgae grown under controlled cultivation conditions (temperature = 24 ± 1 ºC, illumination 150 µmol photons m-2 s-1, photoperiod of 12 h) in terms of their nutritional quality and lipid profiles. Significant differences in growth characteristics and chemical compositions were observed among the species investigated. High carbohydrate contents (> 25 g 100 g-1) were recorded in various strains of Chlorococcum and the marine microalga Amphidinium carterae; high protein contents (> 35 g 100 g-1) were observed in Scenedesmus acuminatus and Pediastrum tetras; and high lipid contents (> 25 g 100 g-1) in A. carterae and some strains of Chlorococcum sp. (cf. hypnosporum). Chlamydomonas sp. demonstrated the greatest production of carotenoids (64.92 mg g-1), chlorophyll-a (234.74 mg g-1), and chlorophyll-b (59.34 mg g-1). The lipid profiles of Chlorella cf. minutissima, four strains of Chlorococcum sp. (cf. hypnosporum), P. tetras, Planktothrix isothrix, and S. acuminatus indicated the presence of palmitic, oleic (ω-9), linoleic (ω-6) and α-linolenic (ω-3) acids, with more than 50% omegas in the total composition of their fatty acids. In terms of chemical nutrients, the microalgae cited were found to be potential sources of omegas, carotenoids, and chlorophylls that could be used in food industries.

Key words:
Polyunsaturated fatty acids; Carotenoids; Omegas; Microalgae cultivation

RESUMO

Existe uma demanda por pesquisas prospectivas de microalgas isoladas da região Nordeste do Brasil que possam ser potencialmente importantes à indústria de alimentos. Nesta pesquisa foram avaliadas as características da cinética de crescimento sob condições controladas de cultivo (temperatura = 24 ± 1 ºC, iluminação 150 µmol fótons m-2 s-1, fotoperíodo de 12 h), a qualidade nutricional e o perfil lipídico de doze espécies regionais de microalgas visando suprir esta lacuna. Diferenças nas características de crescimento e na composição química foram observadas nas espécies pesquisadas, com maiores teores de carboidratos (> 25 g 100 g-1) registrados em várias cepas de Chlorococcum e na microalga marinha Amphidinium carterae, de proteína (> 35 g 100 g-1) em Scenedesmus acuminatus e Pediastrum tetras e de lipídios (> 25 g 100 g-1) em A. carterae e em algumas cepas de Chlorococcum sp. (cf. hypnosporum). A Chlamydomonas sp. apresentou os maiores teores de carotenoides (64,92 mg g-1), clorofila-a (234,74 mg g-1) e clorofila-b (59,34 mg g-1). O perfil lipídico evidenciou a presença dos ácidos palmítico, oleico (ω-9), linoleico (ω-6) e α-linolênico (ω-3), com mais de 50% de ômegas em sua composição total de ácidos graxos, em Chlorella cf. minutissima, em quatro cepas de Chlorococcum sp. (cf. hypnosporum), P. tetras, Planktothrix isothrix e S. acuminatus. No que concerne aos nutrientes químicos foi observado que as microalgas citadas são fontes potenciais de produção de ômegas, carotenoides e clorofilas para serem utilizados na indústria de alimentos.

Palavras-chave:
Ácidos graxos poli-insaturados; Carotenoides; Ômegas; Cultivo de microalga

INTRODUCTION

Recent research has analyzed the chemical compositions of microalgae and certified their positive contributions to human health and possible uses as food resources (ANDRADE et al., 2018ANDRADE, L. M. et al. Chlorella and Spirulina microalgae as sources of functional foods, nutraceuticals, and food supplements: an overview. MOJ Food Processing and Technology, v. 6, n. 1, 2018.; HAYES et al., 2018HAYES, M. et al. Microalgal proteins for feed, food and health. In: Microalgae-Based Biofuels and Bioproducts, p. 347-368, 2018.; SATHASIVAM; KI, 2018SATHASIVAM, R. K. J. A review of the biological activities of microalgal carotenoids and their potential use in healthcare and cosmetic industries. Marine Drugs, v. 16, p. 26. 2018.). Theoretically, microalgae are capable of producing more lipids than any other conventional crop, and numerous species can synthesize considerable quantities of essential fatty acids (EFA), especially omega-3 (ω-3) and 6 (ω-6) (the two most abundant), as well as α-linolenic acid (ALA, C18:3 ω-3) and linoleic acid (AL, C18:2 ω-6) (BELLOU et al., 2016BELLOU, S. et al. Microbial oils as food additives: recent approaches for improving microbial oil production and its polyunsaturated fatty acid content. Current Opinion in Biotechnology, v. 37, p. 24-35. 2016.; HO et al., 2014HO, S-H. et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnology for Biofuels, v. 7, p. 97-113. 2014.) - both precursors in the human body to long chain (≥ C20) polyunsaturated fatty acids (PUFA) (RINCÓN-CERVERA et al., 2016RINCÓN-CERVERA, M. A. et al. Supplementation with antioxidant-rich extra virgin olive oil prevents hepatic oxidative stress and reduction of desaturation capacity in mice fed a high-fat diet: effects on fatty acid composition in liver and extrahepatic tissues. Nutrition, v. 32, n. 11, p. 1254-1267, 2016.). Those microorganisms could be used as sustainable sources of EFA for human consumption or for use in animal rations as replacements for fish oil, which is now their principal source (RYCKEBOSCH et al., 2014RYCKEBOSCH, E. et al. Nutritional evaluation of microalgae oils rich in omega-3 long chain polyunsaturated fatty acid as alternative for fish oil. Food Chemistry, v. 160, p. 393-400. 2014.).

