Screening of Protease-Producing Microorganisms and Optimization of Fermentation Processes for the Efficient Preparation of Broussonetia papyrifera Feed

With the rapid development of China's livestock industry, the demand for protein feed has concomitantly increased, underscoring the importance of developing and utilizing new types of feed. As a novel and unconventional protein source, fermented <i>Broussonetia papyrifera</i> feed has become a promising alternative to traditional feeds due to its high nutritional value, abundance of bioactive compounds, and wide range of applications. However, the efficient utilization of crude proteins in <i>B. papyrifera</i> has been hindered by the currently used microorganisms and fermentation processes. In the present study, nine proteolytic bacterial strains and eight yeast and fungal strains were isolated from the root zone soil of <i>B. papyrifera</i> and screened using qualitative and quantitative methods. The protease activities of strains <i>Paenibacillus</i> sp. AY and <i>Rhodotorula mucilaginosa</i> FG were determined to be 21.95 and 55.16 U/mL, respectively. Compared with the control group, <i>Paenibacillus</i> sp. AY significantly increased the acid-soluble protein and ammonia nitrogen contents in the fermented feed by 26.7% and 12.2% (<i>p</i> < 0.05), respectively, while <i>R. mucilaginosa</i> FG enhanced them by 43.3% and 24.5%; these isolates' effects were comparable to those of the type strains. Finally, to increase the quality of fermented <i>B. papyrifera</i> feed, the cultivation conditions were further optimized using single-factor experiments and an orthogonal design. Under optimal conditions, the acid-soluble protein content reached 7.63%, which was 27.2% higher than that of the control. Our results provide a basis for developing a novel process to efficiently utilize the crude proteins in <i>B. papyrifera</i> feed and can accelerate the application of this feed in animal production.