Optimization of Fermentative Parameters and In Vitro Safety Evaluation of Amylase Produced by Mucor flavus

Fungal species are a valuable resource for starch-based industries, particularly for amylase production. This study evaluated the amylolytic potential of Mucor flavus under submerged fermentation using agro-industrial substrates and evaluated the safety profile of its crude enzyme extract. The fungal isolates were identified by macroscopic and microscopic characters including brownish fluffy growth with no pigment on reverse side and coenocytic hyphae with sporangium. Molecular identification via PCR produced an amplicon of 290 bps on agarose gel. Screening on starch agar revealed M. flavus as the most potent amylolytic strain, demonstrating the largest zone of hydrolysis. Subsequently, amylase production was optimized under different growth conditions in submerged fermentation, including temperature (22, 28 and 37°C), pH (4.5, 6 and 7) and different natural substrates (maize flour, wheat bran, and rice husk) at varying concentrations (1, 3 and 5%) using one variable at a time. The effect of these parameters was assessed by measuring amylase activity (IU/mL/min) using the dinitrosalicylic acid (DNS) method. The amylolytic potential of M. flavus varied significantly with the change in temperature, pH, substrate and substrate concentration. The maximum amylase activity of 87.90 ± 1.30 IU was achieved at pH 6 and 3% concentration of rice husk followed by wheat bran, which yielded the maximum amylase activity (24.35 IU) at 22°C, pH 7.5 and 5% concentration. In case of maize flour, the maximum amylase activity was 7.33 IU at pH 4.5, 22°C and a substrate concentration of 3%. A linear increase in enzyme yield was recorded up to 3% concentration of maize flour and rice husk, beyond which a decline was observed. The ideal conditions include a temperature of 37˚C, a pH of 6, and rice husk as the most effective substrate. Statistical analysis revealed the significant variations among fermentation parameters (P > 0.05). A safety assessment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed a direct link between fungal supernatant concentration and reduced cell survival. The cell survival percentage dropped below 50% at a concentration of ≥ 31.25 µL/mL. Ultimately, these findings highlight the potential of M. flavus as a potential candidate for starch-based industries due to its rapid growth and exceptional enzyme productivity using cost-effective agro-industrial substrates.