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Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii
Biomedicine & Preventive Nutrition 3 (2013) 139–144
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Original article
Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii Arif Nisha Syad , Karutha Pandian Shunmugiah , Pandima Devi Kasi ∗ Department of Biotechnology, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 30 November 2012 Accepted 12 December 2012 Keywords: Nutritional composition Seaweeds Vitamins Amino acids Fatty acids Minerals
a b s t r a c t Seaweeds or marine algae are rich in minerals and nutrients that are important for most of the biochemical reactions and non-nutrient components like dietary fibres and polyphenols. The present study aims at elucidating the nutritional composition of the two marine algae Gelidiella acerosa (red seaweed) and Sargassum wightii (brown seaweed) and the results revealed that the seaweeds possess high fibre content of 13.45 ± 1.076% and 17 ± 1.19% DW and ash content of 0.103 ± 0.049 g/g DW and 0.25 ± 0.02 g/g DW respectively. Nutritional composition analysis showed that the carbohydrate, protein, lipid, proline and chlorophyll contents of the seaweeds were high. Evaluation of mineral content demonstrates that the concentration of potassium was high in G. acerosa, whereas S. wightii was found to possess high amount of Sodium. Fatty acid profile verified the presence of major fatty acids with high nutritional value including, linolenic acid and ␣-linolenic acid. Amino acid composition showed that both the seaweeds possess most of the essential amino acids including valine, methionine, lysine and phenyl alanine. Vitamin analysis revealed the presence of high amount of vitamin C (an antioxidant) in the seaweeds. The results suggest that both the seaweeds have greater nutritional value and could be used as excellent nutritional supplements. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Seaweeds are one of the living renewable resources of the marine environment with potential food and therapeutic applications [1]. From the past decades, these marine algae have been consumed widely in Asian countries, whereas they have been used as sources of phycocolloids, thickening and gelling agents in food industries [2]. Seaweeds are also rich in polysaccharides, vitamins and minerals and they have become matchless source of chemical compounds that includes wide variety of biologically active secondary metabolites [3]. These bioactive compounds are molecules obtained from synthetic or natural sources, which are assayed biologically for activities in many therapeutic areas. The activity of these bioactive compounds has been linked to good health for many years, and it appears that bioactive food components can alter the genetic expression of a host of cellular events, thereby influencing health outcomes or providing beneficial antioxidant or enzyme inhibitory activities [4]. The marine red alga Gelidiella acerosa is a warm tropical alga, a major source of raw material for the production of superior quality
∗ Corresponding author. Tel.: +91 4565 225215. E-mail address: [email protected] (P.D. Kasi). 2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2012.12.002
of agar [5]. Agar, the hydrophilic colloid extracted from certain seaweeds of the Rhodophyceae class, has been used as a food ingredient for centuries. It is used as a gelling agent most commonly in icings, sugar confectionery, canned meat and fish products, and dairy products [6]. The other seaweed used in the study is Sargassum wightii, brown seaweed, which is a major food source, especially in Japan, where it is added to soups and fermented with the other ingredients in soy sauce to create a specific flavour. S. wightii is a major source of alginic acid (acid polysaccharides) which finds wide application in food, pharmaceuticals, cosmetics, paper and textile industries [7]. Though the seaweeds G. acerosa and S. wightii have been reported to possess excellent food value, detailed analysis of their nutritional composition is not available. Therefore, in the present study the physicochemical properties, proximate composition, mineral content, vitamins, fatty acid and amino acid composition of both G. acerosa and S. wightii were investigated. 2. Materials and methods 2.1. Collection of seaweed samples Seaweeds (G. acerosa and S. wightii) were collected from intertidal region in Gulf of Mannar and identified according to Oza and Zaidu [8] and Krishnamurthy and Joshi [9] and further confirmed
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by Dr. M. Ganesan, Scientist, CSMCRI, Mandapam Camp, Tamil Nadu. The voucher specimen was deposited at Department of Biotechnology, Alagappa University, under the accession number AUDBTGA20100101 and AUDBTSW20100102 for G. acerosa and S. wightii respectively. 2.2. Processing of seaweeds The collected seaweeds were processed to remove the attached specimens on its surface. After that, the samples were washed in tap water and in distilled water. To remove the adhered microflora, the seaweeds were washed with alcohol. The processed seaweeds were stored in airtight zip-lock containers and stored. 2.3. Physicochemical properties of seaweeds [10] 2.3.1. Swelling capacity (SWC) SWC was analyzed by bed volume technique after equilibrating in excess solvent. 200 mg of seaweed was placed in a container with 20 ml of distilled water and vigorously stirred. To measure the effect of temperature on SWC, the sample was left to stand for 24 h in two different temperatures (25 ◦ C and 37 ◦ C). The swelling volume was measured and expressed as ml of swollen sample per g of sample dry weight (DW). 2.3.2. Water Holding Capacity (WHC) WHC was analyzed by modified Centrifugation method. 200 mg of seaweed sample was placed in 20 ml of distilled water in a centrifugation tube and were kept in a shaker for 24 h. To determine the effect of temperature on WHC, the samples were kept at 25 ◦ C and 37 ◦ C. WHC was expressed as weight of gram of water held by 1 g of dry weight of sample. 2.3.3. Oil Holding Capacity (OHC) 3 g of seaweed sample was taken in 10.5 g of corn oil in a centrifugation tube. The tubes were left for 30 min at room temperature with constant agitation. The mixture was centrifuged at 2500 g for 30 min at room temperature. The oil supernatant was removed and used for measurement. The OHC of seaweed was measured as the number of grams of oil held by 1 g of dry weight of sample. 2.4. Proximate composition of G. acerosa and S. wightii 2.4.1. Ash content 5 g of freeze dried seaweed sample was kept at 525 ◦ C for 5 h in blast furnace, the ash content was expressed as g of ash obtained per 100 g of sample dry weight. 2.4.2. Total fibre content analysis The content of total dietary fibre (TDF) in seaweeds was determined according to the AOAC enzymatic gravimetric method (AOAC official methods of Analysis; 2005: 962.09). 2.4.3. Protein extraction from powdered seaweed sample 1 g of powdered seaweed was introduced into centrifuge tubes containing 50 ml of Diethyl ether and water (1:4). The tubes were kept in a shaker for 3 h. The supernatant was discarded and 1N NaOH was added to the sample and kept in a shaker for 3 h. The mixture was centrifuged at 7000 rpm for 10 min and the supernatant was collected and precipitated with 10% solution of TCA at pH 4.0. The samples were kept in ice for 30 min or until visible precipitate appears. The samples were then centrifuged at 7000 rpm for 20 min. The precipitated protein was washed and dried.