Western diets are currently excessive in terms of ω-6, but deficient in ω-3, resulting in a imbalance in the ω-6:ω-3 ratio that can interfere in the conversion of ω-3 ALA into eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6). That lack of conversion can result in increased levels of arachidonic acid (AA, C20:4 ω-6) in the phospholipid membranes that, over time, result in the excessive production of pro-inflammatory eicosanoids and the consequent hardening and contraction of blood vessels, increasing pain transmission, immunosuppression and, the pathogenesis of cardiovascular, inflammatory, and autoimmune diseases, as well as cancer - as increasing levels of ω-3 exert a suppressor effect (RUBIO-RODRÍGUEZ et al., 2010RUBIO-RODRÍGUEZ, N. et al. Production of omega-3 polyunsaturated fatty acid concentrates: a review. Food Science and Emerging Technologies, v. 11, p. 1-12. 2010.; SUBASH-BABU; ALSHATWI, 2018SUBASH-BABU, P.; ALSHATWI, A. A. Effects of increasing ratios of dietary omega-6/omega-3 fatty acids on human monocyte immunomodulation linked with atherosclerosis. Journal of Functional Foods, v. 41, p. 258-267. 2018.). There is not yet a consensus, however, in respect to optimal ω-6:ω-3 ratios, nor sufficient evidence to define the maximum tolerable dose, although some researchers suggest that the ratios between those acids should lie between 4-5:1, but never above 10:1 (CANDELA; LÓPEZ; KOHEN, 2011CANDELA, C. G.; LÓPEZ, L. M. B.; KOHEN, V. L. Importance of a balanced omega 6/omega 3 ratio for the maintenance of health: nutritional recommendations. Nutrición Hospitalaria, v. 26, n. 2, p. 323-329, 2011.; WARNER et al., 2017WARNER, D. et al. ω3-PUFA supplementation reduced ALT levels In mice exposed to a low linoleic acid diet with or without ethanol in a drinking in the dark binge model. The FASEB Journal, v. 31, n. 1, p. 894.4-894.4, 2017.).

The present work sought to characterize and compare the lipid profiles of 12 regional strains of microalgae isolated from marine and Different freshwater environments in northeastern Brazil to determine if they produce significant levels of omegas ω-3, ω-6, and ω-9 and could serve as alternative sources of compounds of interest to the food industry. The nutritional qualities of those algal strains were also investigated in terms of their percentage contents of carbohydrates, proteins, lipids, carotenoid pigments and chlorophyll-a and b, as well as their growth characteristics in mono-specific cultures under controlled conditions.

MATERIALS AND METHODS

Biomass production and growth characteristics

Twelve regional strains of microalgae maintained that the Microalgae Collection of the Laboratory of Reef Environments and Biotechnology with Microalgae (LARBIM/UFPB) were investigated, including 11 freshwaters and one marine species. All of the strains were isolated from distinct aquatic environments in northeastern Brazil: nine from Paraíba State (PB), one from Bahia State (BA), one from Pernambuco State (PE), and one from Rio Grande do Norte State (RN) (Table 1).

Table 1
List of the microalgae investigated in the present project, citing their respective taxonomic groups and origins

The species were cultivated in triplicate in flat bottomed 6 L flasks containing 5 L of Conway media (WALNE, 1966WALNE, P. R.. Experiments in the large scale culture of the larvae of Ostrea edulis. Journal Fishery Investiment, v. 25, n. 4, p. 1-53, 1966.) for the cultivation of the marine microalga, or 5 L of Zarrouk medium (ZARROUK, 1966ZARROUK, C. Contribution à l'étude d'une cyanophycée: influence de divers facteurs physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima (Setch et Gardner) Geitler. 1966. Theises (Ph. D.) - Faculty of Science, Université des Paris, Paris, 1966.) for cultivating Chamydomonas, Pediastrum, and Scenedesmus, or WC medium (GUILLARD; LORENZEN, 1972GUILLARD, R. R. L.; LORENZEN, C. J.. Yellow-green algae with chlorophyllide c. Journal of Phycology, v. 8, p. 10-14. 1972.) for cultivating the other freshwater strains. The microalgae were grown in culture chambers (24 ± 1 ºC) under a light intensity of approximately 150 µmol photons m-2 s-1, furnished by 40 W fluorescent lamps, under a 12 h photoperiod, with continuous forced air (0.1 L min-1) injection.

Culture growth was accompanied by measuring "in vivo" fluorescence (Turner Design Fluorometer) and by cell counts using Fuchs-Rosenthal or Sedgewick-Rafter chambers (for filamentous forms) as viewed under a Leica binocular microscope. Growth curves were prepared for each species, allowing calculations of their growth velocity (k), expressed as the number of cell divisions per day (STEIN 1973STEIN, J. R. Handbook of phycological methods: culture methods and growth measurements. In: Handbook of phycological methods. Cambridge University Press; p. 448. 1973.), the duration (in days) of their log phase, maximum cell density (MCD), culture time, final biomass yield, and biomass productivity per day. Upon reaching their respective stationary phases, the cultures were interrupted and the biomasses produced were concentrated by centrifuging (at 18 ºC), frozen at -30 °C, and subsequently lyophilized. The dry biomasses were weighed using an analytical balance and maintained under refrigeration (9 ºC) until analyzed.

