Multi-enzyme cascade colouring for meat without nitrite at low-temperature: Catalysis by NOS-YkuN-YumC and glucose...

This study investigated the role of a multi-enzyme cascade system in developing the colour of meat products at 10 °C. The system consists of glucose dehydrogenase (GDH) and a fusion enzyme, NOS-YkuN-YumC, which was created through the genetic fusion of the NOS gene to those encoding the flavonoid-binding protein YkuN and the flavonoid reductase YumC. The results showed that recombinant GDH was successfully expressed and purified from Bacillus subtilis 168. The purified GDH exhibited a molecular weight of approximately 32 kDa and a specific activity of 11.40 U/mg, demonstrating high efficiency in nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. Following a 24-h incubation period at 10 °C, the additional supplementation of GDH significantly increased (P < 0.05) the a*-value of the minced meat compared to the NOS-YkuN-YumC alone treatment, whereas no significant difference was observed relative to the nitrite-treated group (P > 0.05). UV-Vis spectroscopy provided further evidence that the incorporation of GDH effectively promoted the formation of nitrosylmyoglobin (NO-Mb). After cooking, the minced meat supplemented with the additional GDH maintained a higher a*-value compared to the sample treated with NOS-YkuN-YumC alone. These results indicated that the addition of GDH promoted NADPH production, which in turn enhanced the catalytic generation of NO by the NOS-YkuN-YumC, thus improving both NO-Mb formation and a*-value in raw meat. And the colour can be stabled in cooked meat. This proof-of-concept study successfully demonstrates the potential of this multi-enzyme cascade system for developing colour in nitrite-free meat products under low-temperature conditions.