Chemical Synthesis and Analysis of Seaweed

Chemical Synthesis and Analysis of SeaweedChapter 1 books review2015IntroductionMarine-microalgae be normally termed as seaweed belonging to the unmannered family of non-f impressionering plant known as thallophyta ( design 1). The sea plant lacks the leaves, stem and root of true plants. The body of the seaweed is called the thallus named after the thallophyta. The flattened leaf portion of the microalgae is called the make and its function is to increase the photosynthesizing surface atomic number 18a.Seaweeds are attached to the bottom by a root-like organise called a holdfast. Seaweeds are autotrophic which grows in the intertidal and sub-tidal region of the sea. Seaweeds grow abundantly where rocks and coral are present as substratum (Marine Fisheries information Services October-November 1998 Kaliaperumal et al., 2004).Figure 1 Shows the general twist of seaweed ( Thallophyta)( Reproduced from http//dtc.pima.edu/blc/183/03_183/03_183answers.html).Seaweed may be classifi ed into two categories namely phaephyceae (Brown) and Rhodophyceae (Red). Seaweeds carry varied types of phycolloids such as alginate, carrageenan, agarose and agar agar.Seaweed is mainly utilize in Asia as food, fodders, fertilizer and to a fault as drugs in the pharmaceutical intentness for its therapeutics properties (Marine Fisheries information Services, October-November, 1998).1.1 Brown seaweed (Phaephyceae)Brown seaweed is assemble in the family of seaweed which is called the phaephyceae. The brown seaweed motif varies according to the species, through out(a) the year and between diametrical habitats.Brown seaweed is sedate mainly of carbohydrate such as alginate, laminaran, mannitol, fucoidan and small traces of cellulose as shown in Table 1. (Fasahati et al., 2012)Table1. Brown seaweed compositionComponentBase design dry out weight (%)Used in simulationdry weight (%) alter23.6325.48Algin27.7827.78Laminaran16.6718.52Mannitol14.8114.81Fucoidan3.7Cellulose6.6Protein7. 417.411.2 AlginateAlginate is a major structural biopolymer found in cell wall and intercellular matrix which provides mechanical strength in brown seaweed. Seaweeds containing alginate are called alginophyte (Yabur et al., 2006). In the natural environment, alginate exists as a compartmentalization of potassium, calcium and atomic number 11 salt (Gomez et al., 2009).1.2.1 Structure of alginateThe structure differs from species, age and from unalike part of seaweed. Alginate consists of two monomers and is linked to to each one other alternately in a linear form. The monomers are namely -D-Mannuronate and -L-Guluronate in Fig 2(B) and 2(D) respectively. The dot form of mannuronate and guluronate, is mannuronic window glass and guluronic blistery (Fig 2(C)) respectively. Figure 2(A) depicts the structure of atomic number 11 alginate in a linear form.Alginate is the salt of alginic deadly and its simplest formula is NaC6H7O6 in Fig 2(A) (Mahmood et al., 2009).. 2(A) 2(B) 2( C) 2(D)Figure 2 Repeat unit of (A) Sodium alginate (B) guluronate, (C) guluronic acid and (D) mannuronate (Falkeborg et al., 2014)The monomers are linked through carbon 1 and 4 and have sequences forming thwarts of MM(Fig 3(A)), GG(Fig 3(A)) or GM blocks in Fig 3(A) (Arzate-Vzquez et al., 2012, Morais et al., 2013). Mannuronates and guluronates both have carboxylic root word on the carbon 5 of their respective structures (Falkeborg et al., 2014). (A)G G M M (B)G G M M GMMMMGGGGGGGMGMGMGMGMMMMG (C) MM blocks GG blocks MG blocks MM blocksFigure 3 (A) Alginate structure with G and M take over units, (B) showing the repeat of G and M joined together (C) block fractions of alginate polymer (Yuemei Lin et al., 2010)1.3 Species of brown seaweedsAlginate are commercially manufactured mainly from genus Laminaria hyperborea, Macrocystis pyrifera, Laminaria digitata, Ascophyllumnodosum, Laminaria japonica, Eclonia maxima, Lessonia nigrescens, Durvillea Antarctica, Turbinaria conoides, Turbi anria ornata and Turbinaria decurrens, Sargassum, Turbinaria ,Hormophysa, Cystoseira and Sargassum sp (Bertagnolli et al.,2014 Chennubhotla et al., 2013 Kaliaperumal et al., 1974).1.4 Extraction of alginateGomez et al., (2009) described the pre-treatment and the lineage of alginate from brown seaweed. The seaweed was number 1-pitched and was added to urine to moisten the latter. 