Biochemical Composition of Some Seaweed From Hurghada Coastal Along Red Sea Coastal, Egypt

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1 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:14 No:01 29 Biochemical Composition of Some Seaweed From Hurghada Coastal Along Red Sea Coastal, Egypt Shimaa M. EL-Shafay Botany Department, Faculty of science, Tanta University, Egypt 1- Corresponding Author: Dr. Shimaa Mohamed El-Shafay Current address: Botany Department, Faculty of science, Tanta University, Egypt Contacts: Phone: , Abstract-- Variations in protein, carbohydrate, lipid, ash, moisture, fatty acids, amino acids, β-carotene and total phenolic compounds content of the seaweeds Ceramium rubrum, Sargassum vulgare, Sargassum fusiforme and Padina pavonia were studied. C. rubrum characterized by the highest protein content, while the highest lipid and carbohydrates contain was observed in S. vulgare. The highest concentration of essential amino acid and β-carotene were observed in P. pavonia. S. fusiforme characterized by high concentration of total phenolic compound and total unsaturated fatty acids. Index Term-- Seaweeds, Protein, Carbohydrates, Lipid, Fatty acid, β-carotene, Total phenol 1.0 INTRODUCTION Seaweeds have been used since ancient times food, fodder and fertilizer and as source of medicinal drugs. Seaweeds are one of the commercially important marine living and renewable resources. Variability in chemical components and growth of algae may be inter specific, intra-annual or inter-annual. Certain seaweeds contain significant quantities of protein, lipids, minerals and vitamins, w h i l e although nutrient contents vary with species, geographical location, season and temperature (Haroon 2000). Protein, carbohydrates and Lipids, are the most important biochemical components of algae. A few studies were done on fatty acids of microalgae and seaweeds. Macroalgal biomass can store large amounts of oil which can be exploited for the production of biodiesel John and Anisha (2011). Seaweeds belonging to the Rhodophyta possess high levels of proteins (10 30% DW) (Darcy-Vrillon 1993). In some red seaweed, such as Palmaria and Porphyra tenera, the protein contents are 35 and 47% DW, respectively. These levels are even comparable to that of the soybeans (35% DW). Some evidences suggest that fatty acids and sterol composition may be useful for taxonomic purposes (Herbreteau et al. 1997). Rhodophyta are characterized by high content of C 20 polyunsaturated fatty acids, mainly arachidonic and eicosapentaenoic acids. Other abundant fatty acids in this class are palmitic and oleic acids. The amino a c i d s composition in free or bound form has been studied in several species of marine algae (Qasim 1991), whereas pronounced differences were observed in protein and amino acids between different algal groups. Carotenoids are an important group of natural pigment with specific applications as colourants, feed supplements, nutraceuticals, and medicine, cosmetic and biotechnological purposes. Despite the avail ability of a variety of natural and synthetic carotenoids, only a few have been exploited commercially, including b -carotene, lycopene, astaxanthin, canthaxanthin, lutein and capxanthin (Bhosale 2004). The β- carotene accumulation and the rate of synthesis depend on certain environmental parameters (Shariati and Hadi 2011). Antioxidants in particular carotenoids, help to prevent the free radicals damage associated with the aging process itself. The review by the National research council (NRC 1982) concluded the epidemiological evidences sufficient to suggest that food rich in carotenes or vitamin A are associated with a reduced risk of cancer. Thus foods rich in carotenoids in this case β-carotene may not only be able to prevent but also reverse cancers. Phenolic compound are one of the most abundant class of phytochemicals present in algae. Among natural antioxidants, phenolic antioxidants are commonly found in plants, including seaweeds. Presence of phenolic compounds in any medicinal preparation may attribute to various medicinal properties, earlier many algae have been reported to possess a variety of phenolic compounds with antioxidant and hepatoprotective properties (Song el al. 2003). Phenolic compounds have been reported to have several biological effects including antioxidant,antiapoptosis, anti-aging, anti carcinogen and have been highly considered for their important dietary roles as antioxidant and chemoprotective agents. The aim of this work is to investigate the biochemical composition (in protein, carbohydrate, lipid, ash, moisture, fatty acids, amino acids, β-carotene and total phenolic compounds) in Ceramium rubrum, Sargassum vulgare, Sargassum fusiforme and Padina pavonia