Chemical analyses

Determinations (in triplicate) were made of: proteins, following Lowry et al. (1951)LOWRY, O. H. et al. Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, v. 193, n. 1, p. 265-75, 1951.; carbohydrates, following Kochert (1978)KOCHERT, G. Carbohydrate determination by the phenol-sulfuric method. In: HELLEMBUST, J. A., CRAIGIE, J. S. (Ed.). Handbook of phycological methods. Cambridge: Cambridge University, 1978. p. 95-97.; total lipids, following Folch, Lees and Stanley (1957)FOLCH, J.; LEES, M.; STANLEY, G. H. S.. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, v. 226, p. 497-509. 1957.; total carotenoids, following Strickland and Parsons (1968)STRICKLAND, J. D. H., PARSONS, T. R. A practical handbook of seawater analysis. 2. ed. Ottawa: Fisheries Research Board of Canada Bull, 1968. 293 p.; and chlorophyll-a and b, following Jeffrey and Humphrey (1975)JEFFREY, S. W., HUMPHREY, G. F. New spectrophotometric equation for determining chlorophyll a, b, c1 and c2. Biochemistry and Molecular Biology of Plant Hormones, v. 167, p. 194-204. 1975.. The fatty acid profiles of the species were determined using gas chromatography, following Menezes et al. (2013)MENEZES, R. S. et al. Avaliação da potencialidade de microalgas dulcícolas como fonte de matéria-prima graxa para a produção de biodiesel. Química Nova, v. 36, n. 1, p. 10-15, 2013., and quantified by normalization of the areas of the methyl esters and expressing them as percentage areas (%).

Statistical analyses

All of the data obtained from the analyses were submitted to statistical treatments using Statistica 7.0 software, at a 5% level of significance. The homoscedasticity of the variances of all of the variables analyzed were confirmed using the Levene test. The differences among the variables analyzed among the species were compared using one-way ANOVA and the Tukey HSD a posteriori test.

RESULTS AND DISCUSSION

Kinetic growth characteristics

The kinetic growth characteristics of the microalgae species investigated here are listed in Table 2. The statistical analyses verified significant differences in terms of their maximum cell density values (MCD) (F = 363,7311; gl = 11; p<0.01; Table 2), final yields (F = 907,6998; gl = 11; p<0.01; Table 2), and biomass productivity (F = 1294,2997; gl = 11; p<0.01; Table 2). The growth velocities (k) did not significantly differ between the different species analyzed (F = 0.7561; gl = 11; p = 0.6802; Table 2).

Table 2
Kinetic characteristics of the growth of the 12 microalgae species investigated

Not all of the species that demonstrated high k values produced large quantities of biomass or rapidly reached their log phase. Similarly, not all of the species with large k values produced the highest cell concentrations (Table 2). Those data indicated that each species responded differently to the culture conditions, and reinforced the importance of determining the unique behaviors of the different strains potentially useful for biotechnological applications under controlled conditions. Significant differences were observed between clones of the same species, such as clones D28Z, D37Z, D65Z and D76Z of Chlorococcum sp. (cf. hypnosporum) (Table 2), which demonstrated wide variations in their kinetic growth characteristics - reinforcing the idea that various factors (possibly genetic) can affect microalgae growth in addition to the physical and/or chemical conditions in the environment.

The values encountered here corroborated data published by other authors in terms of the biomass productivities of various microalgae. The maximum biomass production of 98.73 mg L-1 d-1 for the chlorophyte Chlorococcum sp. D106Z seen here was greater than that reported by Rodolfi et al. (2009)RODOLFI, L. et al. Microalgae for oil: strain, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnology Bioengery, v. 102, p. 100-112, 2009. (21.8 mg L-1 d-1). Those authors also evaluated S. acuminatus, and encountered values greater than those reported here (35.1-53.9 mg L-1 d-1). Chiu et al. (2008)CHIU, S. Y. et al. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresource Biotechnology, v. 99, p. 3389-3396. 2008. cultivated C. minutissima and obtained a biomass productivity of 143 mg L-1 d-1, while Nakanishi et al. (2014)NAKANISHI, A. et al. Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. Bioresource Technology, v. 152, p. 247-252, 2014. reported the productivity of Chlamydomonas sp. as 169.1 mg L-1 d-1, both values greater than those reported here. The data encountered in the literature for different microalgae confirm the concept that different intrinsic and extrinsic factors can alter the kinetics of microalgae growth.

Chemical analyses

Carbohydrate analyses revealed values varying between 14.50 g 100 g-1 of biomass (S. acuminatus D115WC) to 53.81 g 100 g-1 (Chlorococcum sp. D106Z), with expressive values (greater than 25 g 100 g-1) noted for Chlorococcum sp. (cf. hypnosporum) strains D28Z, D37Z, D65Z and D76Z and for the microalga A. carterae M18C (F = 250.4351; gl = 11; p<0.01; Table 3).

Table 3
Carbohydrate, protein, and lipid contents in the biomasses of the 12 species of microalgae investigated

In terms of carbohydrates, Kiran et al. (2015)KIRAN, B. et al. Growth pattern and biofuel production potential of newly isolated microalga, Chlorococcum sp. IM-03 under nitrogen limited conditions. Journal of Chemical Technology and Biotechnology, v. 90, 2015. reported values inferior to those encountered here with Chlorococcum sp. when exposed to different culture media nitrogen concentrations, and they obtained yields of 18 g 100 g-1 in culture medium containing 100 mg of sodium nitrate per liter (equivalent to the nitrogen concentration in the Zarrouk medium used here). The yields obtained in the present work for that microalga were greater, varying from 26.32 to 53.81 g 100 g-1. The carbohydrate contents of microalgae are important not only in terms of producing supplements and rations for animals, but also for the production of biofuels through fermentation (CHEW et al., 2017CHEW, K. W. et al. Microalgae biorefinery: high value products perspectives. Bioresource Technology, v. 229, p. 53-62. 2017.).

