Macroalgae as an Alternative Source of Nutrients and Compounds with Bioactive Potential

: The consumption of macroalgae has increased in occidental countries, favored by the ex-cellent nutritional properties of their food products and the bioactive properties attributed to them. The present work aims to analyze the nutritional values, the composition in fatty acids, organic acids and carotenoids of several macroalgae species: Codium tomentosum ; Himanthalia elongata ; Laminaria ochroleuca .; Saccharina latissima ; Undaria pinnatifida ; Porphyra sp. and Palmaria palmata. Further-more, the antioxidant and antimicrobial properties were assessed. Regarding the composition of the macroalgae, the levels of proteins (which ranged between 6 and 30 g/100 g of dry weight (DW)) and the low levels of lipids (below 1 g/100 g DW for all the species) stand out. In the case of carotenoids, lycopene and chlorophyll a were detected in all samples. Regarding antioxidant activity, OxHLIA assay was employed. EC50 values varied between 1.7 and 650 µg/mL for L. ochroleuca and P. palmata . H. elongata presented the greatest antibacterial potential (0.5–2 mg/mL) while L. ochroleuca showed the best antifungal effects (2–4 mg/mL). These species have good nutritional values and present interesting bioactivities. Thus, the incorporation of this macroalgae into the daily diet could provide nutritional and health benefits to the consumers. In addition, they could be used as a source of compounds for the nutraceutical, cosmetic and pharmaceutical industries.


Introduction
Macroalgae have been consumed by humans since ancient times, principally in the oriental countries, such as China, Japan or Indonesia. However, in the last decades, the consumption of edible algae in western countries has been increasing exponentially, mainly due to the current consumer preference and demand for organic products with high nutritional values and health benefits [1,2]. In general, dried macroalgae are foods with a low-calorie content, being rich in non-digestible polysaccharides, proteins and essential amino acids, vitamins, interesting minerals (such as sodium, chlorine, potassium and iodine) and phenolic compounds. Despite the low lipid content, macroalgae are rich in polyunsaturated fatty acids [2][3][4].
According to the literature, macroalgae are described as a source of bioactive compounds, with nutritional and pharmacological benefits on human health, such as dietary fibers, carotenoids or phenolic compounds [4][5][6]. However, the chemical and nutritional  properties of the seaweed product may differ depending on different factors, such as species, the harvest conditions (season and environmental factors), geographical region and the manufacturing process of the product [1][2][3].

Sample Preparation
Commercial samples of the abovementioned macroalgae were provided by the Algas Atlánticas Algamar S.L. (based in Pontevedra, Spain) company. The macroalgae were recollected in the natural environment in Pontevedra province coasts (Galicia, Spain) and washed with distilled water in a mechanical roller system. Then, the samples were dried in a force air stove (Pazos de Borbén, Pontevedra, Spain) for 48 h at 40 °C, and finally, were reduced to a fine dried powder (~20 mesh), mixed to obtain a homogeneous sample and package-protected from light, until further analysis.

Nutritional Characterization of Macroalgae
The contents of protein, fat, carbohydrates and ash, were determined in the seven commercial macroalgae species according the AOAC methods [7] and following a protocol previously reported by [8]. The total carbohydrates were calculated by difference and the energic value was calculated using the equation: Energy (kcal) = 4 × (g protein + g carbohydrates) + 9 × (g fat).

Chemical Composition of Macroalgae
The fatty acids were evaluated according to a methodology previously described by [8]. The determination was performed through a gas chromatography coupled with a flame ionization detector (GC-FID, DANI model GC 1000, Contone, Switzerland) and were identified by comparing the relative retention times of FAME peaks from samples with commercial standards (FAME reference standard mixture, standard 47885-U, Sigma-Aldrich, St. Louis, MO, USA).
The content of organic acids of seven macroalgae samples was determined following a methodology previously described by [8], using an Ultra-Fast Liquid Chromatography (UFLC, Shimadzu 20A series, Kyoto, Japan) and a photodiode array detector. The quantification of compounds was made using calibration curves obtained from commercial standards.
The concentration of carotenoids were determined used a method previously described by [9].

Preparation of Extracts
The studied extracts, obtained from dried macroalgae, were prepared through a maceration, adding 50 mL of ethanol/water (80:20 v/v) to 1 g of dried sample. The mixture was left under stirring at room temperature for 1 h and then filtered. The residue was re-extracted with additional 50 mL of the same solution, under the same conditions. Both extracts were evaporated at 40 °C in a rotary evaporator (Buзchi R-210, Germany) to remove the alcoholic fraction. Finally, the aqueous phase was frozen and lyophilized (FreeZone 4.5, Labconco, Kansas City, MO, USA) to obtain a dry extract [10].

Evaluation of Antioxidant Activity
For the evaluation of antioxidant activity, dried extracts were re-dissolved (100 mg/mL) in ethanol/water (80:20 v/v) solution and successively diluted to determine their respectively EC50 values. The oxidative hemolysis inhibition assay (OxHLIA) was carried out using sheep blood samples, as previously described by [11].

Evaluation of Antimicrobial Activity
The dried extracts obtained from macroalgae were dissolved in water (10 mg/mL) and the antibacterial potential was evaluated applying a methodology previously described by [12]. In this assay, three Gram-negative bacteria strains: Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 13311) and Enterobacter cloacae (ATCC 35030) and three Gram-positive bacteria strains: Staphylococcus aureus (ATCC 6538), Bacillus cereus (clinical isolate) and Micrococcus flavus (ATCC 10240), were used. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined, using streptomycin and ampicillin as positive controls.