2.4.4. Estimation of total protein content Estimation of crude protein content was determined by Lowry et al. [11]. 0.5 ml of standard and test sample was taken in a test tube and made to 2 ml by adding water. BSA was used as standard solution (50 mg/50 ml). 5 ml of solution C (2% Na2 CO3 in 1N NaOH and 0.5% CuSO4 in 1% sodium potassium tartarate) was added to the tubes and incubated at room temperature for 10 min. Then, 0.5 ml of solution D (Folin’s Ciocalteau reagent 1:2) was added and incubated in dark for 45 min. The samples were then read at 660 nm in UV-Vis spectrophotometer. 2.4.5. Extraction of crude lipid Crude lipids were extracted from the powdered seaweed sample using Soxhlet apparatus [10]. The solvent mixture used for extraction is chloroform and methanol in the ratio of 2:1 (v/v). The contents of the crude lipids were determined gravimetrically after oven-drying (80 ◦ C) the extract overnight. 2.4.6. Estimation of total carbohydrate content Total carbohydrate estimation was done by Phenol-sulphuric acid method [12]. 200 mg of sample was added to 5 ml of 2.5 N HCl and the sample was hydrolyzed by keeping in boiling water bath for 3 h. The solution was neutralized by adding solid Na2 CO3 until effervescence ceases. The volume was then made to 50 ml and centrifuged at 8000 rpm for 10 min. The supernatant was collected and about 0.5 ml (or) 1 ml of it was aliquoted in a test tube. The sample was made to 1 ml with distilled water and 1 ml of phenol solution was added to the sample along with 5 ml of 96% sulphuric acid. The solution was mixed well and placed in water bath for 20 min at 25 ◦ C. The absorbance was measured at 490 nm using UV-Vis spectrophotometer. 2.4.7. Analysis of moisture content Moisture content was determined by moisture analyzer (Karl Fischer oven 860 KF thermoprep). The moisture content was expressed as percentage by weight of sample [10]. 2.5. Nutritional profile of G. acerosa and S. wightii 2.5.1. Proline content Proline content of the G. acerosa and S. wightii was determined by Bates [13]. 1.25 g of ninhydrin was added to 30 ml of glacial acetic acid in a test tube containing 20 ml of 6 M of phosphoric acid. The mixture was agitated till it dissolved and the solution was kept at 4 ◦ C, which is stable for 24 h. 0.5 g of seaweeds were placed in 10 ml of 3% aqueous sulphosalicylic acid and filtered with Whatman no 1 or 2 filter paper. 2 ml of filtrate and 2 ml of acid ninhydrin was mixed with 2 ml of glacial acetic acid. The mixture was kept at 100 ◦ C for 60 min. The reaction was terminated by placing the mixture in ice bath. After that, the mixture was extracted with toluene. The solution was mixed vigorously with the test tube stirrer. Chromophore containing toluene was collected from the aqueous phase and warmed at room temperature and the absorbance was measured at 520 nm using UV-Vis spectrophotometer. Proline was used as standard and the experiments were done in triplicates. 2.5.2. Estimation of chlorophyll content [14] Required amount of seaweed samples of G. acerosa and S. wightii were homogenized with 96% methanol (50 ml for each g) and centrifuged at 1000 rpm for 1 min. The homogenate was then filtered and centrifuged at 2500 rpm for 10 min. The supernatant was collected and the absorbance was measured at 400–700 nm in UV-Vis spectrophotometer. The formula used to calculate chlorophyll A and B content was as follows: Chlorophyll A = 15.65 (A666 ) − 7.340 (A653 )
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Chlorophyll B = 27.05 (A653 ) − 11.21 (A666 ) The amount of chlorophyll obtained was expressed as g/g of fresh weight. 2.5.3. Evaluation of mineral contents by flame atomic absorption spectroscopy [15] 2 g of seaweed was taken in a glass container. 10 ml of perchloric acid was added to it and left without disturbance for 5 min (to remove the organic constituents present in it). Then, 10 ml of concentrated nitric acid was added to it and incubated for 5 min and then added with 10 ml of HCl. The mixture was allowed to evaporate and the final residue was dissolved in 10 ml of Concentrated HCl. The filtrate was subjected to analysis in atomic absorption spectrophotometer (Perkin Elmer Analyst 800 with flame furnace). The minerals analyzed were Sodium, Potassium, Calcium, Iron, Magnesium, Lead and Zinc and the results were expressed as ppm. 2.5.4. Determination of fatty acid composition in G. acerosa and S. wightii 75 mg of lipid samples were dissolved in 1 ml of toluene. To the sample mixture, 2 ml of 1% H2 SO4 (prepared in methanol) was added and the required esters were extracted twice with 5 ml of hexane. The hexane layer was separated and washed with 4 ml of 2% potassium bicarbonate. The mixture was then dried over anhydrous Na2 SO4 and filtered. The organic solvent was removed in evaporator. The FAME thus obtained was subjected to Gas Chromatography [16]. GC was performed in 6890N system for GC Agilent Technologies. HP-5 capillary column was used and it is equipped with Electron impact ionization. Initial temperature was 70 ◦ C and then increased to 250 ◦ C (10 ◦ C/min) and the injection temperature employed was 220 ◦ C. Helium was used as carrier gas at flow rate of 1 l/min. FAME peaks were identified by comparison of their retention times with those of standard FAME mix (Supelco; Sigma Aldrich). 2.5.5. Amino acid analysis The amino acid composition of seaweed samples was determined according to Gratzfeld-Huesgen [17]. 2 g of powdered seaweed samples were mixed with PO4 buffer (pH-7.0) and centrifuged at 3000 rpm for 20 min at 4 ◦ C. The proteins present in the supernatant were precipitated separately using 10% TCA. The pellet was resuspended in 1N NaOH and subjected to acid hydrolysis by incubating with 6N HCl in boiling water bath for 24 h. The samples were then centrifuged at 3500 rpm for 15 min. The supernatant obtained was filtered and neutralized with 1N NaOH. The filtered solution was diluted to 1:100 of the volume with milli-Q water and subjected to HPLC analysis (HP-1101 Agilent Technologies with UV and Fluorescent detectors). 2.5.6. Vitamin analysis Vitamin analysis was done for the seaweeds G. acerosa and S. wightii. The fat soluble vitamins analyzed were vitamin A and E using the standards beta carotene and alpha tocopherol respectively. The seaweed samples after processing were subjected to
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HPLC analysis using n-hexane and orthophosphoric acid: methanol in the ratio 95:5 and 0.1 M potassium acetate (pH 4.9) as mobile phase for vitamin A and E. The liquid chromatograph was equipped with a 254-nm detector and an 8-mm × 10-cm column. The flow rate was about 2 ml/min. Retinyl acetate and retinyl palmitate (7.5 g/ml), ␣-tocopherol (2 mg/ml) were used as standards for vitamin A and E respectively. Water soluble vitamins analyzed were vitamin B1, vitamin B2 and vitamin C using the standards thiamine hydrochloride (10 g/ml), riboflavin (12 g/ml) and ascorbic acid (1 mg/ml) respectively. Vitamin C was analyzed by HPLC method using acetonitrile-water (50:50) as mobile phase whereas vitamins B1 and B2 were analyzed spectrophotometrically according to the method of Bradbury and Singh [18]. 2.6. Statistical analysis Except for the fatty acid profiles, all analyses were performed in triplicates. All the data were represented as Mean ± S.D. The statistical analysis was performed using SPSS software package (Version 17.0). Paired t-test was employed in the evaluation of physicochemical properties to compare the data obtained at different temperatures. P-value < 0.05 was regarded as significant. 3. Results and discussion In the present study, the nutritional composition of the marine red alga G. acerosa and brown alga S. wightii were analyzed. Various parameters including physicochemical properties, proximate composition, amino acids, vitamins and mineral content of the seaweeds were evaluated. The physicochemical properties determine the physiological effects of the dietary fibres. These dietary fibres are resistant to digestion, which provides bulk to faeces, holds water, acts as a site for ion-exchange, and binds organic molecules [2]. The centrifugation method was employed to determine the physicochemical properties like SWC, WHC and OHC and the results are illustrated in Table 1. The effect of temperature on SWC and WHC were investigated. At 25 ◦ C, SWC of S. wightii was 8.75 ± 1.76 ml/g of dry weight (DW) and a slight increase in the value (10 ± 0) was observed after incubating at 37 ◦ C. Interestingly, upon incubation at 37 ◦ C, the WHC of the seaweed increased significantly (P < 0.05) from 4.75 ± 0.14 (at 25 ◦ C) to 5.72 ± 0.14. In the case of G. acerosa, the SWC and WHC was 4 ± 0 ml/g DW and 3.06 ± 0.14 ml/g DW respectively at 25 ◦ C and when incubated at 37 ◦ C, the values increased slightly to 5 ± 1.4 ml/g DW (SWC) and 3.08 ± 0.14 ml/g DW (WHC). This increase can be attributed to the increase in solubility of fibres and proteins. Similarly, oil holding capacity (OHC) is another important property of food ingredients. The entrapment of oil by capillary attraction is generally represented as OHC of the seaweeds [10]. OHC depends on the polar side chains of the amino acids on the surface of their protein molecules. The OHC of G. acerosa and S. wightii was found to be 0.91 ± 0.02 g/g and 1.32 ± 0.08 g/g DW respectively (Table 1). The proximate composition of G. acerosa and S. wightii were investigated and the results are tabulated in Table 2. In general, the ash content represents the total mineral content of the seaweeds. The ash content of G. acerosa and S. wightii was
Table 1 Physicochemical properties of the seaweeds G. acerosa and S. wightii. Seaweed
G. acerosa S. wightii a *
SWC (ml/g DW)a
WHC (g/g DW)a
OHC (g/g DW)a
25 ◦ C
37 ◦ C
25 ◦ C
37 ◦ C
4±0 8.75 ± 1.76
5 ± 1.41 10 ± 0
3.06 ± 0.14 4.75 ± 0.14
3.08 ± 0.14 5.72 ± 0.14*
Results are expressed as Mean ± SD (n = 3). P < 0.05.
0.91 ± 0.02 1.32 ± 0.08
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Table 2 Proximate composition, proline content and chlorophyll content of the seaweeds G. acerosa and S. wightii. S. No
Composition
G. acerosaa
1. 2. 3. 4. 5. 6. 7. 8. 9.
