Optimization of the simultaneous production of proteases and bioactive peptides by Aspergillus oryzae in solid-state fermentation of Tenebrio molitor

The consumption of insects as an alternative protein source has generated increasing interest due to their high nutritional value and potential application in functional foods. Among edible insects, Tenebrio molitor stands out for containing up to 50% protein on a dry basis, in addition to essential amino acids, fatty acids, vitamins, and minerals. Biotechnological strategies, such as solid-state fermentation (SSF), have been explored to enhance the nutritional value of these substrates, promoting biotransformations capable of increasing protein digestibility, reducing antinutritional compounds, and generating bioactive peptides. In this context, experimental designs constitute effective tools to optimize fermentative processes and identify critical variables. The present study aimed to evaluate, using a central composite rotational design (CCRD), the effect of moisture content (ranging from 30 to 50%), temperature (from 25 to 35 °C), and initial inoculum level (1 × 10⁵ to 1 × 10⁷ spores/g) on the production of proteases and bioactive peptides through solid-state fermentation of T. molitor flour with Aspergillus oryzae CCFF 3999. Fermentations were conducted for 48 h and 72 h, and the responses evaluated were protease activity and antioxidant properties, assessed by ABTS and DPPH radical inhibition methods and the ferric reducing antioxidant power (FRAP) assay. The results demonstrated that the operational conditions significantly (p < 0.10) influenced protease production and the antioxidant activity of the fermented products. The overall data analysis indicated that the most suitable condition for SSF corresponded to assay 9, conducted with 30% initial moisture, incubation temperature of 30 °C, inoculum of 5.05 × 10⁶ spores/g, and fermentation time of 48 h, under which the highest values of protease productivity (21.46 U/g/h) and antioxidant capacity assessed by the ABTS method (86.71 µmol TE/g) were obtained. For the FRAP and DPPH methods, the best results were observed at 72 h of fermentation, with values of 13.37 µmol TE/g and 13.59 µmol TE/g, respectively. Overall, the responses indicated that lower moisture values combined with lower temperatures favored the production of proteases and antioxidant compounds, confirming the potential of the optimization process by CCRD. It is concluded that the solid-state fermentation of T. molitor flour by A. oryzae represents a promising strategy for obtaining protein ingredients enriched with bioactive peptides and antioxidant activity, aligning with current demands for functional and sustainable foods.