Nutrient Composition of Dried Seaweed Gracilaria gracilis

The nutrient composition of dried red seaweed Gracilaria gracilis collected from Barru waters, South Sulawesi including proximate, dietary fiber, minerals, fatty acid and amino acid profile has been investigated. The objective of this study was to evaluate the various nutritional parameters of G. gracilis for utilization in human nutrition. Results show that the content of moisture (19.045), protein (10.86%), ash (6.78%), fat (0.18%), carbohydrate (63.13%) and dietary fiber (27.48%) basis on the dry weight. The content of calcium (429.11 mg.100 g-1), sodium (290.89 mg.100 g-1), phosphor (57.01 mg.100 g-1), iron (15.20 mg.100 g-1) and potassium (1380.42 mg.100 g-1). Leucine was the major essential amino acid found to be 9374.22 mg.kg-1, while glutamic acid was the major nonessential amino acid found to be 10848.98 mg.kg-1. Palmitic acid was the major saturated fatty acid found to be 0.08%, while oleic acid was the major unsaturated fatty acid found to be 0.05%. The nutrient composition of G. gracilis was discussed in this study and suggested that the seaweed species have potentially be used as raw material or ingredient of a healthy food for human.


Introduction
Seaweeds have been utilized globally for different purposes (Nazni and Deepa, 2015). Currently, seaweeds are consumed as part of modern diet in the western countries. Changing of food patterns increase in Asia-style food and people become more comfortable consuming edible seaweeds, particularly Porphyra and Undaria spp. that are commonly found in Korea and Japanese dishes (Smith et al., 2010). Especially in China, Gracilaria originally were utilized as food and as binding material in the preparation of lime for painting walls. The use of seaweed as food spreads to several Asian countries, until the content of agar was discovered by the western countries and the Japanese (Santelices, 2014).
Seaweeds (fresh or dried form) are extensively consumed, especially by people living in the coastal region. Seaweeds are generally suitable for making cool, concoctions or gelatinous dishes. The nutrient composition of seaweeds varies and are affected by geographical area, species, temperature, of water and season of the year (Jensen, 1993).
However, there are no published data on the nutrient composition of the dried red seaweed G. gracilis from Barru waters, South Sulawesi. This paper presents data on the various nutrient composition of G. gracilis, including proximate, dietary fiber, minerals, fatty acid and amino acid profile. The potential of G. gracilis as a source of healthy food nutrients is discussed.

Materials and Methods
The red seaweed G. gracilis was collected from Barru waters, South Sulawesi during low tide. The seaweed was picked by hand and cleaned immediately using sea water to remove debris, sand, epiphytes and other unnecessary matter and transported to the laboratory. The sample was sorted and thoroughly cleaned by rinsing distilled water. The sample was dried under the sun for 6 days and then ground in a blender. The powdered samples were kept in the dark container and stored in the room temperature for future analysis.

Proximate analysis
The moisture content was determined by drying 2 g G. gracilis in an oven at 105 o C for 3 hours. The dried sample was put into a desiccator and weighed (AOAC, 1990). The ash content was determined by heating 2 G. gracilis in a muffle furnace at 550 o C for 4 hours. The sample was put into a desiccator and weighed immediately (AOAC, 1990).
The fat content was determined by loosely wrapping 2 g G. gracilis with a filter paper and then put into the thimble which was fitted to a clean round bottom flask containing 120 ml of petroleum ether. The sample was heated and allowed to a reflux for 5 hours. The thimble with the spent sample was kept and weighed (AOAC, 2000). The protein content was determined by calculating from the elemental N using the nitrogen-protein factor of 6.25 (AOAC, 2000). The carbohydrate content was determined by difference: 100 -(moisture + ash + protein + fat) %. The dietary fiber content was determined by putting 2 G. gracilis and 1 g asbestos into 200 ml H2SO4 1.25% and boiled for 30 min. The solution was filtered by Buchner funnel and the residue was put into 200 ml boiled NaOH for 30 min and then filtered. The residue was washed twice with alcohol and continued with petroleum ether. The residue was put in a clean crucible and dried in an oven and weighed (AOAC, 1990).

