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
Teixeira et al. (Wed,) studied this question.