The species studied here showed statistically different protein concentrations (F=73,1832; gl=11; p<0.01; Table 3). The species S. acuminatus D115WC (37.73 g 100 g-1) and P. tetras D121WC (35.40 g 100 g-1) had the highest protein concentrations, while P. isothrix D39Z (22.99 g 100 g-1) and Chlorococcum sp. D106Z (24.84 g 100 g-1) had the lowest. Expressive values (greater than 30 g 100 g-1) were encountered in the marine microalga A. carterae M18C and in the freshwater chlorophytes Chlamydomonas sp. D132WC and Chlorococcum sp. (cf. hypnosporum) strains D28Z and D65Z. It is important to note that carbohydrate and protein contents were significantly different between the D28Z and D37Z clones of Chlorococcum sp. (cf. hypnosporum) that had been isolated from the same locality, showing that different lineages of the same species can demonstrate significant differences in their metabolisms.

Lipid analyses demonstrated differences between the species investigated (F = 76,4348; gl = 11; p<0.01, Table 3), with the highest lipid percentages being found in A. carterae M18C and Chlorococcum sp. (cf. hypnosporum) strains D28Z, D37Z, and D65Z. Those results were similar to those described by Mahapatra and Ramachandra (2013)MAHAPATRA, D. M.; RAMACHANDRA, T. V. Algal biofuel: bountiful lipid from Chlorococcum sp. proliferating in municipal wastewater. Current Science, v. 105, n. 1, 2013. for Chlorococcum sp. (30.55 g 100 g-1), by Ho et al. (2014)HO, S-H. et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnology for Biofuels, v. 7, p. 97-113. 2014. for Chlamydomonas sp. (15.3 g 100 g-1), and by Lemahieu et al. (2013)LEMAHIEU, C. et al. Impact of feed supplementation with different omega-3 rich microalgae species on enrichment of eggs of laying hens. Food Chemistry, v. 141, p. 4051-4059. 2013. for Chlorella (14.7 g 100 g-1). It is important to note, however, that culture conditions and the phase of development at harvesting can be manipulated to direct microalgae metabolism to produce certain desired metabolites. Nitrogen depletion, for example, will force microalgae metabolism to diminish protein or peptide concentrations but increase the percentages of energy-rich compounds such as carbohydrates and lipids, or polysaccharides and fatty acids (HO et al., 2014HO, S-H. et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnology for Biofuels, v. 7, p. 97-113. 2014.).

Significant differences (F=146,0379; gl=11; p<0.01; Table 4) were also observed between the 12 species in terms of their productions of carotenoids, with minimum and maximum amounts of those compounds being produced by Chlorococcum sp. D106Z (1.57 mg g-1) and Chlamydomonas sp. D132WC (64.92 mg g-1) respectively.

Table 4
Carotenoid and chlorophyll-a and b contents in the biomasses of the 12 microalgae species investigated

In terms of chlorophyll-a and b production, Chlorococcum sp D106Z demonstrated significantly lower concentrations (3.61 mg g-1 of chlorophyll-a and 1.18 mg g-1 of chlorophyll-b) than Chlamydomonas sp. D132WC (234.74 and 59.34 mg g-1 respectively) (chlorophyll-a: F = 191,8190; gl = 11; p<0.01 and chlorophyll-b: F = 77,0232; gl = 8; p<0.01; Table 4).

According to Bouman et al. (2018)BOUMAN, H. A. et al. Photosynthesis-irradiance parameters of marine phytoplankton: synthesis of a global data set. Earth System Science Data, v. 10, p. 251. 2018., the photosynthetic pigments present in microalgae reflect chromatic adjustments needed to maximize energy capture under distinct solar irradiation conditions. From a human health point of view, carotenoids (including both carotenes and xanthophylls) act as antioxidants, providing protection against oxidative stress (SATHASIVAM; KI, 2018SATHASIVAM, R. K. J. A review of the biological activities of microalgal carotenoids and their potential use in healthcare and cosmetic industries. Marine Drugs, v. 16, p. 26. 2018.). Some xanthophylls, such as violaxanthin, antheraxanthin, zeaxanthin, neoxanthin, and lutein occur both in microalgae and higher plants, although microalgae also produce different types of xanthophylls, such as loroxanthin, astaxanthin, and canthaxanthin (synthesized by green algae), and diatoxanthin, diadinoxanthin, and fucoxanthin (synthesized by brown algae or diatoms) (BARREDO, 2012BARREDO, J. L. Microbial carotenoids from bacteria and microalgae. In: Methods and protocols. New York: Humana Press, 2012. p. 5.). Chlorophylls likewise have beneficial effects for human health due to their anticancer properties and anti-inflammatory and anti-oxidant activities, and can help prevent arteriosclerosis as well as atherothrombotic cardiovascular diseases (PEMMARAJU et al., 2018PEMMARAJU, D. et al. Chlorophyll rich biomolecular fraction of A. cadamba loaded into polymeric nanosystem coupled with photothermal therapy: a synergistic approach for cancer theranostics. International Journal of Biological Macromolecules, v. 110, p. 383-391. 2018.).