Ash content Total dietary fiber content Crude protein content Crude lipid content Total carbohydrate content Moisture content Proline content Chlorophyll A Chlorophyll B
0.103 13.45 0.61 0.028 1.05 12.15 83.7 1.583 1.896
a
± ± ± ± ± ± ± ± ±
S. wightiia
0.049 g/g DW 1.076 % DW 0.07 mg/g DW 0.14 g/g DW 0.031 g/g DW 0.85 % 7.4 mol/g of DW 0.049 g/g FW 0.10 g/g FW
0.25 17 1.482 0.0272 0.095 22.4 44.26 6.56 3.76
± ± ± ± ± ± ± ± ±
0.02 g/g DW 1.19 % DW 0.20 mg/g DW 0.36 g/g DW 0.03 mg/g DW 1.79 % 8.2 mol/g of DW 0.47 g/g FW 0.56 g/g FW
Results are expressed as Mean ± SD (n = 3).
found to be 0.103 ± 0.04 g/g and 0.25 ± 0.02 g/g of DW respectively. Analysis of dietary fibre content shows that the total dietary fibre content of G. acerosa and S. wightii was 13.45 ± 1.076% and 17 ± 1.19% DW respectively. Recent studies have demonstrated that the algal dietary fibres exhibit important functional activities such as antioxidant, anti-mutagenic and anti-coagulant, anti-tumor activity and a major role in the modification of lipid metabolism [19]. The amount of crude protein content observed was 0.61 ± 0.07 mg/g and 1.482 ± 0.20 mg/g of DW for G. acerosa and S. wightii respectively. Lipids were the minor components of seaweeds and in the present study, the total crude lipid content observed was 0.028 ± 0.14 g/g and 0.0272 ± 0.36 g/g of DW respectively for G. acerosa and S. wightii. Total carbohydrate content was determined by phenol-sulphuric acid method and G. acerosa and S. wightii was found to possess 1.05 ± 0.031 g/g and 0.095 ± 0.03 g/g DW respectively. One of the most fundamental and analytical procedures which need to be performed is the moisture content determination. Moisture is a quality factor in the preservation of some products and affects stability of food materials. Moisture is also used as quality factor and often it is specified in compositional standard [20]. The moisture content of the red seaweed G. acerosa and brown seaweed S. wightii was observed as 12.15 ± 0.85% and 22.4 ± 1.79% respectively. The extraordinary nutritional composition of algae in terms of fibre, protein, minerals make algae a nutritive, low-energy food which represents an important food alternative. In addition to the major nutrient elements, the evaluation of proline and chlorophyll content has become an important aspect of nutritional profiling. Several studies have demonstrated that proline elicits stress-stimulated phenolic biosynthesis and stimulation of antioxidant enzyme response pathways [21]. Moreover, at higher concentrations, chlorophyll possesses excellent antioxidant activity and the possible mechanism of action behind its antioxidant activity might be either the protection of linoleic acid against oxidation or prevention of the decomposition of hydroperoxides [22]. Therefore evaluating the proline and chlorophyll content of the seaweeds will be an added advantage in the facet of therapeutics. The proline content of G. acerosa and S. wightii was observed as 83.7 ± 7.4 mol/g and 44.26 ± 8.2 mol/g of DW respectively. Determination of chlorophyll content by spectrophotometric method reveals that about 1.583 ± 0.049 g/g Fresh weight (FW) of chlorophyll A and 1.896 ± 0.10 g/g FW of chlorophyll B was observed in G. acerosa. In the case of S. wightii, 6.56 ± 0.47 g/g FW of chlorophyll A and 3.76 ± 0.56 g/g FW of chlorophyll B was present in it. Essential minerals and trace elements required for human nutrition are the major constituents of seaweeds which ranges from 8–40% [15]. Since seaweeds are a rich source of minerals when compared to land plants, the mineral content of G. acerosa and S. wightii were evaluated in the present study using atomic absorption spectrophotometer. The results reveal that the red seaweed G. acerosa contain high amount of Potassium (K)
Table 3 Mineral composition determined by atomic absorption spectrophotometer in G. acerosa and S. wightii. S. No
Name of the element
1. 2. 3. 4. 5. 6. 7.
Sodium Potassium Calcium Iron Magnesium Lead Zinc
Observed concentration (ppm)a G. acerosa
a
129.05 522.43 450.04 1.549 0.203 0.001 0.350
± ± ± ± ± ± ±
S. wightii 12.79 22.24 24.5 0.10 0.012 0.0003 0.028
4090.2 2830.37 787.48 0.168 1.031 0.006 0.007
± ± ± ± ± ± ±
29.02 141.51 47.24 0.01 0.05 0.00015 0.0005
Results are expressed as Mean ± SD (n = 3).
(522.43 ± 22.24 ppm), whereas S. wightii contains high amount of Sodium (Na) (4090.2 ± 29.02 ppm) (Table 3). Both Sodium and Potassium play an indispensable role in electrical conductivity of the brain and facilitates the improvement of brain functions. Next to Sodium and Potassium, Calcium was found to be the abundant mineral in both the seaweeds (450.04 ± 24.5 ppm in G. acerosa and 787.48 ± 47.24 ppm in S. wightii). Whereas the amount of Mg present was 1.031 ± 0.05 ppm and 0.203 ± 0.012 ppm in S. wightii and G. acerosa respectively (Table 3). Recent reports suggest that low levels of Magnesium contribute to the heavy metal deposition in the brain that precedes Parkinson’s, multiple sclerosis and Alzheimer’s disease. Thus Mg is essential in regulating central nervous system excitability and normal functions [23]. In addition to these major elements, marine algae could be interesting candidates to explore as Fe sources, especially in countries where the algal production is feasible [24]. The amount of Iron (Fe) present in the seaweed G. acerosa and S. wightii was 1.549 ± 0.10 ppm and 0.168 ± 0.01 ppm respectively. Apart from these major elements, G. acerosa and S. wightii was found to possess trace elements like Lead (Pb) and Zinc (Zn) in the concentrations of 0.001 ± 0.0003 (Pb); 0.350 ± 0.028 (Zn) and 0.006 ± 0.00015 (Pb); 0.007 ± 0.0005 (Zn) ppm and respectively (Table 3). Fatty acids were found to possess beneficial effects like cardio-protective, cytotoxic, antimitotic, anticancer, antiviral and anti-mutagenic activities [25,26]. The data on fatty acid composition of G. acerosa and S. wightii is given in Table 4. Total fatty acids present in the red seaweed G. acerosa were found to be 0.5324% (w/w). A mixture of both saturated and unsaturated fatty acids Table 4 Fatty acid composition of G. acerosa and S. wightii. Fatty acids
G. acerosa(% w/w)
S. wightii(% w/w)
Palmitic acid (16:0) Margaric acid (17:0) Stearic acid (18:0) Oleic acid (18:1) Linolenic acid (18:2) Alpha linolenic acid (18:3)
0.1045 0.0011 0.1345 0.0934 0.1034 0.0834
1.23 In traces 1.998 1.3434 1.3454 1.11
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Table 5 Vitamin composition of G. acerosa and S. wightii. S. No
Vitamins
G. acerosa (mg/g of DW)a
S. wightii (mg/g of DW)a
1. 2. 3. 4.
Vitamin A Vitamin E Vitamin C Vitamin B1
5.
Vitamin B2
0.0034 ± 0.0002 1.33 ± 0.07 5.0718 ± 0.202 Below detectable level Below detectable level
0.0040 ± 0.00012 1.35 ± 0.08 5.069 ± 0.40 Below detectable level Below detectable level
a
Results are expressed as Mean ± SD (n = 3).
