where Y is the predicted response; β0 is the intercept; β1, β2 and β3 are the linear coefficients, β11, β22 and β33 are the square coefficients and β12, β13 and β23 are the interaction coefficients. To predict the optimal point, a second order polynomial function was fitted to correlate the relationship between the independent variables and the response values (dependent variables; PNSBsi and ORP). The optimal conditions for stimulating PNSB (PNSBsi) were obtained by solving the regression equations and also by analyzing overlay interaction plots.