Fatty acid compositions

The fatty acid profiles of the microalgae examined here are presented in Tables 5 and 6. Fatty acids with carbon chains varying from C8 to C24, both saturated and unsaturated (with 1 to 6 unsaturated bonds), were identified. The most frequent fatty acids were: myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1, ω-9), linoleic (AL, C18:2 ω-6), α-linolenic (ALA, C18:3 ω-3), and stearidonic (STD, C18:4 ω-3). Among those, palmitic acid stood out as the principal saturated fatty acid (SFA), with the microalgae examined producing that fatty acid at concentrations varying from 11.3% (Chlorococcum sp. (cf. hypnosporum) D65Z) to 38.0% (S. nidulans D112Z) of all fatty acid methyl esters (FAME) produced. The same pattern of palmitic acid predominance was reported by Nakanishi et al. (2014)NAKANISHI, A. et al. Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. Bioresource Technology, v. 152, p. 247-252, 2014. for Chlamydomonas sp., by Ambrozova et al. (2014)AMBROZOVA, J. V. et al. Influence of extractive solvents on lipid and fatty acids content of edible freshwater algal and seaweed products, the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. Molecules, v. 19, p. 2344-2360. 2014. for eight microalgae, and by Campos et al. (2010)CAMPOS, V. B.; BARBARINO, E.; LOURENÇO, S. O.. Crescimento e composição química de dez espécies de microalgas marinhas em cultivos estanques. Ciência Rural, v. 40, n. 2, p. 339-347, 2010. for 10 species of marine microalgae.

Table 5
Fatty acid profiles of the microalgae species cultivated (percentage values in relation to the total fatty acid methyl esters)
Table 6
Fatty acid profiles of the microalgae species cultivated (percentage values in relation to the total fatty acid methyl esters)

The unsaturated fatty acids (USFA) present in all of the microalgae studied were oleic (ω-9), AL (ω-6), and ALA (ω-3), with percentages varying from 2% (A. carterae M18C) to 32.5% (P. tetras D121WC), 0.3% (A. carterae M18C) to 30% (P. isothrix D39Z), and 1.7% (P. isothrix D39Z) to 34% (Chlorococcum sp. (cf. hypnosporum) D28Z) of the total FAME produced respectively. The species that produced the highest levels of oleic acid were Chlorococcum sp. (cf. hypnosporum) strains D37Z and D76Z, C. minutissima D101Z, S. acuminatus D115WC, P. tetras D121WC, and Chlamydomonas sp. D132WC. In terms of AL, the highest levels were observed in P. isothrix D39Z, Chlorococcum sp. (cf. hypnosporum) D65Z, Chlorococcum sp. D106Z, and S. nidulans D112Z. In terms of ALA, the species that demonstrated the highest concentrations were Chlorococcum sp. (cf. hypnosporum) strains D28Z, D37Z, D65Z, and D76Z, C. minutissima D101Z, and Chlorococcum sp. D106Z (Tables 5 and 6). Humans do not synthesize ALA or AL as they do not produce the enzymes Δ-12 and Δ-15 desaturase, and those acids (precursors of ω-3 and ω-6 respectively) must be obtained in our diet (LEE et al., 2016LEE, J. M. et al. Fatty acid desaturases, polyunsaturated fatty acid regulation, and biotechnological advances. Nutrients, v. 8, p. 23. 2016.).

Radmann and Costa (2008)RADMANN, E. M.; COSTA, J. A. V. Conteúdo lipídico e composição de ácidos graxos de microalgas expostas aos gases CO2, SO2 e NO. Química Nova, v. 31, n. 7, p. 1609-1612, 2008. analyzed S. nidulans and reported low percentages of AL (2.71%) and AA (0.49%), and slightly higher percentages of ALA (7.61%). Evaluations of Chlamydomonas sp. cultivated at different nitrate concentrations by Ho et al. (2014)HO, S-H. et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnology for Biofuels, v. 7, p. 97-113. 2014. showed percentages of oleic acid varying from 9.1-26.6%, of AL varying from 21.7-25.3%, and of ALA varying from 14.4-5.4%. Campos, Barbarino and Lourenço (2010)CAMPOS, V. B.; BARBARINO, E.; LOURENÇO, S. O.. Crescimento e composição química de dez espécies de microalgas marinhas em cultivos estanques. Ciência Rural, v. 40, n. 2, p. 339-347, 2010. reported 4.7% AL, 17.5% ALA, and 0.8% EPA in C. minutissima. Mahapatra and Ramachandra (2013)MAHAPATRA, D. M.; RAMACHANDRA, T. V. Algal biofuel: bountiful lipid from Chlorococcum sp. proliferating in municipal wastewater. Current Science, v. 105, n. 1, 2013. reported 1.59%, 14.3% and 7.07% of oleic acid, AL, and ALA in Chlorococcum sp respectively; in that same report, those same fatty acids varied between 5.2-22.5%, 8.6-30%, and 1.7-28.3%, respectively, in all of the strains of Chlorococcum sp.