4. Conclusion
Fig. 1. Amino acid composition of G. acerosa and S. wightii. The values were represented as Mean ± SD.
has been found to be present. The saturated fatty acids present were palmitic acid (0.1045% w/w), margaric acid (0.0011% w/w) and stearic acid (0.1345% w/w). The unsaturated fatty acids include oleic acid (0.0934% w/w), Linolenic acid (0.1034% w/w) and ␣Linolenic acid (0.0834% w/w). S. wightii contained a total of 8.341% of fatty acids, of which 3.228% (w/w) was saturated fatty acids and 3.798% (w/w) was unsaturated fatty acids. The results of fatty acid analysis reveal that both G. acerosa and S. wightii are rich in MUFA and PUFA, which possess important health benefits. In particular, oleic acid (MUFA) and ␣-linolenic acid (PUFA) present in both the seaweeds might help in lowering the blood cholesterol, act as excellent antioxidants, strengthen the cell membrane, repair the damaged cells and tissues, improve the functioning of heart and fight against cancer. Quantitative determination of amino acid concentrations was conducted by HPLC and the results are illustrated in Fig. 1. Fourteen amino acids were detected. Almost all of the essential amino acids including methionine, leucine, lysine, phenylalanine, tyrosine, arginine, isoleucine, threonine and valine and five non-essential amino acids like aspartic acid, glutamic acid, serine, alanine and histidine were found to be present in both G. acerosa and S. wightii. One exception is that among the amino acids, serine, histidine and threonine were absent in G. acerosa. The red alga G. acerosa was found to possess high amount of glutamic acid (13.67 ± 0.95 mg/g of protein), next to that, the amount of tyrosine was high (4.12 ± 0.16 mg/g of protein). Whereas aspartic acid, alanine, arginine, valine, methionine, phenyl alanine, isoleucine, lysine, leucine were found to be present at 1.09 ± 0.087, 1.30 ± 0.07, 2.83 ± 0.25, 1.93 ± 0.11, 2.80 ± 0.16, 1.59 ± 0.11, 0.86 ± 0.06, 2.14 ± 0.17, 1.62 ± 0.08 mg/g of protein respectively. In the case of brown alga, S. wightii, glutamic acid constitutes 18.34 ± 1.65 mg/g of protein, whereas histidine, threonine, arginine, serine, alanine, tyrosine and aspartic acid constitutes about 7.44 ± 0.44, 5.42 ± 0.43, 7.31 ± 0.43, 3.95 ± 0.31, 3.19 ± 0.19, 4.12 ± 0.32, 2.82 ± 0.28 mg/g of protein respectively. Analysis of fat and water soluble vitamins (Table 5) reveals that vitamin C, the major antioxidant was found to be abundant in both the seaweeds, which constitutes to about 5.0718 ± 0.202 mg and 5.069 ± 0.40 mg/g of DW in G. acerosa and S. wightii respectively. The fat soluble vitamins like vitamin A and vitamin E were analyzed by HPLC methods. The results show that vitamin E was found abundantly (next to vitamin C) in both the seaweeds G. acerosa (1.33 ± 0.07 mg/g of DW) and S. wightii (1.35 ± 0.08 mg/g of DW). Vitamin A constitutes about 0.0034 ± 0.0002 mg and 0.0040 ± 0.00012 mg/g DW in G. acerosa and S. wightii respectively, whereas Vitamin B1 and B2 were present only in trace amount.
In the present study, the nutritional composition of marine macro algae G. acerosa and S. wightii were evaluated. The outcome of the present study suggests that both the seaweeds could be employed as potential food supplements and may be used in the food industry as a source of ingredients with high nutritional value. Since both the seaweeds were found to be good source of essential nutrients, their commercial value can be enhanced by improving their quality and further increasing the range of seaweed-derived products. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements K.P.D. wishes to thank UGC, India [F.No.36-6/2008 (SR)] and ICMR, India [IRS ID 2007-02200] for the financial support. S.A.N. wishes to thank UGC-MANF for the Junior Research Fellowship provided. The authors gratefully acknowledge the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by Department of Biotechnology, Government of India; Grant No. BT/BI/25/001/2006). References [1] Kumar M, Gupta V, Kumari P, Reddy CRK, Jha B. Assessment of nutrient composition and antioxidant potential of Caulerpaceae seaweeds. J Food Compost Anal 2011;24:270–8. [2] Ruperez P, Calixto FS. Dietary fibre and physicochemical properties of edible Spanish seaweeds. Eur Food Res Technol 2001;212:349–54. [3] Villarreal-Gómez LJ, Soria-Mercado IE, Guerra-Rivas G, Ayala-Sánchez NE. Antibacterial and anticancer activity of seaweeds and bacteria associated with their surface. Rev Biol Mar Oceanog 2010;45:267–75. [4] MacArtain P, Christopher IR, Brooks GM, Campbell R, Rowland IR. Nutritional value of edible seaweeds. Nutr Rev 2007;65:535–43. [5] Prasad K, Siddhanta AK, Ganesan M, Ramavat BK, Jha B, Ghosh PK. Agars of Gelidiella acerosa of west and southeast coasts of India. Bioresour Technol 2007;98:1907–15. [6] Kaliaperumal N. Products from seaweeds. SDMRI Res Publ 2003;3:33–42. [7] Vijayaraghavan K, Prabu D. Potential of Sargassum wightii biomass for copper (II) removal from aqueous solutions: Application of different mathematical models to batch and continuous biosorption data. J Hazard Mater 2006;137:558–64. [8] Oza RM, Zaidu A. Revised Checklist of Indian Marine Algae. Bhavnagar, India: Central Salt and Marine Chemicals Research Institute; 2003, p. 1–296. [9] Krishnamurthy V, Joshi HY. A Check List of Indian Marine Algae. Bhavnagar, India: Central Salt and Marine Chemicals Research Institute; 1970, p. 1–36. [10] Wong KH, Cheung PCK. Nutritional evaluation of some subtropical red and green seaweeds. Part I – proximate composition, amino acid profiles and some physicochemical properties. Food Chem 2000;71:475–82. [11] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [12] Fournier E. Colorimetric Quantification of Carbohydrates Unit E1.1.1. In: Wrolstad RE, Reid DS, Smith DM, Penner MH, Decker EA, Sporns P, editors. Handbook
144
[13] [14]
[15] [16]
[17]
[18] [19]
A.N. Syad et al. / Biomedicine & Preventive Nutrition 3 (2013) 139–144 of food analytical chemistry. New York, USA: Hoboken, N.J. John Wiley and Sons Inc.; 2005. p. 653–60. Bates LS. Rapid determination of free proline for water-stress studies. Plant Soil 1973;39:205–7. Dere S, Günes T, Sivaci R. Spectrophotometric determination of chlorophyll - A, B and total carotenoid contents of some algae species using different solvents. Turk J Bot 1998;22:13–7. Ruperez P. Mineral content of edible marine seaweeds. Food Chem 2002;79:23–6. Yayli N, Kiran Z, Seymen H, Genc H. Characterization of lipids and fatty acid methyl ester contents in leaves and roots of Crocus vallicola. Turk J Chem 2001;25:391–5. Gratzfeld-Huesgen A. Sensitive and reliable amino acids analysis in protein hydrolisates using the Agilent 1100 Series HPLC. Technical Note by Agilent Technologies; 1999. Publication Number 5968-5658E. Bradbury JH, Singh U. Thiamin, Riboflavin, and Nicotinic acid contents of tropical root crops from the South Pacific. J Food Sci 1986;51:1563–4. Tierra M. Treatment based on the six methods of traditional Chinese medicine. Chapter 5. In: Treating cancer with herbs. Wisconsin, USA: Michael Tierra, Lotus press; 2003, p. 111.
[20] Nielsen SS. Moisture and total solid analysis. In: Food Analysis Laboratory Manual. Fourth edition Springer; 2010, p. 85–104. [21] Shetty K. Role of proline-linked pentose phosphate pathway in biosynthesis of plant phenolics for functional food and environmental applications: a review. Process Biochem 2004;39:789–803. [22] Lanfer-Marquez UM, Barros RMC, Sinnecker P. Antioxidant activity of chlorophylls and their derivatives. Food Res Int 2005;38:885–91. [23] Murck H. Magnesium and affective disorders. Nutr Neurosci 2002;5: 375–89. [24] García-Casal MN, Ramírez J, Leets I. Bioavailability from electrolytic and reduced iron in humans is enhanced by NaFe-EDTA and vitamin A in corn and wheat flours Effect of serum retinol status. Afr J Food Sci 2009;3: 131–8. [25] Prabhakar V, Anandan R, Aneesh TP, Jayasree NB, Nair SV, Halima OA. Fatty acid composition of Sargassum wightii and Amphiroa anceps collected from the Mandapam coast Tamil Nadu. J Chem Pharm Res 2011;3:210–6. [26] Ortiz J, Romero N, Robert P, Araya J, Lopez-Herna’ndez J, Bozzo C, Navarrete E, Osorio A, Rios A. Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea Antarctica. Food Chem 2006;99:98–104.
