Optimization of sodium alginate extraction conditions from an Iranian brown algae (Nizimuddinia zanardini) using response surface methodology

Introduction:Macroalgae are source of many polysaccharides. Based on their pigments content they are divided into brown, green and red algae having specific polysaccharide contents. Among them, brown algae (Phaeophyceae) are source of alginates and/or fucans or fucoidans (Percival and McDowell 1990, Torres et al., 2007). Alginates are anionic polysaccharide that existences in mixer of salt forms (Na⁺, Mg²⁺ and Ca²⁺) in cell walls of brown algae makes the tissues flexible and strong. They consist of (1,4) linked β-d-mannuronic acid (M) with ⁴C₁ ring conformation and a-l-guluronic acid (G) with ¹C₄ ring conformation, the two uronic acids being in pyranosic conformation(Pawar and Edgar 2012, De Sousa et al., 2007). They offer three varying sequences identified as the blocks MG consisting of nearly equal proportion of both monomers with a high number of MG (or GM) dimmers, the blocks GG and the blocks MM (Haug and Larsen 1962). M/G ratio, length and distribution of sequences depend on the algae species but also on their growth conditions and geographic origins. It also determines the chemical and physical properties of alginates (Karaki et al., 2013). Indeed, their gelling properties are dependent on guluronic acid content and explained by the structural features of GG blocks where selective alkaline earth metal multivalent cations and notably Ca2+ take place by chelation. This phenomenon is known and explained as the “egg-box” model. The formed gel is not soluble in water. However, sodium alginate is a water-soluble polymer having generally pseudo plastic properties in solution (Draget and Taylor 2011). Polysaccharides have wide range of pharmacological activities and some of them are considered as antitumor, antihypertensive, immunomodulator as well as antioxidant agents. Their radical scavenger’s role can be used to inhibit oxidative damage in foods and improve their nutritional quality (Venugopal 2011, Yang et al., 2011).
Material and methods: Twenty-five gram of dried algae (Nizamuddinia zanardinii) was soaked under steering at room temperature for 24 h in 800 mL of 2% (v/v) formaldehyde to remove phenolic compounds and pigments. After that, the insoluble fraction was washed 3 times with MilliQ water and supplemented by 800 mL of 0.2 M HCL before to be incubated at different levels(25-60°C) for different extraction time periods(0.5-3 h) under stirring (250 rpm).After optimizing of acidic step, the suspension was then centrifuged (10000 g for 20 min at 20°C) and pellets with alginic acid were washed 3 times with MilliQ water before to be socked in a range from (2-4 % (w/v)) Na2CO3 solution for different levels (2-4 h) at different extraction time periods (60-90 °C). The mixture was centrifuged (10000 g for 30 min at 20°C) and the supernatant was precipitated with 3 volumes of ethanol 96 % (v/v). Pellets containing sodium alginate were dissolved in MilliQ water and precipitated with ethanol as described above. This step was repeated two times before to collect and freeze dried the sodium alginate. Response surface methodology (RSM) was used to study the effect of extraction temperature; X₁ extraction time; X₂ for acidic step and concentration; X₁ , extraction time; X₂,; extraction temperature X₃ for alkaline step. A D-optimal design was employed for designing experimental data using Design-Expert 10.0.3 trial software (Stat-Ease Inc., Minneapolis, MN, USA).
Results and discussion: Both temperature and time factors have a significant effect on the extraction efficiency and the effect of temperature on extraction efficiency is higher than the effect of time in acidic step. There is also a significant interaction between the two variables of temperature and time. The extraction temperature had an increasing effect on alginate extraction efficiency. Chloridric acid disrupts the cell wall and releases alginate and also converts alginate salts in the cell wall into arginic acid. Increasing temperature causes further cellular breakdown and alginate extraction efficiency increases. With increasing time, the extraction efficiency increased. This increase can be due to the fact that the acid needs time to penetrate the cell wall, and as the time increases, the permeability increases, resulting in the extraction efficiency, so that the permeability is complete and the highest efficiency is obtained and After that, the extraction efficiency remains constant and even due to the long-lasting effect of acid, it causes de-polymerization and decreases the extraction efficiency. Increasing temperature increased the effect of time factor, meaning that the interaction of temperature and time had a significant effect on extraction efficiency. At low temperatures, the effect of time on the extraction efficiency is low because of the breakdown of the cell wall, but at higher temperatures, which contribute to the thermal breakdown of the cell wall, the effect of time variation is more evident. In alkaline step the effect of concentration on extraction efficiency was considered as a quadratic equation and as interaction with extraction time. By increasing the concentration of sodium carbonate, the extraction efficiency increases with an ascending slope. In other words, the amount of efficiency increases with an increase in direct concentration. Sodium carbonate causes the sodium substitution in carboxylic acid arginine groups and sodium alginate salts are obtained. Therefore, increasing concentration results in more calcium ion replacement and alginate extraction efficiency increases. This increase in extraction efficiency is increased to the extent that all carboxylic groups are replaced by sodium ion, and after that the extraction efficiency remains constant, and even due to the high concentration and de-polymerization of the alginate, it reduces the extraction efficiency. With increasing time of extraction with sodium carbonate, the extraction efficiency decreases with a second order gradient. Sodium ion has been completely replaced in argonic acid groups during the initial extraction hours, and if the extraction time is increased due to the prolonged contact, sodium alginate is obtained with an excess of sodium carbonate which has an alkaline pH and because of the adverse effect of this pH Extraction efficiency is reduced. The effect of temperature on extraction efficiency was considered as a first-order equation and as interaction with extraction time. As the temperature rises due to breaking of the bonds that may lead to the separation of sodium ions and also cause de-polymerization of the alginate, the extraction efficiency decreases.
Conclusion: Chloridric acid disrupts the cell wall and releases alginate and also converts alginate salts in the cell wall into alginic acid. the acid needs time to penetrate the cell wall. The results showed that in acidic step temperature and time are two major effective factors on yield and quality of extracted alginate. Sodium carbonate causes the sodium substitution in carboxylic acid arginine groups and sodium alginate salts are obtained. Sodium ion has been completely replaced in argonic acid groups during the initial extraction hours, in high temperature may lead to the separation of sodium ions and also cause de-polymerization of the alginate so in alkaline step, all factors have effect on yield, polyphenol and apparent viscosity of extracted polymer. The optimized conditions of extraction of sodium alginate from brown algae (Nizimuddinia zanardini)were as follows: temperature: 60 ^oC, time: 3 h (acidic step) and temperature: 60 ^oC, time: 3 h and concentration 3 % w/v (alkaline step).