Materials and methods

Collection of samples: Seaweed samples were collected in the coast of Salinas Bay (2°15’0” S & 80°56’0” W), located in the west coast of Santa Elena Province, Ecuador. These algae were taxonomically identified by the biologists Teodoro Cruz Jaime and Raúl Rincones (BIORMA Aquaculture CA) and subjected to a cleaning process with abundant distillated water. Excess water was allowed to drip off under sunlight; then the algae was dried under shade at room temperature for 96 h, and subsequently dried in a stove (Memmert SNB 400 with air flow) at 40 °C for 24 h. Afterwards, the dried seaweeds were pulverized with a grinder (Lab. Mill serial No. 56969, Type AR 400 Erweka®, Germany), hermetically sealed in Ziploc bags and kept at room temperature until further analyses.

Four of the seven species collected, such as Acanthophora spicifera (M. Vahl) Børgesen 1910 (Rhodomelaceae), Centroceras clavulatum (C. Agardh) Montagne 1846 (Ceramiaceae), Hypnea spinella (C. Agardh) Kützing 1847 (Cystocloniaceae) and Kappaphycus alvarezii (Doty) Doty ex P.C.Silva 1996 (Solieriaceae), are red seaweeds (Rhodophyta); two species, Padina pavonica (Linnaeus) Thivy 1960 (Dictyotaceae) and Spatoglossum scroederi (C. Agardh) Kützing 1859 (Dictyotaceae), are brown seaweeds (Ochrophyta); and the last species, Ulva lactuca Linnaeus 1753 (Ulvaceae), is a green seaweed (Chlorophyta) (^{Guiry & Guiry, 2018}). Samples of the different species were deposited in the Pharmacy Pilot Plant of Universidad Técnica de Machala with the records PPFAS022, PPFCC023, PPFHS024, PPFKA025, PPFPP026, PPFSS027 and PPFUL028, respectively.

Proximate analysis: Moisture and ash contents of dried seaweeds were obtained by gravimetric determinations according to ^{World Health Organization (1998)}, using an oven at 105 °C and a furnace at 750 °C, respectively. Both analyses were carried out at Pharmacy Pilot Plant, Universidad Técnica de Machala, Ecuador. Crude lipid, fiber and protein contents were determined using the methodology reported by ^{Chen, Liu, Zhang, Chen, & Wang (2012}), the NTE INEN-ISO 6865 protocol (^{INEN, 2014}), and Kjeldahl (^{Avilés, 2002}), respectively. These last analyses were performed at “Centro de Investigaciones Biotecnológicas del Ecuador”, Escuela Superior Politécnica del Litoral. Carbohydrates content was calculated by difference as:

% _Carbohydrate = 100 - (% _Moisture + % _Ash + % _Fat + % _Fiber + % _Protein) (Eq. 1)

While the caloric energy of seaweeds was calculated as:

Energy = (9 kcal g^-1 × % _Fat) + (4 kcal g^-1 × % _Protein) + (4 kcal g^-1 × % _{Carbohydrates}) (Eq. 2)

All data was expressed in terms of mean ± standard deviation. To calculate mean percentage and standard deviation, Statistical Package for Social Science software (SPSS) Version 23.0 for windows (^{IBM Corp. Released}) was used. To determine whether there were any differences amongst the means, one way Analysis of Variance (ANOVA) and Duncan’s multiple range tests were applied to the results and P values < 0.05 were considered significant. Test of normality was previously applied to the dates, which showed being approximately normal.

Results

Table 1 shows the proximate composition of the dried seaweeds collected from Santa Elena, Ecuador. The water content of the seaweeds ranged from 90.74 to 95.97 %, which indicates that the dry weight was lower than 10 % of the fresh organisms. A higher water content implies a lower dry biomass. The lowest water content was found for Acanthophora spicifera (90.74 %), followed by Hynea spinella (90.89 %). While the highest content of water was found for Kappaphycus alvarezii (95.97 %), followed by Ulva lactuca (92.31 %).

The brown seaweed Spatoglossum schroederi showed the lowest value of moisture (12.84 %) after drying, while the rest of the organisms showed around 20 % of moisture (18.61-23.54 %). Ash content ranged from 25.49 to 36.69 %, being the brown seaweed Padina pavonica and the red seaweed Centroceras clavulatum the ones which showed the lowest and the highest values, respectively. Fat content oscillated between 0.33 and 3.06 %, being U. lactuca and S. schroederi the species that exhibited the lowest and highest content of fat, respectively. The highest protein content (8.02 %) was found in the red seaweed H. spinella, and the lowest value (4.78 %) was found in the red seaweed C. clavulatum, while the rest of the organisms showed protein contents that oscillated around 4.86-5.54 %. Fiber content ranged between 0.96 % and 4.94 %. P. pavonica was found to have the highest value, followed by H. spinella, and S. schroederi, while U. lactuca showed the lowest.

Carbohydrate contents were between 32.24 and 45.52 %, C. clavulatum and U. lactuca showed the lowest and the highest values, respectively. Finally, the caloric energy of seaweeds ranged from 1 547 kcal kg^-1 (for C. clavulatum) to 2 085 kcal kg^-1 (for S. schroederi). According to ANOVA (Table 2), proximate compositions were significantly different among the species (P < 0.05) with some exceptions. Duncan’s multiple range tests indicated five, six or seven homogeneous sub-conjuncts among different parameters.