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Original article
Seaweeds as nutritional supplements: Analysis of nutritional profile, physicochemical properties and proximate composition of G. acerosa and S. wightii Arif Nisha Syad , Karutha Pandian Shunmugiah , Pandima Devi Kasi ∗ Department of Biotechnology, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 30 November 2012 Accepted 12 December 2012 Keywords: Nutritional composition Seaweeds Vitamins Amino acids Fatty acids Minerals
a b s t r a c t Seaweeds or marine algae are rich in minerals and nutrients that are important for most of the biochemical reactions and non-nutrient components like dietary fibres and polyphenols. The present study aims at elucidating the nutritional composition of the two marine algae Gelidiella acerosa (red seaweed) and Sargassum wightii (brown seaweed) and the results revealed that the seaweeds possess high fibre content of 13.45 ± 1.076% and 17 ± 1.19% DW and ash content of 0.103 ± 0.049 g/g DW and 0.25 ± 0.02 g/g DW respectively. Nutritional composition analysis showed that the carbohydrate, protein, lipid, proline and chlorophyll contents of the seaweeds were high. Evaluation of mineral content demonstrates that the concentration of potassium was high in G. acerosa, whereas S. wightii was found to possess high amount of Sodium. Fatty acid profile verified the presence of major fatty acids with high nutritional value including, linolenic acid and ␣-linolenic acid. Amino acid composition showed that both the seaweeds possess most of the essential amino acids including valine, methionine, lysine and phenyl alanine. Vitamin analysis revealed the presence of high amount of vitamin C (an antioxidant) in the seaweeds. The results suggest that both the seaweeds have greater nutritional value and could be used as excellent nutritional supplements. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Seaweeds are one of the living renewable resources of the marine environment with potential food and therapeutic applications [1]. From the past decades, these marine algae have been consumed widely in Asian countries, whereas they have been used as sources of phycocolloids, thickening and gelling agents in food industries [2]. Seaweeds are also rich in polysaccharides, vitamins and minerals and they have become matchless source of chemical compounds that includes wide variety of biologically active secondary metabolites [3]. These bioactive compounds are molecules obtained from synthetic or natural sources, which are assayed biologically for activities in many therapeutic areas. The activity of these bioactive compounds has been linked to good health for many years, and it appears that bioactive food components can alter the genetic expression of a host of cellular events, thereby influencing health outcomes or providing beneficial antioxidant or enzyme inhibitory activities [4]. The marine red alga Gelidiella acerosa is a warm tropical alga, a major source of raw material for the production of superior quality
∗ Corresponding author. Tel.: +91 4565 225215. E-mail address: [email protected] (P.D. Kasi). 2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2012.12.002
of agar [5]. Agar, the hydrophilic colloid extracted from certain seaweeds of the Rhodophyceae class, has been used as a food ingredient for centuries. It is used as a gelling agent most commonly in icings, sugar confectionery, canned meat and fish products, and dairy products [6]. The other seaweed used in the study is Sargassum wightii, brown seaweed, which is a major food source, especially in Japan, where it is added to soups and fermented with the other ingredients in soy sauce to create a specific flavour. S. wightii is a major source of alginic acid (acid polysaccharides) which finds wide application in food, pharmaceuticals, cosmetics, paper and textile industries [7]. Though the seaweeds G. acerosa and S. wightii have been reported to possess excellent food value, detailed analysis of their nutritional composition is not available. Therefore, in the present study the physicochemical properties, proximate composition, mineral content, vitamins, fatty acid and amino acid composition of both G. acerosa and S. wightii were investigated. 2. Materials and methods 2.1. Collection of seaweed samples Seaweeds (G. acerosa and S. wightii) were collected from intertidal region in Gulf of Mannar and identified according to Oza and Zaidu [8] and Krishnamurthy and Joshi [9] and further confirmed
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by Dr. M. Ganesan, Scientist, CSMCRI, Mandapam Camp, Tamil Nadu. The voucher specimen was deposited at Department of Biotechnology, Alagappa University, under the accession number AUDBTGA20100101 and AUDBTSW20100102 for G. acerosa and S. wightii respectively. 2.2. Processing of seaweeds The collected seaweeds were processed to remove the attached specimens on its surface. After that, the samples were washed in tap water and in distilled water. To remove the adhered microflora, the seaweeds were washed with alcohol. The processed seaweeds were stored in airtight zip-lock containers and stored. 2.3. Physicochemical properties of seaweeds [10] 2.3.1. Swelling capacity (SWC) SWC was analyzed by bed volume technique after equilibrating in excess solvent. 200 mg of seaweed was placed in a container with 20 ml of distilled water and vigorously stirred. To measure the effect of temperature on SWC, the sample was left to stand for 24 h in two different temperatures (25 ◦ C and 37 ◦ C). The swelling volume was measured and expressed as ml of swollen sample per g of sample dry weight (DW). 2.3.2. Water Holding Capacity (WHC) WHC was analyzed by modified Centrifugation method. 200 mg of seaweed sample was placed in 20 ml of distilled water in a centrifugation tube and were kept in a shaker for 24 h. To determine the effect of temperature on WHC, the samples were kept at 25 ◦ C and 37 ◦ C. WHC was expressed as weight of gram of water held by 1 g of dry weight of sample. 2.3.3. Oil Holding Capacity (OHC) 3 g of seaweed sample was taken in 10.5 g of corn oil in a centrifugation tube. The tubes were left for 30 min at room temperature with constant agitation. The mixture was centrifuged at 2500 g for 30 min at room temperature. The oil supernatant was removed and used for measurement. The OHC of seaweed was measured as the number of grams of oil held by 1 g of dry weight of sample. 2.4. Proximate composition of G. acerosa and S. wightii 2.4.1. Ash content 5 g of freeze dried seaweed sample was kept at 525 ◦ C for 5 h in blast furnace, the ash content was expressed as g of ash obtained per 100 g of sample dry weight. 2.4.2. Total fibre content analysis The content of total dietary fibre (TDF) in seaweeds was determined according to the AOAC enzymatic gravimetric method (AOAC official methods of Analysis; 2005: 962.09). 2.4.3. Protein extraction from powdered seaweed sample 1 g of powdered seaweed was introduced into centrifuge tubes containing 50 ml of Diethyl ether and water (1:4). The tubes were kept in a shaker for 3 h. The supernatant was discarded and 1N NaOH was added to the sample and kept in a shaker for 3 h. The mixture was centrifuged at 7000 rpm for 10 min and the supernatant was collected and precipitated with 10% solution of TCA at pH 4.0. The samples were kept in ice for 30 min or until visible precipitate appears. The samples were then centrifuged at 7000 rpm for 20 min. The precipitated protein was washed and dried.
