Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 8 (3 and 4): 147-151, 2012 2012 Elias Academic Publishers

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Chemical and Spectral Characterization of Cell Wall Polysaccharide of Gracilaria edulis (Gmelin) Silva
B. Madhaiyan and N.Rajasulochana*
Department of Plant Biology and Plant biotechnology, Presidency College, Chennai-600 005, Tamil Nadu, India.

Received: 12 January, 2012; revised received: 26 May, 2012

Abstract
The red alga Gracilaria edulis (Gmelin) Silva (Gigartinales) was collected from Rameswaram coast of Tamil Nadu. Proximate analysis was carried out. After extracting the phycocolloid, the yield and physico-chemical properties were determined. The gel strength of agar was found to be dependent on higher 3,6anhydrogalactose content and inversely related to the sulphate. The cell wall polysaccharides extracted from G. edulis was analysed by FTIR spectroscopic method. Keywords: Gracilaria edulis, cell wall polysaccharide, phycocolloid ,anhydrogalactose.

Introduction
Seaweeds contain large amount of carbohydrates, proteins, minerals, trace elements and vitamins. Gracilaria species are also utilized as human food, mostly in salads and soups and also as feed for marine animals. Having this in mind, proximate analysis of Indian seaweeds were carried out by several algologists in different Coasts (SumitraVijayaraghavan et al., 1980; Chennubhotla et al., 1990; Kaliaperumal et al., 1994;Ganesan and Kannan, 1994; Sarojini and Subbarangaiah, 1999; Roslin,2003). The major value of some seaweed lies in their relatively large content of hydrophilic colloidal polysaccharides such as agar, algin and carrageenan. Agar is of great importance in the food and drug industries and has found several applications in bacteriology, plant tissue culture, biochemistry and molecular biology. Despite its high price, increasing with its degree of purity, agarose cannot be challenged by other colloids (alginates, carrageenans, etc.) in its specific uses (Armisen and Galatas, 1987).The polysaccharide agar is composed of repeated units of 3-linked -D-galactose and 4-linked -galactose units. The production of agar by seaweeds shows considerable variations in gel quality and yield from different algal species and strains (Cote and Hanisak, 1986) due to seasonal fluctuations. Agar has many applications depending on its quality or properties. The yield and physico-chemical properties of phycocolloids obtained from various species of agarophyteswere recorded in earlier works (Duckworth et al., 1971; Oza ,1978; Chennubhotla et al., 1986; Kaliaperumal et al.,1990; Sasikumar et al., 1999; Vimalabai et al., 2003). The alkali treated agar showed low sulphate and with the high 3, 6-anhydrogalactose content in Gracilaria sp. (Villanueva et al., 1999; Praiboon et al., 2006). FTIR spectroscopy has been used as an effective tool to identify the type of polysaccharides.The spectral analysis of the agar from Gelidium madagascariense, Gelidiella acerosa and Gracilaria millardetii showed the presence of agar polymers, with an intense peak at 890 cm-1, characteristic of a non sulphatedosidic ring as found in agar (Stancioff and Stanley, 1969; Villanueva et al., 1999; Rajasulochana, 2009). Most of the 4 linked units are found as 3, 6 anhydrogalactose, resulting in an intense peak at 930 cm-1.As the information available is very scanty on the spectral studies of agars in India, the present study is focused to investigate the physico-chemical properties and FTIR spectroscopy of polysaccharide extracted from Gracilaria edulis (Gmelin) Silva.
*Corresponding author; Email address: rajasulochana_n@ymail.com

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Materials and Methods

Thalli of Gracilaria edulis(Gmelin) Silva(Gigartinales) were collected from the coast of Rameswaram, TamilNadu, India. The samples were washed in seawater to remove sand, mud and epiphytes and then shade dried. The dried algae were powdered and used for estimation of total carbohydrates (Dubois et al., 1956), proteins (Lowry et al., 1951) and lipids (Folch et al., 1957). Extraction of phycocolloid was done by the method adopted by Visweswara Rao et al., (1965). The polysaccharide so obtained was dried, powdered, transferred to air tight glass bottle and stored for quality analysis. The percentage yield of the dried polysaccharide was calculated from the dry weight of the original sample (raw material).Gelling temperature, melting temperature and gel strength were determined for 1.5% phycocolloid (Hellebust and Craigie, 1978). Total galactosewas estimated by the phenol sulphuric acid method (Dubois et al., 1956;). 3, 6 anhydrogalactose (3, 6 AG) was estimated following the acetal - resorcinol method of Yaphe and Arsenault (1965) and sulphate by following method of Verma, (1977). Values were expressed as percentage of polysaccharide dry wt (% ps dry wt). Spectrum was recorded using Bruker Vector 20 FT-IR double beam Spectrophotometer in the range of 4000-600 cm-1, under identical conditions and compared with spectrum of commercial agar.

