Characterizing the Three Different Alginate Type of Sargassum siliquosum

This research was aimed to identify the brown seaweed, to characterize the acid, sodium and calcium alginate, and to examine the alginate yield. The identification was done phaenotypically. The extraction method was pretreated by ethanol depigmentation, followed by the extraction of Na2CO3/EDTA andCaCl2 and presipitated with absolute ethanol. The characterization of alginate was done by FT-IR spectroscopy and Thin Layer Chromatographyby comparing the samples with standard alginate (Sigma, USA). The key of identification showed that the species was Sargassum siliquosum. There are similarities in signal vibration and TLC spots among the samples and the standard. The TLC test was also showed that those alginates contain mannuronic and guluronic acid. The highest yield was produced by Sodium alginate (40.34% + 0,21), followed by Acid alginate (11.51% + 0,15) and Calcium alginate (4.8% + 0,09).


Introduction
Biopolymers, that originally come from marine natural products, has been studied very progressively lately (Fawzy et al., 2017).Moreover, those application in industry have been devepoled very well.Alginate is one of the biopolymer which can be applicated in cosmetics, foods, drugs (Falkeborg et al., 2014) as well as in marine culture (Isnansetyo et al., 2014;Yudiati et al., 2016).These biopolymers was kept in the brown seaweed cell wall such as Turbinaria sp., Sargassum sp., and Padina sp.
Alginate is a linear polysaccharide which constructed as α-L-guluronate (G) dan C5 epimer β-D-mannuronate (M), homopolymeric blocks (polymannuronate and polyguluronate), and threaded as heteropolymeric blocks (alternative GM blocks or G/M blocks).Guluronate and mannuronate are uronate with carboxyl groups at C5, every configuration shows the difference between two pyranose (Pawar & Edgar, 2011).Alginate is an insolute part of algae (Draget & Taylor, 2011) and take place in intercellular matrix as a gel which consist Natrium, Calcium, Magnesium, Strontium and Barium ions.The ionic composition was determined by the balance of ionic exchange and seawater (Pawar & Edgar, 2011).
Alginate have a high abundant of free hydroxil and carboxyl group bonds which isdistributed along the backbone of their polymers chain.These two functional groups is enable to modify and shifting differently from the original coumpound (Yang et al., 2011).Na2CO3 is an alcalic compounds which can modify the carboxyl groups, so therefore, shift the conformation.CaCl2 which is added in the Calcium Alginate extraction will build an "egg box" for more stabilized alginate (Yang et al., 2009).Both compounds, will be expected producing more yield by EDTA addition (Jork et al., 2000).
The brown algae Sargassum siliquosum is abundant in South Sea, Gunungkidul, Yogyakarta, Indonesia and still unexploitated.The simple and unexpensive extraction methods of this local n tropical algae will pursue some new information by targetting the higher yield of alginate and the prosperous of Indonesian alginate industry in the future.

Identification of Brown Seaweed
The key of phaenotypic characters of brown algae was determined by the main axis, vesicle, receptacle, and thallus (Nguyen, 2015).Sargassum genus from Phaeophyta is one of the largest species all over the world (Nguyen et al., 2013).Consequently, this species is ecologically important in the marine ecosystem including Indonesia.Is has been reported that there was around 400 species of Sargassum spread over the world and 14 of these have been found in Indonesia : Sargassum duplicatum, S. histrix, S. echinocarpum, S. gracilimun, S. obtusifolium, S. binderi, S. policystum, S. crassifolium, S. microphylum, S. aquofilum, S. vulgare, S. polyceratium dan S. siliquosum (Rachmat, 1999).Due to the morphological plasticity of Sargassum, the genus identification is slighly difficult (Stiger & Payri., 1999).
Extraction ofAcid, Sodium andCalcium Alginate of S. siliquosum S. siliquosum was collected from Sundak Coast, Gunungkidul, Yogyakarta, Indonesia.The collection were then brought to the laboratory for cleaning up andwas rinsed with tapwater dan then dried up in room temperature without sun light exposure.
The acid, calcium and sodium alginate extract was prepared based on the methods of Jork et al. (2000), Davis et al. (2004), andKim et al. (2004),respectively.Prior to the extraction, the dried seaweed was depigmented with 85% ethanol until colourless.The preparation of acid alginate was done by 2 hr waterbath extraction of S. siliquosum with 2% Na2CO3at 70 o C. Filtration was then administered, continued byprecipitation of HCl at pH<1, and followed by centrifugation at 3.500 rpm for 20 min.The supernatant was then discharged and the pellet was washed in absolute ethanol (1:1) and then filtered.The sodium alginat was prepared by overnight magnetic stirrer extraction with 5% Na2CO3/ 50mM EDTA.The pellet was then filtered, 0.13 M KCl was added and followed by 96% ethanol in 1:1 volume, stirred well.Centrifugation was then performed at 3.500 rpm for 5 min.The extraction of Calcium alginate was prepared by 60 o C waterbath immersion with 0,2 N HCl for 2 hrs.The pellet was then filtered at pH=7, followed by washing with absolute ethanol and then centrifugation was administered at 3000 rpm for 15 min and added with aquadest until pH=2.Finally, the all types ofalginate were collected and then dried overnight in the oven at 60 o C.The three types of alginate yield were then determined by comparing the dried weight before and after extraction (%).