2.4.4. Estimation of total protein content Estimation of crude protein content was determined by Lowry et al. [11]. 0.5 ml of standard and test sample was taken in a test tube and made to 2 ml by adding water. BSA was used as standard solution (50 mg/50 ml). 5 ml of solution C (2% Na2 CO3 in 1N NaOH and 0.5% CuSO4 in 1% sodium potassium tartarate) was added to the tubes and incubated at room temperature for 10 min. Then, 0.5 ml of solution D (Folin’s Ciocalteau reagent 1:2) was added and incubated in dark for 45 min. The samples were then read at 660 nm in UV-Vis spectrophotometer. 2.4.5. Extraction of crude lipid Crude lipids were extracted from the powdered seaweed sample using Soxhlet apparatus [10]. The solvent mixture used for extraction is chloroform and methanol in the ratio of 2:1 (v/v). The contents of the crude lipids were determined gravimetrically after oven-drying (80 ◦ C) the extract overnight. 2.4.6. Estimation of total carbohydrate content Total carbohydrate estimation was done by Phenol-sulphuric acid method [12]. 200 mg of sample was added to 5 ml of 2.5 N HCl and the sample was hydrolyzed by keeping in boiling water bath for 3 h. The solution was neutralized by adding solid Na2 CO3 until effervescence ceases. The volume was then made to 50 ml and centrifuged at 8000 rpm for 10 min. The supernatant was collected and about 0.5 ml (or) 1 ml of it was aliquoted in a test tube. The sample was made to 1 ml with distilled water and 1 ml of phenol solution was added to the sample along with 5 ml of 96% sulphuric acid. The solution was mixed well and placed in water bath for 20 min at 25 ◦ C. The absorbance was measured at 490 nm using UV-Vis spectrophotometer. 2.4.7. Analysis of moisture content Moisture content was determined by moisture analyzer (Karl Fischer oven 860 KF thermoprep). The moisture content was expressed as percentage by weight of sample [10]. 2.5. Nutritional profile of G. acerosa and S. wightii 2.5.1. Proline content Proline content of the G. acerosa and S. wightii was determined by Bates [13]. 1.25 g of ninhydrin was added to 30 ml of glacial acetic acid in a test tube containing 20 ml of 6 M of phosphoric acid. The mixture was agitated till it dissolved and the solution was kept at 4 ◦ C, which is stable for 24 h. 0.5 g of seaweeds were placed in 10 ml of 3% aqueous sulphosalicylic acid and filtered with Whatman no 1 or 2 filter paper. 2 ml of filtrate and 2 ml of acid ninhydrin was mixed with 2 ml of glacial acetic acid. The mixture was kept at 100 ◦ C for 60 min. The reaction was terminated by placing the mixture in ice bath. After that, the mixture was extracted with toluene. The solution was mixed vigorously with the test tube stirrer. Chromophore containing toluene was collected from the aqueous phase and warmed at room temperature and the absorbance was measured at 520 nm using UV-Vis spectrophotometer. Proline was used as standard and the experiments were done in triplicates. 2.5.2. Estimation of chlorophyll content [14] Required amount of seaweed samples of G. acerosa and S. wightii were homogenized with 96% methanol (50 ml for each g) and centrifuged at 1000 rpm for 1 min. The homogenate was then filtered and centrifuged at 2500 rpm for 10 min. The supernatant was collected and the absorbance was measured at 400–700 nm in UV-Vis spectrophotometer. The formula used to calculate chlorophyll A and B content was as follows: Chlorophyll A = 15.65 (A666 ) − 7.340 (A653 )
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Chlorophyll B = 27.05 (A653 ) − 11.21 (A666 ) The amount of chlorophyll obtained was expressed as g/g of fresh weight. 2.5.3. Evaluation of mineral contents by flame atomic absorption spectroscopy [15] 2 g of seaweed was taken in a glass container. 10 ml of perchloric acid was added to it and left without disturbance for 5 min (to remove the organic constituents present in it). Then, 10 ml of concentrated nitric acid was added to it and incubated for 5 min and then added with 10 ml of HCl. The mixture was allowed to evaporate and the final residue was dissolved in 10 ml of Concentrated HCl. The filtrate was subjected to analysis in atomic absorption spectrophotometer (Perkin Elmer Analyst 800 with flame furnace). The minerals analyzed were Sodium, Potassium, Calcium, Iron, Magnesium, Lead and Zinc and the results were expressed as ppm. 2.5.4. Determination of fatty acid composition in G. acerosa and S. wightii 75 mg of lipid samples were dissolved in 1 ml of toluene. To the sample mixture, 2 ml of 1% H2 SO4 (prepared in methanol) was added and the required esters were extracted twice with 5 ml of hexane. The hexane layer was separated and washed with 4 ml of 2% potassium bicarbonate. The mixture was then dried over anhydrous Na2 SO4 and filtered. The organic solvent was removed in evaporator. The FAME thus obtained was subjected to Gas Chromatography [16]. GC was performed in 6890N system for GC Agilent Technologies. HP-5 capillary column was used and it is equipped with Electron impact ionization. Initial temperature was 70 ◦ C and then increased to 250 ◦ C (10 ◦ C/min) and the injection temperature employed was 220 ◦ C. Helium was used as carrier gas at flow rate of 1 l/min. FAME peaks were identified by comparison of their retention times with those of standard FAME mix (Supelco; Sigma Aldrich). 2.5.5. Amino acid analysis The amino acid composition of seaweed samples was determined according to Gratzfeld-Huesgen [17]. 2 g of powdered seaweed samples were mixed with PO4 buffer (pH-7.0) and centrifuged at 3000 rpm for 20 min at 4 ◦ C. The proteins present in the supernatant were precipitated separately using 10% TCA. The pellet was resuspended in 1N NaOH and subjected to acid hydrolysis by incubating with 6N HCl in boiling water bath for 24 h. The samples were then centrifuged at 3500 rpm for 15 min. The supernatant obtained was filtered and neutralized with 1N NaOH. The filtered solution was diluted to 1:100 of the volume with milli-Q water and subjected to HPLC analysis (HP-1101 Agilent Technologies with UV and Fluorescent detectors). 2.5.6. Vitamin analysis Vitamin analysis was done for the seaweeds G. acerosa and S. wightii. The fat soluble vitamins analyzed were vitamin A and E using the standards beta carotene and alpha tocopherol respectively. The seaweed samples after processing were subjected to
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HPLC analysis using n-hexane and orthophosphoric acid: methanol in the ratio 95:5 and 0.1 M potassium acetate (pH 4.9) as mobile phase for vitamin A and E. The liquid chromatograph was equipped with a 254-nm detector and an 8-mm × 10-cm column. The flow rate was about 2 ml/min. Retinyl acetate and retinyl palmitate (7.5 g/ml), ␣-tocopherol (2 mg/ml) were used as standards for vitamin A and E respectively. Water soluble vitamins analyzed were vitamin B1, vitamin B2 and vitamin C using the standards thiamine hydrochloride (10 g/ml), riboflavin (12 g/ml) and ascorbic acid (1 mg/ml) respectively. Vitamin C was analyzed by HPLC method using acetonitrile-water (50:50) as mobile phase whereas vitamins B1 and B2 were analyzed spectrophotometrically according to the method of Bradbury and Singh [18]. 2.6. Statistical analysis Except for the fatty acid profiles, all analyses were performed in triplicates. All the data were represented as Mean ± S.D. The statistical analysis was performed using SPSS software package (Version 17.0). Paired t-test was employed in the evaluation of physicochemical properties to compare the data obtained at different temperatures. P-value < 0.05 was regarded as significant. 3. Results and discussion In the present study, the nutritional composition of the marine red alga G. acerosa and brown alga S. wightii were analyzed. Various parameters including physicochemical properties, proximate composition, amino acids, vitamins and mineral content of the seaweeds were evaluated. The physicochemical properties determine the physiological effects of the dietary fibres. These dietary fibres are resistant to digestion, which provides bulk to faeces, holds water, acts as a site for ion-exchange, and binds organic molecules [2]. The centrifugation method was employed to determine the physicochemical properties like SWC, WHC and OHC and the results are illustrated in Table 1. The effect of temperature on SWC and WHC were investigated. At 25 ◦ C, SWC of S. wightii was 8.75 ± 1.76 ml/g of dry weight (DW) and a slight increase in the value (10 ± 0) was observed after incubating at 37 ◦ C. Interestingly, upon incubation at 37 ◦ C, the WHC of the seaweed increased significantly (P < 0.05) from 4.75 ± 0.14 (at 25 ◦ C) to 5.72 ± 0.14. In the case of G. acerosa, the SWC and WHC was 4 ± 0 ml/g DW and 3.06 ± 0.14 ml/g DW respectively at 25 ◦ C and when incubated at 37 ◦ C, the values increased slightly to 5 ± 1.4 ml/g DW (SWC) and 3.08 ± 0.14 ml/g DW (WHC). This increase can be attributed to the increase in solubility of fibres and proteins. Similarly, oil holding capacity (OHC) is another important property of food ingredients. The entrapment of oil by capillary attraction is generally represented as OHC of the seaweeds [10]. OHC depends on the polar side chains of the amino acids on the surface of their protein molecules. The OHC of G. acerosa and S. wightii was found to be 0.91 ± 0.02 g/g and 1.32 ± 0.08 g/g DW respectively (Table 1). The proximate composition of G. acerosa and S. wightii were investigated and the results are tabulated in Table 2. In general, the ash content represents the total mineral content of the seaweeds. The ash content of G. acerosa and S. wightii was
Table 1 Physicochemical properties of the seaweeds G. acerosa and S. wightii. Seaweed
G. acerosa S. wightii a *
SWC (ml/g DW)a
WHC (g/g DW)a
OHC (g/g DW)a
25 ◦ C
37 ◦ C
25 ◦ C
37 ◦ C
4±0 8.75 ± 1.76
5 ± 1.41 10 ± 0
3.06 ± 0.14 4.75 ± 0.14
3.08 ± 0.14 5.72 ± 0.14*
Results are expressed as Mean ± SD (n = 3). P < 0.05.
0.91 ± 0.02 1.32 ± 0.08
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Table 2 Proximate composition, proline content and chlorophyll content of the seaweeds G. acerosa and S. wightii. S. No
Composition
G. acerosaa
1. 2. 3. 4. 5. 6. 7. 8. 9.