Nutritional Characterization
The nutritional composition of the seven dried macroalgae samples (Porphyra sp., P. palmata, L. ochroleuca, S. latissima, H. elongata, U. pinnatifida and C. tomentosum) is showed in Table 1. In general, the macronutrient present in the highest concentration were carbohydrates, ranging in the values between 76.6 ± 0.3 and 43.9 ± 0.8 g/100 g DW for S. latissima and L. ochroleuca, respectively. In contrast, the evaluation of lipid content showed the lowest concentrations in all studied species, with evidenced values oscillating between 0.29 ± 0.01 and 3.12 ± 0.13 g/100 g DW in P. palmata and C. tomentosum, respectively. The protein content showed great heterogeneity between the algae species, with higher concentrations in Porphyra sp (30.2 ± 0.1 g/100 g DW) and lower concentrations in S. latissima (6.7 ± 0.1 g/100 g DW). The amount of ash showed concentrations between 7.8 ± 0.04 and 46.1 ± 1.3 g/100 g DW for Porphyra sp. and L. ochroleuca, respectively. For the energic value, amounts between 218.5 ± 3.8 (for L. ochroleuca) and 370.9 ± 0.1 Kcal/100 g DW (for Porphyra sp.) were obtained.
In general, the nutritional results obtained in the present study are similar to those reported previously [2,3,[14][15][16][17]. Red macroalgae had the highest protein content, followed by green and finally, brown algae, except U. pinnatifida, which has been reported to have intermediate protein levels [2]. In the present study, the protein content of U. pinnatifida was lower than the expected. Regarding lipid content, most of the studies agreed with the results obtained, demonstrating the low lipid content of the algae. Ash content and carbohydrate content agree with the mentioned studies, except for the carbohydrate content of S. latissima, which was higher than previously reported. The differences between studies could be associated to diverse factors that affect macroalgae composition, such as the region, season or the environmental factors during the harvest [1,3].
Regarding the organic acids, oxalic, malic and citric acids were identified. However, only oxalic acid was present in all the species with values between 0.2 g/100 g (DW) and 4.3 g/100 g (DW) for U. pinnatifida and L. ochroleuca, respectively. Malic acid was present in H. elongata and Porphyra sp., with values of 0.42 g/100 g (DW) and 3.66 g/100 g (DW), respectively. Finally, citric acid was found in H. elongata (1.57 g/100 g (DW)), U. pinnatifida (0.83 g/100 g (DW)) and Porphyra sp. (5.47 g/100 g (DW)). The total concentration of organic acids of the macroalgae fluctuate between 0.59 g/100 g (DW) for U. lactuca and 10.61 g/100 g (DW) for Porphyra sp. According to the research in the literature, there are no other studies that report the organic acid content in the macroalgae selected in this study, except for C. tomentosum [19] and Porphyra sp [20].
The β-carotene, lycopene and chlorophyll a and b content were evaluated in the seven macroalgae. β-carotene was only detected in the brown macroalgae U. pinnatifida, with a value of 0.78 mg/100 g DW. In the case of lycopene, the compound was detected in all the samples, ranging between 11.2 and 0.32 mg/100 g DW for C. tomentosum and Porphyra sp., respectively. Finally, C. tomentosum showed the highest content of chlorophyll a and b (56.3 and 47 mg/100 g DW), while P. palmata had the lowest (1.5 and 0.58 mg/100 g DW). Chlorophyll b was not detected in U. pinnatifida. Several studies have evaluated the content of β-carotene, chlorophyll a and b content of the selected macroalgae [21][22][23][24]. The differences observed with previous studies may be attributed to the fact that, like other previous parameters, the pigment content varies throughout the year, depending on environmental factors, such as light, salinity and temperature. Other factors may have affected, such as the extraction solvent [23]. Finally, although it has been described that some macroalgae may contain lycopene [23], to our knowledge, there are no studies that have reported the presence of lycopene in the selected species.

Bioactive Potential
The EC50 values ranged between 1.7 and 650 µg/mL for L. ochroleuca and P. palmata, respectively. S. latissima and U. pinnatifida did not show antioxidant effects, as no reduction of the hemolysis was observed. As it could be observed, C. tomentosum, H. elongata and L. ochroleuca displayed great antioxidant activity, presenting a EC50 much lower that the antioxidant control (46 µg/mL). To our knowledge, no previous studies have employed OxHLIA to evaluate the antioxidant activity of the seven selected macroalgae.
Regarding antibacterial activity, the tested extracts of all macroalgae showed antibacterial activity with inhibitory and bactericidal potential against several studied strains. The MBC values ranged between 2 and 4 mg/mL for C. tomentosum, 1 and 4 mg/mL for H. elongata, 2 and >8 mg/mL for L. ochroleuca, 1 and 8 mg/mL for S. latissima, 2 and 8 mg/mL for U. pinnatifida and Porphyra sp. and 2 and >8 mg/mL for P. palmata. Finally, antifungal results showed that MFC values ranged between 4 and 8 mg/mL for C. tomentosum, H. elongata, U. pinnatifida, Porphyra sp. and P. palmata, while L. ochroleuca and S. latissima showed values ranging between 2 and 4 mg/mL and 1 and 8 mg/mL, respectively. The antimicrobial properties of C. tomentosum [25], H. elongata, S. latissima and P. palmata [26], and L. ochroleuca [27] were consistent with previous reports. To our knowledge, no studies have evaluated previously the antibacterial and antifungal properties of U. pinnatifida and Porphyra sp. extracts.