Optimization of succinoglycan hydrocolloid production by Agrobacterium radiobacter grown in sugar beet molasses and investigation of its physicochemical characteristics
Graphical abstract
Introduction
Succinoglycans are hetropolysaccharides synthesized by variety of bacterias from Rhizobiaceae (Rhizobium, Agrobacterium), as well as by other microorganisms such as Alcaligenes faecalis var. myxogenes and Pseudomonas spp. (Zevenhuizen, 1997). They are acidic polysaccharides composed of octasaccharide repeating units in which galactose and glucose monomers occur in a molar ratio of 1–7 (Simsek, Mert, Campanella, & Reuhs, 2009). Natural and chemically modified succinoglycans show high stability even under drastic operational conditions such as high temperature and pressure, extreme pH, as well as, high shear rates. These properties make the polymer suitable for a wide range of applications in food, pharmaceutical and other industries such as thickening, stabilizing, emulsifying, texturizing and gelling agents (Nampoothiri, Singhania, Sabarinath, & Pandey, 2003). The succinoglycan characteristics in aqueous solutions have shown reversible pseudoplastic behavior (Balnois et al., 2000, Gravanis et al., 1990; Moosavi-Nasab et al., 2012, Ridout et al., 1997). The bacterial origin of the polysaccharide and the degree and type of non-carbohydrate substituent determine the rheological properties of succinoglycan solution (Cesaro, Gamini, & Navarini, 1992). Solution of succinoglycans exhibit a temperature dependent sharp order-disorder transition when examined by 1H NMR (Gravanis et al., 1990), differential scanning calorimetry (DSC) (Boutebba, Milas, & Rinaudo, 1997), optical rotation (RO), conductivity measurements and viscosity studies (Cesaro et al., 1992a, Gravanis et al., 1990). However, the high cost of the used sources, mainly sugars such as glucose, sucrose and fructose, which have direct impact on production costs, limits the market potential of these biogums (Kumar et al., 2007, Sutherland, 2001). Moreover, the main use of exopolysaccharides is to modify the rheological characteristics of food products which also help optimization of process parameters. Therefore, it is crucial to search for low cost carbon sources such as sugar beet molasses and characterize its properties in order to decrease the production costs of exopolysaccharides and, at the same time, facilitate the development of new products and processes.
Due to its high level of sugar, molasses are suitable for production of succionoglycan biogum. Molasses is a co-product of sugar production, both from sugar beet as well as from sugar cane, and is defined as the runoff syrup from the final stage of crystallization, from which further crystallization of sugar is uneconomical (Higginbotham & McCarthy, 1998). Despite their similarities, beet and cane molasses exhibit significant differences with regards to nitrogenous compounds, fermentable sugars, ash and vitamin content. Sugar beet molasses, therefore, is a solution of sugar, organic and inorganic matter in water with a dry substance of 74–77% (w/w). Total sugars (mainly sucrose) constitute approximately 47–48% (w/w) of molasses, ash 9–14% (w/w) and total nitrogen containing compounds (mainly betain and glutamic acid) 8–12% (w/w). Sugar beet molasses is widely used as a substrate in fermentation since it constitutes a valuable source of growth substances such as pantothenic acid, inositol, trace elements and, to a lesser extent, biotin. However, the occurrence of undesired volatile nitrogenous pyrazines, pyrroles as well as furans and phenols have also been reported. In addition, other contaminants such as heavy metals, biocides and organic acids have been detected but most of them at concentration levels lower than that needed to affect process inhibition. Nevertheless, in processes with molasses as a sole substrate or when used in high amounts as a co-substrate, specific inhibitors are partly removed upon pre-treatment (Stoppok & Buchholz, 1996). Microbial production of succinoglycan in industrial scale has been traditionally restricted to Agrobacterium spp.; information on fermentation conditions and the patented strains presently utilized are available in European patent application 0138225, 0251638, and 0527061 (Stredansky, Conti, Bertocchi, Matulova, & Zanetti, 1998). In the production of succinoglycan processes, where there are multiple independent variables affecting the responding factor, it is likely to use an optimization method that can determine all the factors. In addition, the possibility of interactions between the independent variables should be considered in order to determine the optimal experimental conditions. Response surface methodology (RSM) has been reported to be an effective tool for optimization of a process when the independent variables have a combined effect on the desired response. RSM is a collection of statistical and mathematical system that has been successfully used for developing, improving and optimizing such processes (Shih and Shen, 2006, Shih et al., 2002). Lastly, potential applications of the succinoglycan in food and non-foods products are related to functional properties of these systems associated with their gelling capacity. In this study, the optimization of succinoglycan biogum production by Agrobacterium radiobacter from sugar beet molasses as functions of substrate, time, total soluble solid and agitation rate of fermentation was investigated. Also, physico-chemical and rheological properties of the produced succinoglycan gums were examined.
Section snippets
Composition of molasses
Total sugar, protein, ash content, pH and total soluble solids (brix) were quantitatively determined according to the AOAC methods in triplicate (AOAC, 1984).
Preparation of molasses
The initial solution of molasses was prepared by adding molasses (Brix 90) in distilled water and then mixed, thoroughly, using a blender (Mammonlex, Model 242, Taiwan) for 3 min at high speed. Using distilled water, the resultant solution was adjusted at 20°Brix. In order to remove all insoluble solids, the extracted solutions were
Yield of exopolysaccharide production
The production of exopolysaccharide in fermentation media containing sucrose and molasses (4, 5.5, 7, 8.5 and 10°Brix, pH = 7), within 120 h of incubation at 28 °C was studied. The raw molasses were obtained from a sugar factory with Brix 80 and contained 13.6% protein, 51.2% total sugar and 12.04% Ash. Fermentation medium containing molasses at different conditions of fermentation exhibited greater influence on succinoglycan production compare to fermentation medium containing sucrose
Conclusion
This study reported the isolation and characterization of extracted exopolysaccharides using a strain of A. radiobacter, which was obtained from PTCC 1654. The structural and rheological properties of succinoglycan exopolysaccharide (EPS) as a function of substrate (sucrose or molasses), time (24–120 h), brix (4–10) and agitation (200–300 rpm) were investigated. Using Fourier transform infrared (FT-IR) spectroscopy, Proton nuclear magnetic resonance (1H NMR) spectroscopy, thin-layer
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