2 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:14 No: MATERIAL AND METHODS 2.1. Sampling Four marine algal species were collected during March 2013 these collected species including the one species from Rhodophyta (Ceramium rubrum) and three species from Phyophyta (Sargassum vulgare, Sargassum fusiforme and Padina pavonia). The algae were collected from Hurghada coastal along the Red sea coastal, Egypt. All samples were brought to laboratory in plastic bags containing sea water to prevent evaporation. Epiphytic and extraneous matter were removed by washing first with sea water and then washed with distilled water to separate potential contaminants. The samples were dried in the air at the room temperature and kept in plastic bags for biochemical analysis Protein estimation The protein fraction (% of DW) was calculated from the elemental N determinations using the nitrogen-protein conversion factor of 6.25 according to AOAC (1995) Carbohydrate estimation The total carbohydrate was estimated by following the Phenol-sulphuric acid method of Dubois et al. (1956) using glucose as standard. 2.4 Lipid estimation The extraction of lipid was done by the chloroformmethanol mixture (2:1 v/v). The lipids in chloroform were dried over anhydrous sodium sulfate, and then the solvent was removed by heating at 80 º C under vacuum (AOAC 2000) Estimation of β-carotene: β-carotene was determined in diethyl ether at 440 nm as mentioned in the method of Neeld and Pearson (1963), and expressed as mg/100g dry wt Total phenols: Total content of phenolic compounds of algal extracts was determined spectrophotometrically at 725 nm using Folin- Ciocalteu reagent according to the method described in Lim et al. (2002). The total content of phenolic compounds was calculated based on a standard curve of phloroglucinol and expressed in % of dry weight Fatty acids methyl ester The lipids in chloroform were dried over anhydrous sodium sulfate, and then the solvent was removed by heating at 60 o C under vacuum. Fatty acid methyl esters were prepared according to Vogel (1975). The analysis was performed in a gas liquid chromatography (Pye Unicam Series 304 Gas Chromatograph) equipped with dual flam ionization detector and duel channel recorder. The separation of fatty acid methyl ester was performed through a coiled glass column (1.5 X 4 mm) packed with Diatomite ( mesh) and coated with 10% polyethylene glycol adipate (PEGA). The column oven temperature was programmed at 8 o C/min from 70 o C to 190 o C, then isothermally at 190 o C for 25 min with nitrogen at 30 ml/min. Identification of fatty acid methyl esters was accomplished by comparing the retention times of experimental samples to those of known standards. The fatty acids analysis was performed in the National Institute of Oceanography and Fisheries, Alexandria, Egypt Ash content Ash content was estimated by ashing the ground dried samples overnight in muffle furnace at 525 º C Estimation of amino acids Algal sample of 3 g was prepared for hydrolysis according to Walker (1996) before determination of amino acids. Amino acid analyses were carried out using amino acid analyzer LC 3000 eppendorf / Biotronik using column type H 125 x in the National Institute of Oceanography and Fisheries, Alexandria, Egypt. 3. STATISTICAL ANALYSIS The given data were expressed in terms of the mean of three replicates ± SD (standard deviation). In addition, data concerning the environmental parameters, content of proteins, carbohydrates, lipids, ash and moisture for the algal species were analyzed by Duncan s multiple range test at p < 0.05 by using SPSS Inc. program version15 (2006). 4. RESULTS AND DISCUSSION The biochemical contents of the four algal species are documented in Table I. The Ash contents were considerably low, and Sargassum vulgare have the lowest ash content when compared to the tree other species, however, Ceramium rubrum have the highest ash content. As shown in table I there were significant relation between the all studied algae. The high element contents in seaweed are shown by their ash contents, which are in the range of 8 40% DW. Sanchez-Machado et al. (2002) f o u n d ash of 24.21% in Caulerpa lentillifera against 17.58% in U. reticulate, while comparable a m o u n t s w e r e m e a s u r e d i n Himanthalia elongata (26.78%), Laminaria ochroleuca (29.47%) and Porphyra sp. (19.07%). The differences in ash contents depend on seaweed species, physiological factors, environmental changes, methods of mineralization and type of processing. Results showed that, the protein content in species C. rubrum and S. fusiforme was almost higher than in S.vulgare and P.pavonia (table I). S. vulgare demonstrated the minimum protein contain as compared to the other algal species. The statistic analysis showed that the relationship between the different studied species of algae was significant (one-way ANOVA, p < 0.05) Variation in protein content of seaweeds can be due to different species, seasonal period and geographic area (Haroon 2000, Ratana-arporn and Chirapart 2006).