The microalgae examined in the present work did not demonstrate good ω-6/ω-3 ratios (Table 5 and 6) when compared to the suggested healthy ratio of 4-5:1 (CANDELA; LÓPEZ; KOHEN, 2011CANDELA, C. G.; LÓPEZ, L. M. B.; KOHEN, V. L. Importance of a balanced omega 6/omega 3 ratio for the maintenance of health: nutritional recommendations. Nutrición Hospitalaria, v. 26, n. 2, p. 323-329, 2011.; WARNER et al., 2017WARNER, D. et al. ω3-PUFA supplementation reduced ALT levels In mice exposed to a low linoleic acid diet with or without ethanol in a drinking in the dark binge model. The FASEB Journal, v. 31, n. 1, p. 894.4-894.4, 2017.); even microalgae that demonstrated high concentrations of ω-6 had inadequate ratios, as observed in Chlamydomonas sp. D132WC (1.05), P. isothrix D39Z (28.24), and S. nidulans D112Z (2.44). The same low ω-6/ω-3 ratios were observed by Ryckebosch et al. (2014)RYCKEBOSCH, E. et al. Nutritional evaluation of microalgae oils rich in omega-3 long chain polyunsaturated fatty acid as alternative for fish oil. Food Chemistry, v. 160, p. 393-400. 2014., varying from 0.053 to 2.0 among nine different species examined, although the ratio seen in fish oil is 0.071, indicating that even the current principal source of omegas has a low ratio of those compounds.

PUFA/SFA ratios can be used to rapidly evaluate the fatty acid profiles of the microalgae analyzed. According to Ambrozova et al. (2014)AMBROZOVA, J. V. et al. Influence of extractive solvents on lipid and fatty acids content of edible freshwater algal and seaweed products, the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. Molecules, v. 19, p. 2344-2360. 2014., the larger that value, the greater will be the potential benefits to human health. The highest ratios were observed here in the species C. minutissima D101Z; Chlorococcum sp. (cf. hypnosporum) strains D28Z, D37Z, D65Z and D76Z, Chlorococcum sp. D106Z, and P. isothrix D39Z (Table 5). Ambrozova et al. (2014)AMBROZOVA, J. V. et al. Influence of extractive solvents on lipid and fatty acids content of edible freshwater algal and seaweed products, the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. Molecules, v. 19, p. 2344-2360. 2014. analyzed the PUFA/SFA fatty acid ratios of numerous microalgae and observed variations between 0.46 and 2.13. Campos, Barbarino and Lourenço (2010)CAMPOS, V. B.; BARBARINO, E.; LOURENÇO, S. O.. Crescimento e composição química de dez espécies de microalgas marinhas em cultivos estanques. Ciência Rural, v. 40, n. 2, p. 339-347, 2010. reported a ratio of 0.88 for C. minutissima; Mahapatra and Ramachandra (2013)MAHAPATRA, D. M.; RAMACHANDRA, T. V. Algal biofuel: bountiful lipid from Chlorococcum sp. proliferating in municipal wastewater. Current Science, v. 105, n. 1, 2013. reported a value of 0.32 for Chlorococcum sp. ; Radmann and Costa (2008)RADMANN, E. M.; COSTA, J. A. V. Conteúdo lipídico e composição de ácidos graxos de microalgas expostas aos gases CO2, SO2 e NO. Química Nova, v. 31, n. 7, p. 1609-1612, 2008. reported a high value for S. nidulans (2.66); and Ho et al. (2014)HO, S-H. et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnology for Biofuels, v. 7, p. 97-113. 2014. reported PUFA/SFA ratios between 1.0-1.21 for Chlamydomonas sp.

Based on the results obtained with other species of microalgae, it is evident that differences in fatty acid profiles can be found even within the same species, indicating the existence of intraspecific differences presumably due to the fact that the environments from which they were isolated molded their metabolic natures. The ability of those same microalgae to survive under different and extreme conditions, however, demonstrates their diversity and the unique lipid profiles of those organisms (PALIWAL et al., 2017PALIWAL, C. et al. Abiotic stresses as tools for metabolites in microalgae. Bioresource Technology, v. 244, p. 1216-1226. 2017.).

Higher concentrations of saturated and monounsaturated fatty acids and generally lower percentages of polyunsaturated fatty acids where found in the microalgae examined here (Figure 1); only Chlorococcum sp. (cf. hypnosporum) strains D28Z (75%), D37Z (56.9%), and D76Z (51.8%) and Chlorococcum sp. D106Z (58.9%) showed high concentrations of PUFA.

Figure 1
Percentage composition by degree of saturation of the microalgae species examined.

M18C = Amphidinium carterae M18C, D132WC = Chlamydomonas sp. D132WC, D101Z = Chlorella cf. minutissima D101Z, D28Z = Chlorococcum sp. (cf. hypnosporum) D28Z, D37Z = Chlorococcum sp. (cf. hypnosporum) D37Z, D65Z = Chlorococcum sp. (cf. hypnosporum) D65Z, D76Z = Chlorococcum sp. (cf. hypnosporum) D76Z, D106Z = Chlorococcum sp. D106Z, D121WC = Pediastrum tetras D121WC, D39Z = Planktothrix isothrix D39Z, D115WC = Scenedesmus acuminatus D115WC, D112Z = Synechococcus nidulans D112Z


