Phytase Assay Kit

Oat and barley bran are high in dietary fiber (β-glucans), minerals, and antioxidants, have high activity of enzymes, but possess also antinutrients. This study aimed to investigate the influence of high-intensity ultrasound on enzyme and antioxidant activities, phytic acid content, as well as functional and rheological properties of oat and barley bran. Ultrasonic treatment was performed at 24 kHz on a 15% bran water suspension, at three specific energies (87, 217.5, and 348 kJ/kg), with or without pulsation (5 sec every 10 sec). Bran was assessed for β-glucanase and phytase activity, phytic acid and total phenolic content, antioxidant activity, hydration, and rheological properties. β-glucanase from oat bran was inactivated up to 82% and from barley bran up to 55%, in dependence of ultrasound specific energy and pulsation. In both bran types, phytase activity increased by 40-44% after treatments with 87 kJ/kg but decreased by 89-94% at 348 kJ/kg. Phytic acid was reduced on average in oat bran by 17% and in barley bran by 39%. Depending on the energy and pulsation, the ultrasonication of both bran types reduced total phenolic content (27-55%), antioxidant activity (by 28-48%), complex viscosity (62-71%) and maximum stress tolerated by the sample (46-68%). In contrast, water swelling (42-48%) and water retention capacity (44–59%) increased for both bran types. Hence, high-intensity ultrasound is a useful technique in reducing antinutrients, while altering the enzyme activity and functional properties of the bran. These results could help wider application of bran in food production.

Objectives: This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). Results: In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 10⁶ spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005–0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. Conclusions: The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.