0.1 N Hydrochloric acid was added to the sample and the solution was affected in order to reach a pH value of 4. This fulfill was continued for 15 min at style temperature and the supernatant was eliminated. The pre-treatment was carried out three times for each sample using 0.1 N Hydrochloric acid.The extraction was followed by the extension of the pre-treated solution to a beaker with 1 N sodium carbonate (pH of solution 10). The mixture was displace mechanically for a stop of 2 instants at a temperature of 60 oC. To the extracted solution a atomic number 14 based rock was added and was stirred f or 15 minutes. The mixture was and then centrifuged to amaze the sodium alginate supernatant.Sodium alginate can be purified using three opposite routes namely (i) Ethanol route, (ii) Hydrochloric acid route and (iii) Calcium chloride route as summarised in scheme 1.(i) Ethanol pathwayThe ethanol pathway was performed by the direct precipitation of sodium alginate using ethanol. The precipitate was washed then with ethanol under soxhlet condition for 100 hours to obtain the pure biopolymer which was dried-out under vacuum.(ii) Alginic pathwayThe extract alginate was mixed up with 1 N of HCl at room temperature under constant stirring for 1 hour at a pH of 1. To obtain the alginic acid, the mixture was separated by centrifugation by adding peeing and 1 N sodium carbonate. The mixture was left wing at room temperature under stirring for 1 hour in order to obtain the soluble form of sodium alginate. The polymer was precipitated by the addition of 11 volume ratio of ethanol by the mode described in the ethanol pathway.(iii) Calcium chloride pathwayTo the extract of sodium alginate, 1 M calcium chloride was added and the precipitate was thoroughly washed with distilled water using a soxhlet for 64 hours. The mixture was agitated at room temperature and distilled water was then added followed by the addition of 1 N Hydrochloric acid until a pH 2 was reached. The alginic acid which is insoluble was separated from the supernatant by centrifugation and the insoluble alginic acid was washed with 0.05 N HCl and the solution were stirred at room temperature. The precipitation of alginate was obtained by the addition of Na2CO3 to obtain sodium alginate. The purified alginate was collected by the soxhlet as discussed in the ethanol pathway. design1. Extraction of alginate (Gomez et al., 2009)1.5 slacken off of alginateThe yield of alginate is being affected by different factors such as the species, age, period of harvest, temperature of extraction, extracted with e ither alkali or acid and on the national of alginate in brown seaweed in like manner the size of alginate too affects the yield.(Fertah et al., 2014)Seaweeds that are freehanded in summertime show a disgrace content of alginate in the branches and stems (15.1 %) and no trace of alginate in the receptacles (Bertagnolli et al., 2014).Sample SS size is 1mm and BS for 1mmoC (51.8 for SS and 44.01for BS) however an increase in temperature proved to have a drastic mitigate in the yield shown in hedge 2 (Fertah et al., 2014). Table 2. defer of alginate at different size sample and temperatureSize1mmTemperature /o C254060254060 number/%38.3351.843.235.2844.0140.2There are up to 40% of alginate content in brown seaweed and the content of alginate depending