Substrate optimization, characterization and safety evaluation of exopolysaccharide from Rhodotorula minuta BIOTECH 2178

This study was conducted to optimize and partially characterize the exopolysaccharide (EPS) produced by Rhodotorula minuta BIOTECH 2178 and assess its safety as a functional food. The optimization of EPS yield involved screening of the culture conditions using the Placket-Burman experimental design then followed by formulation optimization using the Simplex-Lattice experimental Design. Partial characterization of the optimized crude EPS included the determination of total sugar and protein content. Fourier transform infrared (FTIR) spectroscopy, microscopix analysis, rheological properties of aqueous EPS and molecular weight determination. Toxicological evaluation was done through physical, physiological, biochemical and histophatological analysis of crude EPS during 14 days of administration using male and female ICR mice. Plackett-Burman screening experiment showed that among the variables screened for EPS production, the most effective factors showing high level of significance are as follows: the type of yeast preferring R. minuta BIOTECH 2178, high level of yeast extract and xylose, low level of glucose, Rhodotorulla minuta was found to be a prolific EPS producer. Response surface analysis revealed that the presence of yeast extract in the formulation plays a significant role of the product both linear or an interaction with xylose and glucose. Results of the superimposed plots suggest the optimum region consisted of 50-83% (12.5-20.75 g/L yeasts extract, 0-13% (0.-3.25 g/L glucose and 15.37% (3.75-9.25 g/L) xylose. The recommended optional media (independent variable) formulation was found at 75% or 18.75 g/L yeasts extract, 25% or 6.25 g/L xylose. Verification studies revealed the prediction equation for final pH, total sugar and EPS yield of the optimum region with an accuracy of 94.21% to 97.46%. Partial characterization of the optimized crude EPS from Phodotorula minuta revealed high total sugar levels (59.21-62.47%) and low protein (17.19%). FTIR spectrum of the crude EPS showed prominent characteristic groups for hydroxyl and carboxyl groups, mannans and beta-glycosidic linkages, which strongly affirms the presence of polysaccharide structure. Transmission electron microscopy of Rhodotorula minuta using showed the accumulation of the EPS to the entire surrounding of the cell surface. Furthermore, the morphological structure of crude EPS revealed the presence of smaller pore size of the crude EPS that enables the polymer hold water. The crevices found in the EPS may also aid in fast absorption and interaction of water molecules into the polysaccharide. Thus, the microstructure of the crude EPS indicates its potential use as a thichener, viscofier and stabilizing agent for various food applications. Rheological characteristics of the aqueous crude EPS solutions indicated that it had good shear thinning property similar to pseudoplastic low behavior. The molecular weight of EPS was estimated to be 1.12x10 sup 7 Da. Safety evaluation that based on the daily observation of the EPS-treated mice did not show any physical, physiological and clinical signs of toxicity. Blood chemistry analysis showed that mice fed with crude EPS had comparable BUN, creatinine and ALT values with that of the control. Liver histopathological analysis of the control and treated group showed normal hepatic architecture with hepatic cells, nucleus, sinusoidal spaces and a central vein. Hispathological analysis of the kidney showed normal renal structure of cortex, which showed a normal architecture of renal glomeruli, proximal convoluted tubule and distal convulated tubules. Thus, crude EPS from Rhodotorula minuta BIOTECH 2178 was neither hepatotoxic nor nephrotoxic and considered safe even in the administration of 400mg EPS/kg bodyweight within 14 days consumption. Moreover. Its safety property lays a good foundation for application of EPS in food and pharmaceutical industries.