In terms of the percentages of the omegas ω-3, ω-6, and ω-9 (Figure 2), the microalgae C. minutissima D101Z, Chlorococcum sp. (cf. hypnosporum) strains D28Z, D37Z, D65Z and D76Z, P. tetras D121WC, P. isothrix D39Z, and S. acuminatus D115WC demonstrated more than 50% omegas in the composition of their FAME, with Chlamydomonas sp D132WC showing exactly 50% - indicating a very high potential for utilization in the food industry. Microalgae oils can also be sources of other nutritionally interesting compounds, such as carotenoids, phytosterols, and antioxidants, thus increasing the functionality of microalgae oils as compared to fish oil (RYCHEBOSCH et al., 2014RYCKEBOSCH, E. et al. Nutritional evaluation of microalgae oils rich in omega-3 long chain polyunsaturated fatty acid as alternative for fish oil. Food Chemistry, v. 160, p. 393-400. 2014.) and aggregating value, with carotenoids acting as antioxidants to preserve the relatively unstable PUFA and thus increasing lipidic stability. Fish oil is currently considered the principal source of EFA, although fish have only limited capacities to synthesize PUFA and most of those compounds are bio-accumulated through the food chain (in which microalgae have a principal role) (SAWYER et al., 2016SAWYER, J. M. et al. A general model of polyunsaturated fatty acid (PUFA) uptake, loss and transformation in freshwater fish. Ecological Modelling, v. 323, p. 96-105. 2016.). As such, the possibility of using microalgae to obtain EFA appears quite promising.

Figure 2
Percentage of omega ω-3, ω-6, and ω-9 fatty acids based on the total percentages of fatty acids in the microalgae examined.

M18C = Amphidinium carterae M18C, D132WC = Chlamydomonas sp. D132WC, D101Z = Chlorella cf. minutissima D101Z, D28Z = Chlorococcum sp. (cf. hypnosporum) D28Z, D37Z = Chlorococcum sp. (cf. hypnosporum) D37Z, D65Z = Chlorococcum sp. (cf. hypnosporum) D65Z, D76Z = Chlorococcum sp. (cf. hypnosporum) D76Z, D106Z = Chlorococcum sp. D106Z, D121WC = Pediastrum tetras D121WC, D39Z = Planktothrix isothrix D39Z, D115WC = Scenedesmus acuminatus D115WC, D112Z = Synechococcus nidulans D112Z


CONCLUSIONS

  1. Interspecific variations seen in the growth characteristics and chemical compositions of 12 microalgae indicated that each displayed a different response to standard culture conditions;

  2. The species Chlorella cf. minutissima D101Z, Chlorococcum sp. (cf. hypnosporum) strains D28Z, D37Z, D65Z, and D76Z, Pediastrum tetras D121WC, Planktothrix isothrix D39Z, and Scenedesmus acuminatus D115WC demonstrated elevated percentages of PUFA, including ω-3, ω-6, and ω-9, as well as carotenoids and chlorophylls - nutrients vital to human health - making those species potential alternative sources of metabolites for food industries.

  • 1
    Parte da Tese de Doutorado da primeira autora apresentada ao Programa de Pós-Graduação em Ciência e Tecnologia de Alimentos - Universidade Federal da Paraíba/UFPB

ACKNOWLEDGMENTS

The authors thank the Ministério da Ciência Tecnologia e Inovação (MCTI), Finep (Processo: 2557/09), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Processo: 407519/2013-0) for their financial support; Prof. Dr. Nelson R. Antoniosi Filho of the Laboratório de Métodos de Extração e Separação (LAMES/UFG) for his assistance with the analyses of the fatty acids; and LARBIM/UFPB for the use of their installations and providing the microalgae investigated.

