D-optimal design was employed to optimize the mixture of cross-linking agents formulation: microbial transglutaminase (MTGase) and ribose, and the processing parameters (i.e. incubation and heating time) in the mixture in order to obtain combined-cross-linked bovine serum albumin gels that have high gel strength, pH close to neutral and yet medium in browning. Analysis of variance (ANOVA) showed that the contribution of quadratic term to the model over the linear was significant for pH and L* value, whereas linear model was significant for gel strength. Optimization study using response surface methodology (RSM) was performed to the mixture components and process variables and the optimum conditions obtained were: MTGase of 1.34-1.43 g/100 mL, ribose of 1.07-1.16 g/100 mL, incubation time of 5 h at 40 degrees C and heating time of 3 h at 90 degrees C. (c) 2008, The Society for Biotechnology, Japan. All rights reserved.