The promotion and inhibition of proteolysis in silages during ensiling of citrus waste

Although waste generated from the production of citrus fruit juice is a suitable raw material for ensiling, excessive proteolysis greatly reduces the quality of silages and economic benefits. The driving force and factors influencing proteolysis during ensiling remain poorly understood. In this study, the degree of silage proteolysis was systematically evaluated through the analysis of silage composition, proteolysis promotion and inhibition under various ensiling factors were quantified, and the causes of proteolysis were explored through changes of microbiome. A reduction in soluble protein (SP) and in-solution protein (ISP) contents coupled with an increase in NH₃-N were found after ensiling. A decrease in free amino acid content was evident in upper and middle sections, whereas there was an increase in the bottom section. The strongest proteolysis of silages was observed in the middle section. The main factor accelerating hydrolysis of protein into amino acid (the first stage of proteolysis) was acid protease activity (APA). The hydrolysis of amino acid into NH₃-N (the second stage of proteolysis) was principally inhibited by lactic acids, acetic acids, pH, and soluble carbon, and accelerated by APA, moisture and total carbon. Acid protease was probably constituted mainly microbial protease, able to tolerate a pH range of 3.44–3.77. Bacterial coupled colony (B-CC), Trichocomaoute, and Trichocomaceae-1 presumably promoted the production and activity of acid protease. As inferred, the first stage of proteolysis was promoted by B-CC and fungal coupled colony (F-CC), and inhibited by Trichocomaoute. While the second stage of proteolysis was likely promoted by P. kudriavzevii and F-CC, and inhibited by B-CC and Trichocomaoute. Thus, the use of homofermentative lactic acid bacteria in raw materials is recommended to reduce the degree of proteolysis, particularly in the middle section of the silo.