Development of a greener flow-based analytical tool for the expeditious determination of total soluble proteins

Protein hydrolysates are increasingly used in feed, underscoring the need for analytical tools for the rapid, reliable determination of total protein. This study revisited a merging-zones flow-based spectrophotometric method for minimizing reagent consumption and employs the Biuret reaction for the quantification of total soluble protein in by-products hydrolysates. The analytical method was optimized across different physical and chemical parameters such as: flowrate, reactor length, sample injection volume and reagents concentration. The use of different matrices relevant in the hydrolysis processes (acetate, phosphate, and hydrochloric acid) showed no significant interference (<10%) on the method's performance. Under optimal conditions, the method quantified hydrolysate protein within a dynamic range of 0.100 to 2.00 mg mL⁻¹, with LOD and LOQ of 0.069 and 0.290 mg mL⁻¹, respectively. Analyses of hydrolysates showed no significant differences compared with the reference method (<10%) while reducing analysis time from 30 min to 3 min per sample (triplicate). The method’s greenness, assessed by AGREE software, showed an improved score, from 0.58 to 0.76. The optimized protocol provides a fast, robust, and greener approach for monitoring protein hydrolysates in the food industry. • Flow injection analysis system using Biuret reagent for the expeditious protein quantification • Reduced reagent consumption and waste generation using a double injection approach for the solutions • The developed method achieved a high Analytical Greenness score (AGREE 0.76) • Successfully applied for the determination of total soluble proteins in by-products hydrolysates