REFERENCES

  • AMBROZOVA, J. V. et al Influence of extractive solvents on lipid and fatty acids content of edible freshwater algal and seaweed products, the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. Molecules, v. 19, p. 2344-2360. 2014.
  • ANDRADE, L. M. et al. Chlorella and Spirulina microalgae as sources of functional foods, nutraceuticals, and food supplements: an overview. MOJ Food Processing and Technology, v. 6, n. 1, 2018.
  • BARREDO, J. L. Microbial carotenoids from bacteria and microalgae. In: Methods and protocols New York: Humana Press, 2012. p. 5.
  • BELLOU, S. et al Microbial oils as food additives: recent approaches for improving microbial oil production and its polyunsaturated fatty acid content. Current Opinion in Biotechnology, v. 37, p. 24-35. 2016.
  • BOUMAN, H. A. et al Photosynthesis-irradiance parameters of marine phytoplankton: synthesis of a global data set. Earth System Science Data, v. 10, p. 251. 2018.
  • CAMPOS, V. B.; BARBARINO, E.; LOURENÇO, S. O.. Crescimento e composição química de dez espécies de microalgas marinhas em cultivos estanques. Ciência Rural, v. 40, n. 2, p. 339-347, 2010.
  • CANDELA, C. G.; LÓPEZ, L. M. B.; KOHEN, V. L. Importance of a balanced omega 6/omega 3 ratio for the maintenance of health: nutritional recommendations. Nutrición Hospitalaria, v. 26, n. 2, p. 323-329, 2011.
  • CHEW, K. W. et al Microalgae biorefinery: high value products perspectives. Bioresource Technology, v. 229, p. 53-62. 2017.
  • CHIU, S. Y. et al Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresource Biotechnology, v. 99, p. 3389-3396. 2008.
  • FOLCH, J.; LEES, M.; STANLEY, G. H. S.. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, v. 226, p. 497-509. 1957.
  • GUILLARD, R. R. L.; LORENZEN, C. J.. Yellow-green algae with chlorophyllide c. Journal of Phycology, v. 8, p. 10-14. 1972.
  • HAYES, M. et al Microalgal proteins for feed, food and health. In: Microalgae-Based Biofuels and Bioproducts, p. 347-368, 2018.
  • HO, S-H. et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnology for Biofuels, v. 7, p. 97-113. 2014.
  • JEFFREY, S. W., HUMPHREY, G. F. New spectrophotometric equation for determining chlorophyll a, b, c1 and c2. Biochemistry and Molecular Biology of Plant Hormones, v. 167, p. 194-204. 1975.
  • KIRAN, B. et al Growth pattern and biofuel production potential of newly isolated microalga, Chlorococcum sp. IM-03 under nitrogen limited conditions. Journal of Chemical Technology and Biotechnology, v. 90, 2015.
  • KOCHERT, G. Carbohydrate determination by the phenol-sulfuric method. In: HELLEMBUST, J. A., CRAIGIE, J. S. (Ed.). Handbook of phycological methods Cambridge: Cambridge University, 1978. p. 95-97.
  • LEE, J. M. et al Fatty acid desaturases, polyunsaturated fatty acid regulation, and biotechnological advances. Nutrients, v. 8, p. 23. 2016.
  • LEMAHIEU, C. et al Impact of feed supplementation with different omega-3 rich microalgae species on enrichment of eggs of laying hens. Food Chemistry, v. 141, p. 4051-4059. 2013.
  • LOWRY, O. H. et al Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, v. 193, n. 1, p. 265-75, 1951.
  • MAHAPATRA, D. M.; RAMACHANDRA, T. V. Algal biofuel: bountiful lipid from Chlorococcum sp. proliferating in municipal wastewater. Current Science, v. 105, n. 1, 2013.
  • MENEZES, R. S. et al Avaliação da potencialidade de microalgas dulcícolas como fonte de matéria-prima graxa para a produção de biodiesel. Química Nova, v. 36, n. 1, p. 10-15, 2013.
  • NAKANISHI, A. et al Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. Bioresource Technology, v. 152, p. 247-252, 2014.
  • PALIWAL, C. et al Abiotic stresses as tools for metabolites in microalgae. Bioresource Technology, v. 244, p. 1216-1226. 2017.
  • PEMMARAJU, D. et al Chlorophyll rich biomolecular fraction of A. cadamba loaded into polymeric nanosystem coupled with photothermal therapy: a synergistic approach for cancer theranostics. International Journal of Biological Macromolecules, v. 110, p. 383-391. 2018.
  • RADMANN, E. M.; COSTA, J. A. V. Conteúdo lipídico e composição de ácidos graxos de microalgas expostas aos gases CO2, SO2 e NO. Química Nova, v. 31, n. 7, p. 1609-1612, 2008.
  • RINCÓN-CERVERA, M. A. et al Supplementation with antioxidant-rich extra virgin olive oil prevents hepatic oxidative stress and reduction of desaturation capacity in mice fed a high-fat diet: effects on fatty acid composition in liver and extrahepatic tissues. Nutrition, v. 32, n. 11, p. 1254-1267, 2016.
  • RODOLFI, L. et al Microalgae for oil: strain, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnology Bioengery, v. 102, p. 100-112, 2009.
  • RUBIO-RODRÍGUEZ, N. et al Production of omega-3 polyunsaturated fatty acid concentrates: a review. Food Science and Emerging Technologies, v. 11, p. 1-12. 2010.
  • RYCKEBOSCH, E. et al Nutritional evaluation of microalgae oils rich in omega-3 long chain polyunsaturated fatty acid as alternative for fish oil. Food Chemistry, v. 160, p. 393-400. 2014.
  • SATHASIVAM, R. K. J. A review of the biological activities of microalgal carotenoids and their potential use in healthcare and cosmetic industries. Marine Drugs, v. 16, p. 26. 2018.
  • SAWYER, J. M. et al A general model of polyunsaturated fatty acid (PUFA) uptake, loss and transformation in freshwater fish. Ecological Modelling, v. 323, p. 96-105. 2016.
  • STEIN, J. R. Handbook of phycological methods: culture methods and growth measurements. In: Handbook of phycological methods. Cambridge University Press; p. 448. 1973.
  • STRICKLAND, J. D. H., PARSONS, T. R. A practical handbook of seawater analysis 2. ed. Ottawa: Fisheries Research Board of Canada Bull, 1968. 293 p.
  • SUBASH-BABU, P.; ALSHATWI, A. A. Effects of increasing ratios of dietary omega-6/omega-3 fatty acids on human monocyte immunomodulation linked with atherosclerosis. Journal of Functional Foods, v. 41, p. 258-267. 2018.
  • WALNE, P. R.. Experiments in the large scale culture of the larvae of Ostrea edulis Journal Fishery Investiment, v. 25, n. 4, p. 1-53, 1966.
  • WARNER, D. et al ω3-PUFA supplementation reduced ALT levels In mice exposed to a low linoleic acid diet with or without ethanol in a drinking in the dark binge model. The FASEB Journal, v. 31, n. 1, p. 894.4-894.4, 2017.
  • ZARROUK, C. Contribution à l'étude d'une cyanophycée: influence de divers facteurs physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima (Setch et Gardner) Geitler. 1966. Theises (Ph. D.) - Faculty of Science, Université des Paris, Paris, 1966.

Publication Dates

  • Publication in this collection
    Jan-Mar 2019

History

  • Received
    22 Feb 2016
  • Accepted
    02 Apr 2018
Universidade Federal do Ceará Av. Mister Hull, 2977 - Bloco 487, Campus do Pici, 60356-000 - Fortaleza - CE - Brasil, Tel.: (55 85) 3366-9702 / 3366-9732, Fax: (55 85) 3366-9417 - Fortaleza - CE - Brazil
E-mail: ccarev@ufc.br