FT-IR spectroscopy
The characterization of alginates were determined spectrophotometrically by signal vibrationusing Fourier Transformed-Infra Red.Preparation was done by mixing the samples with KBr in pellets formation (10% w/w).It was then recorded at the 4000-500 cm -1 region using a Thermo Nicolet 380 FTIR (Germany).

Thin Layer Chromatography
Prior to this, the three type of alginate extracts as well as the standard alginate (Sigma, USA) were hydrolised by aquadest dilution and added with TFA (Triflouroacetic acid) and then heated up to100 o C for six hours.
The hydrolised alginate were then spotted to the TLC plate (silica gel as static phase and isopropanol, ethyl acetate and aquadest 7:2:1 vol/vol as mobile phase).Aniline was used as visualisation.The plate was then heated up to 105 o C for five minutes.The alginate and monosaccharide compunds was appeared and the Rf value were then counted.

Brown Algae Identification
The key phaenotypic characters of this brown algae showed that the main axis is silindrical, the leaves periphere isserrated and sharp, the vesicula was ovally without wings.The receptacle was compact and grouped, cilindrically and without spines.The thallus was ovale, and up to 1 cm in long.Based on the phaenotypic characters, it was concluded that the species of this brown seaweed is Sargassum siliquosum.

Alginate Extraction and Yield
Both, acid and sodium alginates, were extracted in Na2CO3.Based on the result the yield obtained from sodium and acid alginates was higher than calcium alginate (Table 1).The addition of EDTA in sodium alginate extraction even reached the highest yield.EDTA (ethylendiaminetetraacetic acid) has known as a chelating agent and this, consequently, was improved the extract yield more than three times.Rahelivao et al. (2013) reported that EDTA addition in three alga species Sargassum sp., Turbinaria sp.dan Hormophysa sp from Madagscar gave the highest yield.The addition of CaCl2 will build some calcium mats called egg box which is the interaction chain mediated by Ca 2+ (Yang et al., 2009).Based on the molecular density, it has also been reported that CaCl2 was also improve the gelling properties (Yang et al., 2011).Eventhogh, the yield of Calcium alginate was the lowest one.(Chandia et al., 2004).The extraction of S. filipendula was done by formalin 0.4% (w/w) maseration, Na2CO3 addition and acid precipitation.The formaline maseration was aimed to eliminate the phenolic compound and produced the brighter colour (Bertagnolli et al., 2014).Hernandez-Carmona et al. (2002) have done the extraction of acid to sodium alginate of Macrocystis pyrifera brown seaweed and resulted 14.3% in yield.The extraction temperature and the sample size had influenced the sodium alginate yield (44.01-51.8%) of Laminaria digitata.The highest result was reached in 40 o C and < 1 mm sample size (Fertah et al., 2014).

Based on
Up to now, 23 tonnes of world commercial alginate is produced from 85 tonnes dries algae.Based on the table above, in fact, the highest yield was produced from this research by Sargassum siliquosum from local tropical Indonesian coast.
The ability of alginate into gel formation is a great potency of being a biomaterial product as well as the matrix of supporting the renewable and regeneration of human tissue.Moreover, the alginates have an ability as a biocampatible, biodegradable, non-antigenic dan chelating agent.There are some application of alginate in biomedical has been reported includes the tissue improvement (Chandika et al., 2015), drugs wrapping, also useful for the enrichment of the cancer stem cells (CSCs) (Xu et al., 2014).Mutia et al. (2011), has also reported that alginate is a fine primary dressing for  et al., 2005;Liu et al., 2006, Yeh et al., 2009;Chung et al., 2011;Yudiati et al., 2016) was quite interested to conduct some expreriments and counteract the problem on marine culture.