Discussion

Fresh seaweeds naturally contain 80 to 90 % of water (^{Fontaine & Bonilla, 1978}), while Ecuadorian fresh seaweeds contained more than 90 % (Table 1). These values are similar or slightly higher than those reported in the literature for other seaweeds (^{Hussain et al., 2009}; ^{Ahmad, Sulaiman, Saimon, Fook, & Matanjun, 2012}; ^{Sivaramakrishnan et al., 2017}). Furthermore, after drying, the seaweeds still had relatively high residual moisture content, approximately 20 % (on a dry weight basis), which indicates that the method used to dry these organisms was possibly not optimal. Other authors have reported minor residual moisture in proximate analysis of seaweeds using methods such as oven drying at 60 °C or freeze-dry (^{Rohani-Ghadikolaei, Abdulalian, & Ng, 2012}; ^{Rodrigues et al., 2015}; ^{Bhuiyan et al., 2016}). Removal of water from seaweeds is a necessary step in maintaining their quality as a food, since it would impede the chemical and biochemical reactions of degradation and delays the growth of microorganisms that can deteriorate them (^{Coenders, 1996}).

On the other hand, ^{Fontaine & Bonilla (1978}) have indicated that seaweeds can contain around 40 % of carbohydrates, from 1 to 3 % of lipids, and from 3 to 38 % of minerals (ash). Results of the proximate composition of Ecuadorian seaweeds (Table 1) are consistent with that information; for instance, carbohydrates were the parameter found in highest percentage (30-45 %), followed by ash content (25-37 %). However, the specie C. clavulatum showed a higher content of ash (36.69 %) followed by carbohydrates (32.24 %). Possibly, this seaweed requires a mayor content of minerals for structural and ecological interactions, while its lower carbohydrate content could be due to the fact that it was in a different stage of growth respect to the other species (^{Marinho-Soriano, Fonseca, Carneiro, & Moreira, 2006}).

Other studies have reported high content of carbohydrate and/or ash in seaweeds (^{Nguyen, Ueng, & Tsai, 2011}; ^{Rohani-Ghadikolaei et al., 2012}; ^{Gokulakrishnan, Raja, Sattanathan, & Subramanian, 2015}; ^{Bhuiyan et al., 2016}; ^{Sivaramakrishnan et al., 2017}), which suggests these two parameters are important in the composition and biochemical functions of the seaweeds. Moreover, the high content of carbohydrates suggests these organisms could be an important source of phycocolloids for industrial uses. In terms of fat content, the values found in five of these organisms were relatively very low (< 1 %), indicating that these seaweeds are an ideal food choice for people that require a low-fat or fat-free diet. Lastly, protein and fiber contents were moderately low compared to known vegetables and other macroalgae (^{Rodrigues et al., 2015}; Bhuiyan et al., 2016; Sivaramakrishnan et al., 2017).

Although the proximate compositions of the seaweeds were very similar, statistical analysis indicated significant differences among the species. Each Ecuadorian macroalgae exhibited a slightly different proximate composition to that reported in other studies. For instance, A. spicifera showed minor ash, protein and lipid contents, and higher carbohydrate content than the values reported by ^{Mohammadi, Tajik, & Hajeb (2013}). A similar case was found for C. clavulatum that showed lower lipid and protein contents, and higher carbohydrate content than the values indicated by ^{Diniz, Barbarino, & Lourenço (2012}).

Fat, protein and ash contents of H. spinella were lower than those reported by ^{Viera et al. (2005}), while carbohydrate content was higher. The proximate composition found for K. alvarezii in this study was similar to the results found by ^{Abirami & Kowsalya (2011}), although different to that reported by ^{Kumar, Ganesan, & Subba (2015}), who demonstrated the seasonal changes in nutritional composition of this species in other region. ^{Manivannan et al. (2009}) and ^{Tabarsa, Rezaei, Ramezanpour, Waaland, & Rabiei (2012}) reported values of protein, fiber, ash and lipid for P. pavonicahigher than the values found in this research. The content of fat and ash found in S. schroederi were higher than values reported for other species belongs to Spatoglossum genus (^{Mwalugha et al., 2015}), while fiber and protein contents found in this study were lower. Ash content found for Ecuadorian U. lactuca was higher than that reported by Abirami & Kowsalya (2011) and Mwalugha et al. (2015); while, the other nutritional parameters, such as carbohydrate, protein, fiber and fat contents, were found in minor proportion in this study. The results obtained confirm the relationship between the chemical composition of the seaweeds and differences in the geographical area, environmental conditions and seasons where they grow, among other aspects.

Caloric profiles of the seaweeds (energy offered by macromolecules such as carbohydrates, fat and protein) were similar to those reported by ^{Admassu, Abera, Abraha, Yang, & Zhao (2018}) for the commercial edible seaweed Porphyra spp.; in the case of Ecuadorian seaweeds, the major caloric contribution is due to the carbohydrate content. In conclusion, seaweeds from the coast of Ecuador have comparable nutritional value to that of terrestrial vegetables and other seaweeds around the world. These showed a low content of lipids and an important fraction of fiber, protein and carbohydrates, reason for which they could be considered a healthy food with low caloric profiles. In this sense, farming of seaweeds might become an economic option for people from this region, contributing not only with a new nutritive food, but also improving the marine ecosystems.

Ethical statement: authors declare that they all agree with this publication and made significant contributions; that there is no conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements. A signed document has been filed in the journal archives