Results and Discussion

Proximate Analysis The moisture and ash content of Gracilaria edulis were observed as 10 % and 21%. The carbohydrate content was recorded maximum(21%) than the protein and lipid in the present study (Table1). The biochemical studies revealed the order of concentration was carbohydrate> protein>lipid. The higher carbohydrate content might be due to the phycocolloid content of the cell wall (Dhargalkar et al., 1980). The carbohydrate and lipid content recorded in the present study is in conformity with earlier reports (Sumitra Vijayaraghavan et al., 1980; Chennubhotla et al., 1990; Ganesan and Kannan, 1994; Sarojini and Subbarangaiah, 1999). It is quiet natural, since the seaweeds usually accumulate smaller quantities of fatty acids and lipids. The protein content of G.edulis is comparable with earlier findings (Kaliaperumal et al., 1994; Roslin ,2003).The chemical composition of seaweeds varies with species, habitats, maturity and environmental conditions.
Table 1: Biochemical analysis of Gracilaria edulis. Parameters Moisture (%) Ash(%) Carbohydrate(%) Protein(%) Lipid(%)

10 21 21 12 2.3

Physico-chemical Properties of Phycocolloid The physico-chemical properties of phycocolloid are given in Table 2. The phycocolloid obtained from G.edulis showed gel formation at room temperature, confirming their agar nature. The yield of agar was obtained from G.edulis (26%).This is comparable with earlier observations (Sasikumar et al., 1999; Vimalabai et al., 2003). The gelling and melting temperature of agar extracted from Gracilaria edulis is 38C and 80C. The gel strength of agar of G. edulis is 106 g/cm2. These are in conformity with earlier report (Chennubhotla et al., 1986). Gelling and melting temperature is dependent on molecular weight distribution. Gelling ability of phycocolloids depends on the molecular weight and level of 3, 6 anhydrogalactose (Murano et al., 1990). The gel strength showed positive significant correlation with melting temperature. The gel strength of phycocolloid is negatively correlated with sulphate content (Villaneuva et al., 1999). Salinity and temperature affect the gel strength of agar from Gracilaria.
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Madhaiyanand Rajasulochana / Characterization of Cell Wall Polysaccharide of Gracilaria edulis Table 2: Physico-chemical properties of Gracilaria edulis. Parameters Yield (%) Gelling temperature( C) Melting temperature( C) Gel strength(g/cm) Galactose(%) 3,6anhydrogalactose(%) Sulphate (%)

26 38 80 106 36 36 2.2

The chemical analysis of G. edulis was performed to determine the composition of total galactose, 3, 6 anhydrogalactose and sulphateIn the present study, the galactose, 3,6 anhydrogalactose and sulphate content of G .edulis is similar to the findings of Duckworth et al., (1971), Sasikumar et al., (1999 ) and Villanueva et al., (1999) in species of Gelidium and Gracilaria. Galactose content of phycocolloid played a significant role in its yield and the 3, 6 anhydrogalactose and sulphate content contributed to its quality viz. gel strength.

Figure 1: FTIR Spectrum of polysaccharide from Gracilariaedulis compared with Difco agar.

FT-IR Spectral Analysis As in Difco agar, spectrum of G. edulis exhibited more or less common constant peaks around 620, 670, 770, 890, 930, 1020, 1080, 1150, 1250, 1380, 1420, 1640, 1720, 2850, 2925, and 3410 cm-1. Some additional peak exhibited in the study sample (Fig.1). The occurrence of peak at the 2925 cm-1 signifies C-H asymmetric stretching. The 1740 cm-1 band galactan is assigned
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to C=O stretching of carboxylic group but this peak is not exhibited in spectrum of G. edulis. This may be due to generic and species variation. Strong and very sharp peak at 1640 cm-1 is observed in the present study for amide I deformation / C=O asymmetric stretching. The peaks at around 1070, 1150, 1250, cm-1 are also due to ester sulphate. This result is similar to earlier observations (Christiaen and Bodard, 1983, Rajasulochana et al., 2009). In the present study the peaks at around 1450 cm-1 and 1370 cm-1 noticed in both the spectra of polysaccharides, which are attributed to asymmetrical and symmetrical stretching of CH3/CH2 groups respectively. This observation is similar to earlier findings (Troung et al., 1988). The additional peaks at 817, 856 and 878 cm-1 were exhibited in G. edulis. The peaks at 817 and 878 cm-1 for ester sulphate in C-6 and peak at 856 cm-1 is ester sulphate in C-4. This is comparable with previous observations (Villanueva and Montano, 1999; Miller, 2003).This may be due to developmental variation associated with enzymes involved in the synthesis of polysaccharides. The peaks of 931and 960 cm-1 very well correspond to the interpretation of Stancioff and Stanley (1969) as due to the vibration of 3, 6 anhydrogalactose. The band at 890cm-1 is typical of non sulphated -D galactopyranose residues of agar. The linking of carbon-sulphur is determined by the peaks 637 and 708cm-1. Similar observation has been made by many workers (Christiaen and Bodard 1983; Villanueva et al., 1999). The OH / NH stretching mode occurs at around 3400-3500 cm-1 with very strong intensity of bands. As the gelling temperature of agar from Gracilaria edulis is high (38C) unlike the general gelling temperature of the bacteriological agar which ranges from 32-36C, hence it can only be recommended as a food grade agar. Further research towards addition of certain chemicals to bring down the gelling temperature may help in making the bacteriological grade of agar.

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