Mineral analysis
Mineral (calcium, sodium, iron, and potassium) content was determined by the standard AOAC method (2000). While the phosphor content was determined by the spectrophotometric method.
Sample preparation for fat extraction (AOAC, 2000): A 5 g G. gracilis was added 4 ml isopropanol and 6 ml n-hexane. The solution was centrifuged for 3 minutes at 9000 RPM. The clear upper solution was moved into a Hach tube and was dried in a water bath. About 0.03-0.04 g fat extract was added 1.5 ml KOH methanol 0.5M and 1.5 ml BF3 20% in methanol. The solution was heated in a water bath at 100 o C for 20 min. A 3 ml saturated NaCl and 0.2ml n-hexane was added to the mixture and then vortexed for 2 min. The mixture was allowed to stand at room temperature for 10 min. The n-hexane methyl ester layer was transferred into 10 ml volumetric flask, diluted with n-hexane and injected to gas chromatography.
Sample preparation: A 0.1 g G. gracilis was added 5 ml HCl 6N. The mixture was hydrolyzed for 22 h at 110 o C. The hydrolyzed mixture was transferred into a 50 ml volumetric flask and diluted to volume with aquadest. The solution was filtered with a 0.45 µm filter. A 500 µl of the filtrate was added 40 µl AABA and 460 µl aquabidest. A 10 µl of the solution was added 70 µl AccQ Fluor Borate and 20 µl reagent fluor A. The solution was incubated for 10 min at 55 o C and then transferred into the UPLC system.
Standard solution preparation: A 40 µl standard solution was mixed with amino acid. A 40 µl internal standard AABA and 920 µl aquabidest was added and then homogenized. A 20 µl standard solution was pipetted and 70 µl AccQ Fluor Borate was added. A 20 µl reagent fluor A was added and then vortexed for 1 min. The solution was incubated for 10 minutes at 55 o C and injected into the UPLC system.

Results and Discussion
Nutrient composition of seaweeds was different and affected by geographical area, species and environmental growth condition (Benjama and Mashiyom, 2012). Metabolic activity of seaweeds is the fundamental one, but it is controlled by temperature and concentration of essential nutrients of the surrounding waters (Nazni and Deepa, 2015 Gracilaria sp. was 25% dry weight. This result was higher than the other species of Gracilaria reported in the previous study such as G. verrucosa (10.17%) (Nazni and Deepa, 2015) and G. cervicornis (14.33%) (Marinho-Sorano et al., 2007).
The moisture content is an important criterion in determining the quality and shelf-life of processed seaweed meals where high moisture may hasten the growth of microorganisms. In addition, by drying and storage of seaweeds are likely to affect the moisture content of the seaweed examined (Rohani-Ghadikolael et al., 2012).
Carbohydrate was the major component of the proximate composition in G. gracilis examined in this study. The carbohydrate content was 63.13%. This result was higher than other species of Gracilaria such as reported by Nazni and Deepa (2015) for G. verrucosa was 33.67%, Robledo and Freile-Pelegrin (1997) for G. cornea was 36.29% and Marinho-Soriano et al. (2006) for G. cervicornis was 63.12%. Carbohydrate is also the most important component of metabolism, mainly in supplies the energy needed for respiration and other metabolic processes (Khairy and El-Sharay, 2013).
The mineral composition of G. gracilis examined in this study was shown in Table 2. Potassium was the major component in G. gracilis. Potassium content was 1380.42 mg.100g -1 . The content of other mineral examined in this study, including calcium (429.11 mg.100g -1 ), sodium (290.89 mg.100g -1 ), iron (15.20 mg.100g -1 ) and phosphor (57.01 mg.100g -1 ). Rohani-Ghadikolaei et al. (2012) reported that the concentration and composition of mineral in seaweeds are affected by location and species where seaweeds are able to selectively absorb minerals from the surrounding seawater and accumulated them in their thalli.
The fatty acid profile was shown in Table 3. The total percentage of identifying saturated fatty acids were 0.12% and unsaturated fatty acids were 0.07%. For individual fatty acids, palmitic acid (C 16:0) was the major saturated fatty acids while lauric acid (C 12:0) and myristic acid (C 14:0) were the same value.

Conclusion
Based on the nutritional composition of Gracilaria gracilis it is suggested that this seaweed species can potentially be used as a raw material or healthy food ingredient for the human diet.