species of brown algae can vary from 10 to 25% (Yabur et al., 2006).The period of collected of seaweed Sargassum filipendula have different yield of alginate. During fall, ring and summer the yield are 17.0 0.1 %, 17.20.3 % and 1 5.1 0.1 % respectively. The yield is lower in summer than spring and fall beca employment summer algae have lower receptacle and branches which are characteristic in their re harvest-feastive period. (Bertagnolli et al., 2015).The yield of alginate is also affected to different species of Sargassum shown in table 3. The highest yield is from S.vulgare is 30.2 % and the lowest S.dentifulum is 3.3 %.Table 3 Yield % of alginate from different species of SargassumSpecies of seaweed% YieldS.vulgare30.2S.polycystum17.1-27.6S.dentifilium3.3S.latifilium17.7S.asperifolium12.4S.oligocystum16.3-20.5S.fuiltans21.1-24.5S.filipendula15.1-17.21.6 Biosynthesis of alginateThe proposed biosynthesis of alginic acid in Fucus gardineri in the presence of bacteria Pseudomonas is shown in Scheme 2 (Lin and Hassid 1966).Mannose 1-phosphate reacts with GTP/ H+ in the presence of mannose 1-phosphate guanyl transferase to produce GDP--D-mannose and the by product is diphosphate. GDP--D-mannose in the presence of enzyme GDP-mannose dehydrogenase react with water and NAD+ giving GDP-Mannuronate and lastly reacting with GDP/H+ to give alginate.Scheme 2 Biosynthesis of alginate in brown seaweed Fucus gardineri and bacteria (Lin and Hassid 1966)1.7 Characterisation of alginateExtracted alginate may be characterised using a variety of techniques such as 1H-NMR, FT-IR, SEC, DSC, CHNS elemental analysis and viscosity.1.7.1 1H-NMR1H-NMR spectroscopy is apply to determine the main structural pattern and composition of alginate. Structures of alginate are chiefly constituted of homopolymeric blocks i.e M and G blocks that can be separated by heteropolymeric of MG blocks. The M/G ratio has a strong establish on the physical properties of alginate.Figure 41H-NMR Spectra for solution of alginate form S. vulgare in D2O (a) SVLV (S.vulgare low viscosity) and (b) SVHV (S.vulgare High viscosity) (Torres et al., 2007)Figure 4 (a) and (b) show the 1H-NMR spectra of alginate extracted from SLHV and SLHV respectively. Peak I (5.06 ppm) was attributed to anomeric proton (G-1) guluronic acid, peak II (4.7 ppm) show the anomeric proton (M-1) of mannuronic acid and the C-5 alternating blocks (GM-5) which is overlapping each other at this peak and peak III (4.4 ppm) is for the anomeric peak of guluronic acid H-5 (G-5) (Torres et al., 2007).1.7.2 FT-IR analysisFT-IR spectroscopy is used to determine the functional groups present in the structure of alginate. The FT-IR spectra of the extracted alginate from brown seaweed Turbinaria ornata is given in (figure 5). The bands which is around 800 cm-1 corresponds to the C-H which is out glance over of the aromatic ring. The peak near 1040 cm-1 is due to the stretching vibrations of C-O bond. The bands at 1230 and 1600 cm-1 like to C-N stretching and N-H bending vibration respectively show the presence of acyclic amine. The O-H alcoholic groups shows stretching vibrations band at around 3400 cm-1 and C=O stretching vibrations due to the carbo xylic acids group present in the structure (Gowtham Sriram et al., 2014).Figure 5 FT-IR spectra of Turbinaria ornata seaweed (Gowtham Sriram et al., 2014)1.7.3 CHNS elemental analysisTable 4 shows the lead for elemental analysis for SVHV and SVLV and both sample proved to have exchangeable results. The nitrogen content is due to the protein found in the seaweed. The % C are 27.7 and 28.1, %H are 5.15 and 4.4 for SVLV and SVHV respectively. The %N (nitrogen) is due to the presence of protein which contains amine groups in seaweed. % S are zero for both SVLV and SVHV(Torres at al., 2007)Table 4 Analytical data of Sargassum Vulgare alginateAnalytical