Metabolites of interest for food technology produced by microalgae from the Northeast Brazil 1 Metabólitos de interesse à tecnologia de alimentos produzidos por microalgas do Nordeste do Brasil

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.


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., 2018;HAYES et al., 2018;SATHASIVAM;KI, 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., 2016;HO et al., 2014) -both precursors in the human body to long chain (≥ C20) polyunsaturated fatty acids (PUFA) (RINCÓN-CERVERA et al., 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., 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 proinflammatory 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., 2010;SUBASH-BABU;ALSHATWI, 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, 2011;WARNER et al., 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.

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).
The species were cultivated in triplicate in flat bottomed 6 L flasks containing 5 L of Conway media (WALNE, 1966) for the cultivation of the marine microalga, or 5 L of Zarrouk medium (ZARROUK, 1966) for cultivating Chamydomonas, Pediastrum, and Scenedesmus, or WC medium (GUILLARD;LORENZEN, 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 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 o C), 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); carbohydrates, following Kochert (1978); total lipids, following Folch, Lees and Stanley (1957); total carotenoids, following Strickland and Parsons (1968); and chlorophyll-a and b, following Jeffrey and Humphrey (1975).The fatty acid profiles of the species were determined using gas chromatography, following Menezes et al. (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.

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).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 characteristicsreinforcing 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) (21.8 mg L -1 d -1 ).Those authors also evaluated S. acuminatus, and encountered values greater than those reported here (35.1-53.9mg L -1 d -1 ).Chiu et al. (2008) cultivated C. minutissima and obtained a biomass productivity of 143 mg L -1 d -1 , while Nakanishi et al. (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.
In terms of carbohydrates, Kiran et al. (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., 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) for Chlorococcum sp.(30.55 g 100 g -1 ), by Ho et al. (2014) for Chlamydomonas sp.(15.3 g 100 g -1 ), and by Lemahieu et al. (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., 2014).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 (1.57mg g -1 ) and Chlamydomonas sp.D132WC (64.92 mg g -1 ) respectively.
According to Bouman et al. (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, 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, 2012).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., 2018).
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, 2011;WARNER et al., 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), 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), 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.

Species
Carotenoid (mg g -1 ) Chlorophyll-a (mg g -1 ) Chlorophyll-b (mg g   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., 2017).
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., 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., 2016).As such, the possibility of using microalgae to obtain EFA appears quite promising.

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

Figure 2 -
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

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

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

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

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