Recovery of nicotine-free proteins from tobacco leaves using phosphate buffer system under controlled conditions

Establishment of an effective, high-throughput processing system to recover protein from tobacco with no nicotine contamination is essential and vital to the development of value-added, alternative applications for tobacco farmers. We have successfully developed a mechanism capable of processing up to 60 kg of tobacco leaves per hour with phosphate buffer (Na(2)HPO(4)-KH(2)PO(4)) simultaneously added to stabilize the protein as the plant was being disintegrated. The optimal processing parameters were identified, including the ratio of buffer to leaf (BLR) at 4.75 (w/w), buffer pH 7.85, and buffer concentration 0.085 mol/L, achieving a maximum yield of soluble protein at 12.85 mg/g fresh leaf. Acetone at -20 degrees C was the most effective among all methods investigated to remove nicotine from protein; however, it also drastically reduced the recovery rate of protein (63.3%). Ultrafiltration was only able to remove about 50% of the residual nicotine, although the protein recovery rate was high (94.7%). The residual nicotine content inherent in the recovered protein was completely removed by rinsing the protein with 85% phosphoric acid at pH 3.5 for three times with a protein recovery of 94.5%. The pilot-scale operation provides a solid foundation for further scale-up to industrial production of nicotine-free tobacco protein that could bring added value to tobacco for nonsmoking applications.