parameter (m/m %)SVLV(S.Vulgare low viscositySVHV(S.Vulgare high viscosityH5.154.49C27.728.1N0.1780.164S001.7.4. Viscosity indispensable viscosity is the measurement of hydrodynamic volume occupied by complex molecule at measureless dilution in specific responses at a given temperature. The innate viscosity depends on the molar mass (Mv), composition and the sequences of M and G residues. indwelling viscosity is represented as comparison 1 = sp/C comparability (1)Where sp is specific viscosity and sp/C is reduced viscosityAnother equating of reduced viscosity is defined asred = compare (2)the intrinsic viscosity was found by the classical method of a plotting solid line graph according to the Huggins Equation (3) to which y-intercept is obtained.red = + kH2 C Equation (3)Graph of reduced viscosity versus alginate concentration for SVLV and SVHV sample shown in figure 6 (Torres et al., 2007). Figure 6 Specific viscosity of SVLV (S.Vulgare low viscosity) and SVHV(S.vulgare high viscosity)The viscosities were performed in 0.1 N NaCl at 25 oC for both SVLV and SVHV. integral viscosites are 6.9 and 4.14 g/dL for SVLV and SVHV respectively (Torres et al., 2007).Table 5 shows different for different species of brown seaweed. High vis The intrinsic viscosities of S.Vulgare algiante ranges from (2.5-15.4) g/dL cosity was found SVLV due to high content of MM blocks. Table 5 Intrinsic viscosity and average molar masses of alginates from different sources(Torres et al., 2007)Alginate source (dL/g)Mwa cv (g/mol)L. hyperborean6.43.05F. vesiculosus2.51.17A. nodosum2.81.32L. japonica15.47.44S. fluitans6.303.00S. dentifolium12.66.06S. asperifolium15.27.34S. latifolium8.74.16S. vulgare This study4.11.94LV (low viscosity )HV (high viscosity)6.93.301.7.4.1. molar(a) massMolar mass (Mv) is determined by using the Mark-Houwink equation (4), = k Mva - Equation (4)where k and a are empirical coefficients that are dependent on the polymer and the solvent-temperature system used.As for alginate, a value ranges from 0.73 to 1.31 can be acquired depending on the composition of alginate and ionic strength. G-rich alginate has higher a values while lower a values are observed in M-rich alginate which is flexible in natureProposed empirical relations for and the weight-average molar mass (Mw) for SVHV and SVLV was computed using equation 4 and the result shown in table 3 = 0.023 Mw 0.984 - Equation 4where is given in dL/g and M in kDaltons.The a value used is 0.984 which is different on at specific solvent used. Average molar mass for SVHV was higher than SVLV and similar values to S.fluitans. SVLV has a lower value than other Sargassum species reported in table 5 (Torres et al., 2007).1.8 Uses of alginateAlginate has many areas of application. For example in food industry and is use to stabilise mixture dispersion and emulsion which increase the viscosity of the gelatine and forms gel such as jam and jellies. Alginate gel films are also utilized in the food industry as edible films on fruits and vegetables and as coatings designed to improve stored meat quality, industrial and pharmaceutics because the water in alginate has the ability to hold and form gel, to form and stabilise emulsion, fabric industry, rubber adhesive paper products, cosmetics and in dental mould (Chennubhotl a et al., 2013 Crossingham et al., 2014).Alginate also protects against carcinogenic substances, it clears the digestive system and protects the membrane of the stomach and intestine. Alginate is used in the manufacturing of soft capsules and is consumed as beverage for lowering blood sugar level. Alginate is used in the textile industry as an additive for textile and has also been investigated recently in the composition in vaginal film venereal infection in pharmaceutical industries and alginate is also used in wound dressings (Paul et al., 2004).