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Medium composition and culture conditions for maximal collagenase production by Penicillium aurantiogriseum URM4622 were optimized using a response surface approach. A full two-level design on three factors (initial medium pH, soybean flour concentration, and temperature) was employed to identify the most significant fermentation parameters for collagenase production, and a subsequent central composite design (CCD) was used to find the optimal levels of the two most significant factors (initial medium pH and soybean flour concentration). The design results indicated that the initial medium pH and the temperature had significant negative main effects, whereas the substrate concentration had a positive effect on the collagenase production. The maximum collagenolytic activity predicted by the fitted response surface was expected to occur at pH 7.21, 1.645% soybean flour concentration and 24°C. Three replicate experiments were run at these conditions and yielded an activity response of 283.36 ± 1.33 U, which not only is the highest obtained in this study but also represents a 5-fold increase over the lowest response observed in the initial design. Since all experiments were carried out with an inexpensive substrate, the final results point out to a cost-effective medium for collagenase production with potential industrial-scale applications.

A 2 3 full factorial design was used to identify the main effects and interactions of pH, collagen concentration and temperature on the degree of collagen hydrolysis (DH) by collagenase from Penicillium aurantiogriseum URM 4622. Increases in both pH and collagen concentration improved DH, and a positive interaction effect was observed for these variables. On the other hand, temperature had a negative main effect on DH. The maximum value of DH (4.65 μg/mL) was achieved at 7.5 mg/mL collagen concentration, pH 8.0 and 25°C. The peptide profile showed several peptides with molecular weights lower than 2 kDa and exhibited antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. An antioxidant activity of 84.7± 0.24 % towards the radical ABTS• + was obtained with 50 mg/mL hydrolysates. This study demonstrated that collagen hydrolysed by P. aurantiogriseum URM 4622 collagenase possesses interesting antibacterial and antioxidant activities.