Optimization of process parameters was carried out using the CCD design with the parameters found to be significant from the Taguchi approach, including pH (X1) and temperature (X2). Table 6 represents the design matrix and the results of the 13 experiments carried out using the CCD design. The data obtained provided the regression

model using ANOVA software. equation(4) Y=6.7014−0.3367(x1)+0.2083(x2)−0..6048(X1×X2)−0.1175(X1×X2)Y=6.7014−0.3367(x1)+0.2083(x2)−0..6048(X1×X2)−0.1175(X1×X2)where X1 and X2 represents pH and temperature respectively. The estimated regression coefficients from response surface analysis of the quadratic regression model ( Table 7) demonstrate that Eq. (4) is a highly significant model with goodness of fit R2 − 0.982 and adjusted R2 − 0.969. These values indicate that the model equation was adequate for GSK126 cost predicting the Proteases inhibitor melanin production under any combination of values of the variables. The graphical representation

provides a method to visualize the relationship between the response and experimental levels of each variable and the type of interactions between the test variables in order to identify the optimum conditions. The interaction effects and optimal levels of the variables were determined by plotting the three dimensional (3D) response surface curves. The response surface curve in Fig. 3a,b represents the interaction between pH and temperature, which showed that the maximum melanin yield was obtained toward neutral pH, while melanin yield was significantly affected with an alkaline pH. Validation was carried out under click here conditions predicted

by the model. The optimum conditions predicted by the model are pH 6.84, Temp −30.7 °C with yield of ∼6.8 mg/mL and the actual yield obtained was 6.96 ± 0.6 mg/mL. The close correlation between the experimental and predicted values signifies the reliability of the response methodology (CCD design) over traditional optimization approach. The increased yield at the optimum conditions were comparable10 to and better than some microbial sources [13] and [22] reported in the literature. As the reported studies utilized relatively expensive media, our results shows the suitability of significant melanin production on a cheaper substrate FWE and has huge scope for larger scale production. The absorption spectrum of natural melanin is shown in Fig. 4a. The UV–visible wavelength scan showed that absorption was highest in the UV region (200–300 nm), but diminished towards the visible region. This phenomenon is characteristic to melanin and was due to the actual complex structure of melanin [1] and [13]. IR spectroscopy is important for the interpretation of the structure binding capacity, affinity and sites of metal ions in melanin. Fig. 4b,c shows strong absorptions at 3500 cm−1, 1700 cm−1, 1300 cm−1 for standard melanin and for bacterial melanin obtained.