Ash content Total dietary fiber content Crude protein content Crude lipid content Total carbohydrate content Moisture content Proline content Chlorophyll A Chlorophyll B
0.103 13.45 0.61 0.028 1.05 12.15 83.7 1.583 1.896
a
± ± ± ± ± ± ± ± ±
S. wightiia
0.049 g/g DW 1.076 % DW 0.07 mg/g DW 0.14 g/g DW 0.031 g/g DW 0.85 % 7.4 mol/g of DW 0.049 g/g FW 0.10 g/g FW
0.25 17 1.482 0.0272 0.095 22.4 44.26 6.56 3.76
± ± ± ± ± ± ± ± ±
0.02 g/g DW 1.19 % DW 0.20 mg/g DW 0.36 g/g DW 0.03 mg/g DW 1.79 % 8.2 mol/g of DW 0.47 g/g FW 0.56 g/g FW
Results are expressed as Mean ± SD (n = 3).
found to be 0.103 ± 0.04 g/g and 0.25 ± 0.02 g/g of DW respectively. Analysis of dietary fibre content shows that the total dietary fibre content of G. acerosa and S. wightii was 13.45 ± 1.076% and 17 ± 1.19% DW respectively. Recent studies have demonstrated that the algal dietary fibres exhibit important functional activities such as antioxidant, anti-mutagenic and anti-coagulant, anti-tumor activity and a major role in the modification of lipid metabolism [19]. The amount of crude protein content observed was 0.61 ± 0.07 mg/g and 1.482 ± 0.20 mg/g of DW for G. acerosa and S. wightii respectively. Lipids were the minor components of seaweeds and in the present study, the total crude lipid content observed was 0.028 ± 0.14 g/g and 0.0272 ± 0.36 g/g of DW respectively for G. acerosa and S. wightii. Total carbohydrate content was determined by phenol-sulphuric acid method and G. acerosa and S. wightii was found to possess 1.05 ± 0.031 g/g and 0.095 ± 0.03 g/g DW respectively. One of the most fundamental and analytical procedures which need to be performed is the moisture content determination. Moisture is a quality factor in the preservation of some products and affects stability of food materials. Moisture is also used as quality factor and often it is specified in compositional standard [20]. The moisture content of the red seaweed G. acerosa and brown seaweed S. wightii was observed as 12.15 ± 0.85% and 22.4 ± 1.79% respectively. The extraordinary nutritional composition of algae in terms of fibre, protein, minerals make algae a nutritive, low-energy food which represents an important food alternative. In addition to the major nutrient elements, the evaluation of proline and chlorophyll content has become an important aspect of nutritional profiling. Several studies have demonstrated that proline elicits stress-stimulated phenolic biosynthesis and stimulation of antioxidant enzyme response pathways [21]. Moreover, at higher concentrations, chlorophyll possesses excellent antioxidant activity and the possible mechanism of action behind its antioxidant activity might be either the protection of linoleic acid against oxidation or prevention of the decomposition of hydroperoxides [22]. Therefore evaluating the proline and chlorophyll content of the seaweeds will be an added advantage in the facet of therapeutics. The proline content of G. acerosa and S. wightii was observed as 83.7 ± 7.4 mol/g and 44.26 ± 8.2 mol/g of DW respectively. Determination of chlorophyll content by spectrophotometric method reveals that about 1.583 ± 0.049 g/g Fresh weight (FW) of chlorophyll A and 1.896 ± 0.10 g/g FW of chlorophyll B was observed in G. acerosa. In the case of S. wightii, 6.56 ± 0.47 g/g FW of chlorophyll A and 3.76 ± 0.56 g/g FW of chlorophyll B was present in it. Essential minerals and trace elements required for human nutrition are the major constituents of seaweeds which ranges from 8–40% [15]. Since seaweeds are a rich source of minerals when compared to land plants, the mineral content of G. acerosa and S. wightii were evaluated in the present study using atomic absorption spectrophotometer. The results reveal that the red seaweed G. acerosa contain high amount of Potassium (K)
Table 3 Mineral composition determined by atomic absorption spectrophotometer in G. acerosa and S. wightii. S. No
Name of the element
1. 2. 3. 4. 5. 6. 7.
Sodium Potassium Calcium Iron Magnesium Lead Zinc
Observed concentration (ppm)a G. acerosa
a
129.05 522.43 450.04 1.549 0.203 0.001 0.350
± ± ± ± ± ± ±
S. wightii 12.79 22.24 24.5 0.10 0.012 0.0003 0.028
4090.2 2830.37 787.48 0.168 1.031 0.006 0.007
± ± ± ± ± ± ±
29.02 141.51 47.24 0.01 0.05 0.00015 0.0005
Results are expressed as Mean ± SD (n = 3).
(522.43 ± 22.24 ppm), whereas S. wightii contains high amount of Sodium (Na) (4090.2 ± 29.02 ppm) (Table 3). Both Sodium and Potassium play an indispensable role in electrical conductivity of the brain and facilitates the improvement of brain functions. Next to Sodium and Potassium, Calcium was found to be the abundant mineral in both the seaweeds (450.04 ± 24.5 ppm in G. acerosa and 787.48 ± 47.24 ppm in S. wightii). Whereas the amount of Mg present was 1.031 ± 0.05 ppm and 0.203 ± 0.012 ppm in S. wightii and G. acerosa respectively (Table 3). Recent reports suggest that low levels of Magnesium contribute to the heavy metal deposition in the brain that precedes Parkinson’s, multiple sclerosis and Alzheimer’s disease. Thus Mg is essential in regulating central nervous system excitability and normal functions [23]. In addition to these major elements, marine algae could be interesting candidates to explore as Fe sources, especially in countries where the algal production is feasible [24]. The amount of Iron (Fe) present in the seaweed G. acerosa and S. wightii was 1.549 ± 0.10 ppm and 0.168 ± 0.01 ppm respectively. Apart from these major elements, G. acerosa and S. wightii was found to possess trace elements like Lead (Pb) and Zinc (Zn) in the concentrations of 0.001 ± 0.0003 (Pb); 0.350 ± 0.028 (Zn) and 0.006 ± 0.00015 (Pb); 0.007 ± 0.0005 (Zn) ppm and respectively (Table 3). Fatty acids were found to possess beneficial effects like cardio-protective, cytotoxic, antimitotic, anticancer, antiviral and anti-mutagenic activities [25,26]. The data on fatty acid composition of G. acerosa and S. wightii is given in Table 4. Total fatty acids present in the red seaweed G. acerosa were found to be 0.5324% (w/w). A mixture of both saturated and unsaturated fatty acids Table 4 Fatty acid composition of G. acerosa and S. wightii. Fatty acids
G. acerosa(% w/w)
S. wightii(% w/w)
Palmitic acid (16:0) Margaric acid (17:0) Stearic acid (18:0) Oleic acid (18:1) Linolenic acid (18:2) Alpha linolenic acid (18:3)
0.1045 0.0011 0.1345 0.0934 0.1034 0.0834
1.23 In traces 1.998 1.3434 1.3454 1.11
A.N. Syad et al. / Biomedicine & Preventive Nutrition 3 (2013) 139–144
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Table 5 Vitamin composition of G. acerosa and S. wightii. S. No
Vitamins
G. acerosa (mg/g of DW)a
S. wightii (mg/g of DW)a
1. 2. 3. 4.
Vitamin A Vitamin E Vitamin C Vitamin B1
5.
Vitamin B2
0.0034 ± 0.0002 1.33 ± 0.07 5.0718 ± 0.202 Below detectable level Below detectable level
0.0040 ± 0.00012 1.35 ± 0.08 5.069 ± 0.40 Below detectable level Below detectable level
a
Results are expressed as Mean ± SD (n = 3).
