Abstract: This study innovatively combined ultrasonic extraction, ultrafiltration, and weakly alkaline isoelectric precipitation technologies to devise a process for the efficient ex-traction of soluble proteins from discarded tobacco leaves. Additionally, a comprehensive evaluation system incorporating physical, chemical, and biological parameters was established. By optimizing the process, an effective protocol was identified as follows. A pH 7.85 phosphate buffer at a concentration of 0.085 mol/L was utilized for ultrasonic ex-traction with a material-to-liquid ratio of 1∶8. Subsequently, fractionated centrifugation and filtration/ultrafiltration were performed, followed by adjustment of the solution to an isoelectric point pH of 5.5 using a 0.11 mol/L [H+] solution. This protocol facilitated the successful extraction of mixed proteins with molecular weights of approximately 55 kDa and exceeding 200 kDa. The amino-acid composition of the extracted proteins satisfied the essential amino-acid requirements of adults. Infrared-spectrum analysis indicated the absence of significant pectin or polysaccharide features. This research introduced a gentle,weakly alkaline isoelectric point extraction approach, combined with physical-extraction techniques. This method not only overcomes the limitations of conventional extraction processes but also exhibits broad applicability. Innovative pathways are presented herein for the resourceful utilization and industrial-scale production of discarded tobacco leaves.

Key words: tobacco leaf protein, phosphate bu?er system, ultra?ltration, nicotine, isoelectric point