3 Algae Parameters C.rubrum S.vulgare S.fusiforme P.pavonia F value P-value Ash 0.53±0.003 a 0.19± b 0.33± c 0.51± d Table I The mean (±SD) contents of ash, moisture, lipid, protein, carbohydrate, β-carotene and total phenolic compound. Moisture ± a ± b ± c ± d Protein 9.27±0.03 a 5.85±0.03 b 8.85± 0.03 c 8.35 ±0.02 d Carbohydrates ±1.17 acd 93.34±1.01 b 90.71±1.06 a ±1.25 a Lipid ± a ± b ± c 0.006± d β-carotene (IU\100mg) ± 3.9 a ±3.45 b ± 0.94 c ± 2.17 d Total phenol (ppm) ± 0.52 a 0.38 ± b ± 0.25 c ± 0.88 d Each value is the mean of three replicates ±SD Values with the same letters in the same column showed insignificant differences (p 0.05) International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:14 No:01 31

4 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:14 No:01 32 In this study, the protein contents of the studied species were higher than that of Sargassum polycystum, Gelidium domingensis and Gelidium birdiae. Similar values recorded for P. capillacea, the red alga Palmaria sp., notably lower values in the green alga U. lactuca, and t h e brown algae, Himanthalia elongata and Laminaria ochroleuca. Haroon (2000) reported range of 9.42 ± 4.62 to ± 5.0 % of DW for Enteromorpha spp., while protein in U. reticulata was three times o f that contained in U. lactuca and slightly lower than that in Porphyra sp. (Sanchez-Machado et ND= not detected al. 2002). Carbohydrate is the most important component for metabolism as it supplies the energy needed for respiration and other metabolic processes. Changes in carbohydrate content at the different studied algae were observed during the present study (Table I). The results showed that, the four studied species contain a very high concentration of carbohydrates ranged from (88.88% in C. rubrum to 93.65% in S. vulgare). According to statistic analyses there were significant relation between C. rubrum and S. vulgare, however the other relations was insignificant. Table II Fatty acid composition Fatty acids C.rubrum S.vulgare S.fusiforme P.pavonia Saturated C8:0 ND ND ND C10: ND ND C11: ND ND C12: C13: C14: C15: C16: C17: C18: C20: C21: ND C22: ND ND ND Sum of saturated Unsaturated C14: C15: C16: C17: C18: C20: ND ND C22: ND ND Sum of mono Polyunsaturated C18: C18: C20; ND ND Sum of poly Total unsaturated

5 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:14 No:01 33 John and Anisha (2011) reviewed that; carbohydrates obtained from macroalgal biomass can be used for fermentative production of bioethanol. Methane and hydrogen can readily be produced from macroalgal biomass through biologically mediated degradation. Anantharaman et al. (2013) found that the carbohydrate content of five studied species of seaweeds ranged from 10.63% and 28.58%, the maximum carbohydrate content was recorded in the green seaweed Enteromorpha intestinalis and the brown seaweed Dictyota dichotoma recorded the minimum value. The total lipid contents in the studied seaweed species were relatively low (Table I), the highest value was found in S. vulgare, however the lowest value was recorded in P. pavonia. The relationship between all algal species was significant (one-way ANOVA, p < 0.05). Typically, seaweeds are not considered to be good source of lipid (Ratana-arporn and Chirapart 2006), and the total lipid content was always found less than 4% (Herbreteau et al. 1997). The present results a r e supported by the findings of Shanmugam and Palpandi (2008) in Sargassum wightii ( %), in Gracilaria spp. ( %), in Enteromorpha intestinalis (1.2 %) and in U. reticulate (1.7%). These differences could b e due to environmental factors affecting the growth of the seaweed. The literature has established that in seaweeds in general the lipid content is less than 4%. The differences could have been due to factors such as climate and geography of development of the seaweed (Herbetreau et al. 1997). As observed from table II, saturated fatty acids were the predominant in types present in the all studied species. Species S. vulgare contain the highest percentage of total saturated fatty acids, while, species S. fusiforme contain the lowest content of total saturated fatty acids. C16:0 was the predominant unsaturated fatty acid among the all studied species. Species S. vulgare contain the higher percentage of C16:0. This results coincides with Shanmugam and Palpandi (2008) who found that the saturated fatty acids c o n s t i t u t e d 70.01% of the total fatty acids, wi t h t h e d o mi n a n c e of C16:0 and C14:0 (50.76% and11.77%, respectively). The palmitic acid as the major fatty acid (85.36%) was also recorded in Gellidum micropterum, 63.19% in Porphyra spp. and Ergrezia menziesii, Chondracanthus canaliculatus, and Ulva lobata. C13:0, C14:0, C15:0 and C18:0 was also present by relatively high percentages