4. Conclusion
Fig. 1. Amino acid composition of G. acerosa and S. wightii. The values were represented as Mean ± SD.
has been found to be present. The saturated fatty acids present were palmitic acid (0.1045% w/w), margaric acid (0.0011% w/w) and stearic acid (0.1345% w/w). The unsaturated fatty acids include oleic acid (0.0934% w/w), Linolenic acid (0.1034% w/w) and ␣Linolenic acid (0.0834% w/w). S. wightii contained a total of 8.341% of fatty acids, of which 3.228% (w/w) was saturated fatty acids and 3.798% (w/w) was unsaturated fatty acids. The results of fatty acid analysis reveal that both G. acerosa and S. wightii are rich in MUFA and PUFA, which possess important health benefits. In particular, oleic acid (MUFA) and ␣-linolenic acid (PUFA) present in both the seaweeds might help in lowering the blood cholesterol, act as excellent antioxidants, strengthen the cell membrane, repair the damaged cells and tissues, improve the functioning of heart and fight against cancer. Quantitative determination of amino acid concentrations was conducted by HPLC and the results are illustrated in Fig. 1. Fourteen amino acids were detected. Almost all of the essential amino acids including methionine, leucine, lysine, phenylalanine, tyrosine, arginine, isoleucine, threonine and valine and five non-essential amino acids like aspartic acid, glutamic acid, serine, alanine and histidine were found to be present in both G. acerosa and S. wightii. One exception is that among the amino acids, serine, histidine and threonine were absent in G. acerosa. The red alga G. acerosa was found to possess high amount of glutamic acid (13.67 ± 0.95 mg/g of protein), next to that, the amount of tyrosine was high (4.12 ± 0.16 mg/g of protein). Whereas aspartic acid, alanine, arginine, valine, methionine, phenyl alanine, isoleucine, lysine, leucine were found to be present at 1.09 ± 0.087, 1.30 ± 0.07, 2.83 ± 0.25, 1.93 ± 0.11, 2.80 ± 0.16, 1.59 ± 0.11, 0.86 ± 0.06, 2.14 ± 0.17, 1.62 ± 0.08 mg/g of protein respectively. In the case of brown alga, S. wightii, glutamic acid constitutes 18.34 ± 1.65 mg/g of protein, whereas histidine, threonine, arginine, serine, alanine, tyrosine and aspartic acid constitutes about 7.44 ± 0.44, 5.42 ± 0.43, 7.31 ± 0.43, 3.95 ± 0.31, 3.19 ± 0.19, 4.12 ± 0.32, 2.82 ± 0.28 mg/g of protein respectively. Analysis of fat and water soluble vitamins (Table 5) reveals that vitamin C, the major antioxidant was found to be abundant in both the seaweeds, which constitutes to about 5.0718 ± 0.202 mg and 5.069 ± 0.40 mg/g of DW in G. acerosa and S. wightii respectively. The fat soluble vitamins like vitamin A and vitamin E were analyzed by HPLC methods. The results show that vitamin E was found abundantly (next to vitamin C) in both the seaweeds G. acerosa (1.33 ± 0.07 mg/g of DW) and S. wightii (1.35 ± 0.08 mg/g of DW). Vitamin A constitutes about 0.0034 ± 0.0002 mg and 0.0040 ± 0.00012 mg/g DW in G. acerosa and S. wightii respectively, whereas Vitamin B1 and B2 were present only in trace amount.
In the present study, the nutritional composition of marine macro algae G. acerosa and S. wightii were evaluated. The outcome of the present study suggests that both the seaweeds could be employed as potential food supplements and may be used in the food industry as a source of ingredients with high nutritional value. Since both the seaweeds were found to be good source of essential nutrients, their commercial value can be enhanced by improving their quality and further increasing the range of seaweed-derived products. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements K.P.D. wishes to thank UGC, India [F.No.36-6/2008 (SR)] and ICMR, India [IRS ID 2007-02200] for the financial support. S.A.N. wishes to thank UGC-MANF for the Junior Research Fellowship provided. The authors gratefully acknowledge the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by Department of Biotechnology, Government of India; Grant No. BT/BI/25/001/2006). References [1] Kumar M, Gupta V, Kumari P, Reddy CRK, Jha B. Assessment of nutrient composition and antioxidant potential of Caulerpaceae seaweeds. J Food Compost Anal 2011;24:270–8. [2] Ruperez P, Calixto FS. Dietary fibre and physicochemical properties of edible Spanish seaweeds. Eur Food Res Technol 2001;212:349–54. [3] Villarreal-Gómez LJ, Soria-Mercado IE, Guerra-Rivas G, Ayala-Sánchez NE. Antibacterial and anticancer activity of seaweeds and bacteria associated with their surface. Rev Biol Mar Oceanog 2010;45:267–75. [4] MacArtain P, Christopher IR, Brooks GM, Campbell R, Rowland IR. Nutritional value of edible seaweeds. Nutr Rev 2007;65:535–43. [5] Prasad K, Siddhanta AK, Ganesan M, Ramavat BK, Jha B, Ghosh PK. Agars of Gelidiella acerosa of west and southeast coasts of India. Bioresour Technol 2007;98:1907–15. [6] Kaliaperumal N. Products from seaweeds. SDMRI Res Publ 2003;3:33–42. [7] Vijayaraghavan K, Prabu D. Potential of Sargassum wightii biomass for copper (II) removal from aqueous solutions: Application of different mathematical models to batch and continuous biosorption data. J Hazard Mater 2006;137:558–64. [8] Oza RM, Zaidu A. Revised Checklist of Indian Marine Algae. Bhavnagar, India: Central Salt and Marine Chemicals Research Institute; 2003, p. 1–296. [9] Krishnamurthy V, Joshi HY. A Check List of Indian Marine Algae. Bhavnagar, India: Central Salt and Marine Chemicals Research Institute; 1970, p. 1–36. [10] Wong KH, Cheung PCK. Nutritional evaluation of some subtropical red and green seaweeds. Part I – proximate composition, amino acid profiles and some physicochemical properties. Food Chem 2000;71:475–82. [11] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [12] Fournier E. Colorimetric Quantification of Carbohydrates Unit E1.1.1. In: Wrolstad RE, Reid DS, Smith DM, Penner MH, Decker EA, Sporns P, editors. Handbook
144
[13] [14]
[15] [16]
[17]
[18] [19]
A.N. Syad et al. / Biomedicine & Preventive Nutrition 3 (2013) 139–144 of food analytical chemistry. New York, USA: Hoboken, N.J. John Wiley and Sons Inc.; 2005. p. 653–60. Bates LS. Rapid determination of free proline for water-stress studies. Plant Soil 1973;39:205–7. Dere S, Günes T, Sivaci R. Spectrophotometric determination of chlorophyll - A, B and total carotenoid contents of some algae species using different solvents. Turk J Bot 1998;22:13–7. Ruperez P. Mineral content of edible marine seaweeds. Food Chem 2002;79:23–6. Yayli N, Kiran Z, Seymen H, Genc H. Characterization of lipids and fatty acid methyl ester contents in leaves and roots of Crocus vallicola. Turk J Chem 2001;25:391–5. Gratzfeld-Huesgen A. Sensitive and reliable amino acids analysis in protein hydrolisates using the Agilent 1100 Series HPLC. Technical Note by Agilent Technologies; 1999. Publication Number 5968-5658E. Bradbury JH, Singh U. Thiamin, Riboflavin, and Nicotinic acid contents of tropical root crops from the South Pacific. J Food Sci 1986;51:1563–4. Tierra M. Treatment based on the six methods of traditional Chinese medicine. Chapter 5. In: Treating cancer with herbs. Wisconsin, USA: Michael Tierra, Lotus press; 2003, p. 111.
[20] Nielsen SS. Moisture and total solid analysis. In: Food Analysis Laboratory Manual. Fourth edition Springer; 2010, p. 85–104. [21] Shetty K. Role of proline-linked pentose phosphate pathway in biosynthesis of plant phenolics for functional food and environmental applications: a review. Process Biochem 2004;39:789–803. [22] Lanfer-Marquez UM, Barros RMC, Sinnecker P. Antioxidant activity of chlorophylls and their derivatives. Food Res Int 2005;38:885–91. [23] Murck H. Magnesium and affective disorders. Nutr Neurosci 2002;5: 375–89. [24] García-Casal MN, Ramírez J, Leets I. Bioavailability from electrolytic and reduced iron in humans is enhanced by NaFe-EDTA and vitamin A in corn and wheat flours Effect of serum retinol status. Afr J Food Sci 2009;3: 131–8. [25] Prabhakar V, Anandan R, Aneesh TP, Jayasree NB, Nair SV, Halima OA. Fatty acid composition of Sargassum wightii and Amphiroa anceps collected from the Mandapam coast Tamil Nadu. J Chem Pharm Res 2011;3:210–6. [26] Ortiz J, Romero N, Robert P, Araya J, Lopez-Herna’ndez J, Bozzo C, Navarrete E, Osorio A, Rios A. Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea Antarctica. Food Chem 2006;99:98–104.