FT-IR Spectroscopic Analysis
The FT-IR spectra from three different type of algiante compared to the standard alginate (Sigma, USA) can be seen in Figure 1 and the vibration signal is shown in Table 3.There is a wide band at 3400 cm -1 shows the signal of O-H stretching vibration, while the signal at 2900 and 1600 cm -1 is interacted to C-H stretching vibration and O-C-O carboxylate bound assymetrically.The absorbance around 1401 cm -1 is correlated to the deformationvibration of C-OH, which is the contribution of O-C-O symetrically stretching vibration from carboxylate group (Mathlouti & Keoning, 1986;Silverstein & Webster, 1991).
The obeserved band at around 1300 cm -1 was predicted from deformation of C-C-H (dan O-C-H) attributes.Furthermore, 1095 band was the stretching from C-O vibration at pyranose ring.The stretching formation from C-C vibration was measured at 1033 cm -1 .The indication of uronic acid wich formated by the C-O group was observed at 946 cm -1 wavelength number (Chandia et al., 2001;2004).Moreover, the recorded signal at around 900 cm -1 shows the existency of assymetric α-L-gulopyranuronate vibration ring.The maruronic acid residue was observed at 815 cm -1 (Mathlouthi & Koenig, 1986;Chandia et al., 2001).
The fingerprint area at 950-750 cm -1 (Tul'chinsky et al., 1976;Mathlouthi et al., 1986) has been mostly discussed.The spectrum band of three types of alginate at 930-940 cm -1 is referred to   C-O stretching of uronic acid residue (Leal et al., 2008).Meanwhile, the band formation at 880-890 cm -1 shows the signal vibration of C1-H deformation at β-mannuronic acid.Wave no at 810.10 cm -1 is the characterisation of mannuronic acid existence (Chandia et al., 2001;2004).The present study of FT-IR analysis showed that spectra of three different types of alginate were fit with that of the standard alginate (Sigma), and positively finger printed at a specific alginate wave number (950-750 cm -1 )

Thin Layer Chromatography Analysis
The monosaccharide compund was analysed using Thin Layer Chromatograpy (TLC) Methods.The TLC analysis showed that there were two spots appeared in Acid, Sodium and Cacium and Standard Alginate (Sigma, USA) compounds.The Rf of standard alginate hydrolisate spot was 0.18 and 0.69.On the other hand, the Rf samples were similar to the Standard alginate, except the Calcium alginate (0,18 dan 0.64).TLC results is shown in Figure 2.
TLC is objected to test the extract purity by comparing the extract's Rf and samples' Rf.The simmilar Rf value shows that the compound is similar.Analysis by Zhang etal. (2006) based on the HPTLC of guluronic acid and mannuronic acid (silica gel as static phase and n-buthanol/ formic acid/ aquadest 4:6:1 (vol/vol) as mobila phase) showed that the Rf value of guluronate acid is higher than marruronic acid.Furthtermore, 0,69 of standard Rf value dan 0.69; 0.64 and 0.69 of Sodium, Calcium and Acid Alginate are guluronic acid.On the other hand, 0.18 is a standard, Sodium, Calcium and Acid Alginate Rf value and these indicate as mannuronic acid.The TLC analysis is shown in Figure 2. By this TLC analysis, it is clearly confirmed that the three alginates samples consist of two monosaccharides ie.guluronic and mannuronic acid.

Conclusion
The S.Siliquosum from Sundak Coast of Gunungkidul, Indonesia have a high yield of the sodium (40.34%), followed with Acid (11.51%) and Calcium alginate (4.8%), respectively.The FT-IR and TLC analysis showed that all three types of alginate have a similar characters compared with standard Alginate (Sigma, USA).The high yield of alginate from Indonesia promising a good opportunities concerning the application of alginate in food, industry, biomedical/pharmacy as well as immunostimulants in marine culture.

Ackonwledgements
Part of this study was financially supported by a grant from the Faculty of Agriculture, Gadjah Mada University.The authors would like to express the appreciation to those who helped support this research at Departement of Fisheries, Gadjah Mada University.Special thanks to students, Ayuningtyas, M. Biotech.and Riska Amelia, S.Pi.for preparing the sample analyses.

Table 1 .
Yield of three different type of alginate of S. siliqosum

Table 2 .
The alginate yield in different species of Sargassum