in the all studied species. As observed from table II, the unsaturated fatty acids present by nearly low concentration in the four studied species, while the monounsaturated fatty acids present by moderate concentration among the all tested species. S. fusiforme contain the highest concentration of total unsaturated fatty acid; however S. vulgare have the lowest concentration of total unsaturated fatty acid. They were the next most common fatty acids as represented by Shanmugam a n d Palpandi ( 2008), and oleic acid demonstrated high concentration in Porphyra spp. In the present study species S. fusiforme was characterized by the highest amount of polyunsaturated fatty acids among the studied algae, and it characterized by the present of EPA (C20:5). As observed in table I the highest concentration of β-carotene was recorded in species P. pavonia however, the lowest concentration was recorded in species S. fusiforme. The main source of β-carotene was red and brown algae (Sachindra et al. 2007). A lot of studies demonstrated the antioxidant properties of algal carotenoids and the role they play in preventing many diseases linked to oxidative stress (Banerjee et al. 2009). The β-carotene (with provitamin A activity) content ranges from 36 to 4,500 ppm dry weight, with Porphyra with the highest content and Palmaria palmata with the second highest of 456 ppm. However, other studies suggest that β-carotene could induce lung cancer in smokers. Furthermore, experimental studies strongly suggest that β- carotene could prevent the onset of cancers, especially lung cancer. However, natural source of β-carotene is highly recommended as they protect against the development of cancer (NRC 1982). Table III Amino acid composition. Amino acids C.rubrum (µm) S.vulgare (µm) S.fusiforme (µm) P.pavonia (µm) Essential Lysine Thereonine Valine 9.66 ND Isoleucine 60.5 ND ND ND Phenylalanine ND Sum essential Non essential ND ND ND Alanine Cystine ND Glutamine ND ND 15 ND Sum non ND= not detected

6 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:14 No:01 34 The present study investigated that the total phenolic content shows high variation through the all studied species. As shown in table II, total phenol present by very high concentration in S. fusiforme, on the other hand it recorded a very low concentration in S. vulgare. Phenolic compounds are commonly found in plants, including seaweeds, and have been reported to have a wide range of biological activities including antioxidant properties (Wang et al. 2009). Chandini et al. (2008) reported that brown seaweed extracts had a phenolic content of and mg/g of seaweed extract. Wang et al. (2009) reported the total phenolic content in different Icelandic seaweeds ranging from 4 to 242 mg/g extract. Table III shows the different profiles of amino acids in the four studied species of seaweeds. The results showed that Phenylalanine was the major amino acid in the all studied species except in species C. rubrum. Isoleucine was found only in C. rubrum with high concentration. Amino acids exhibit some pronounced differences among the species of Rhodophyceae, Pheophyceae and Chlorophyceae. Christine et al. (2007) examining different seaweed and found all essential amino acids forming over 30% of the total amino acids. Other studies recorded 37±38 % in Porphyra tenera, Grateloupia turuturu, Ulva pertusa and Codium fragile; % in U. lactuca and Gelidium amansii; 36.5± 38.6% in Ulva rigida and U. rotundata and % in Kappaphycus alvarezii and Hypnea musciformis. The studied species featured unique high concentrations of essential amino acids, while nonessential amino acids present with low concentration in the all studied species. These results coincide with Ratana-arporn and Chirapart (2006) who reported that Caulerpa lentillifera and U. reticulata proteins were of high quality because the essential amino acids represented almost 40% of total amino acids. The comparison of the amino acid composition of seaweeds with FAO reference pattern (FAO 1981) and those of other food proteins allows us to estimate of the nutritional value of seaweed proteins. It is evident that most of the seaweeds seem to be able to contribute by adequate levels of total essential amino acids in the food requirement. CONCLUSION In, Egypt, development of noval foods such as functional foods could be a new possibility for use this algae Ceramium rubrum, Sargassum vulgare, Sargassum fusiforme and Padina pavonia, especially for the protein rich species, in human nutrition. On the other hand the highest carbohydrate contents obtained from the studied species can be used for fermentative production of bioethanol. In addition to polyunsaturated fatty acids which present in the all studied species. 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