Development of an In vitro Assay for Evaluating Protease-mediated Protein Solubility in Soybean Meal

Abstract

Background and Objective: Soybean meal is a major protein source in animal nutrition but its nutritional value may be limited by antinutritional factors and processing conditions affecting protein solubility. This study aimed to develop and apply an in vitro assay to evaluate the effects of proteolytic enzymes on protein solubility in soybean meal and to assess the potential interaction between enzymes under controlled conditions.

Materials and Methods: Soybean meal samples were incubated with trypsin, an exogenous commercial protease (Sfericase), or their combination under standardized conditions. The assay involved controlled enzymatic hydrolysis followed by separation and quantification of the soluble protein fraction using total nitrogen analysis by the combustion (Dumas) method. Protein solubility was determined before and after enzymatic treatment.

Results: The baseline protein solubility of untreated soybean meal was 7.73%. Enzymatic treatment with trypsin or the exogenous protease increased protein solubility to 14.68% and 16.70%, respectively. The combined treatment resulted in a protein solubility of 26.69%, indicating a greater increase than that observed with either enzyme alone and suggesting a potential complementary effect between the enzymes under the conditions tested.

Conclusion: The proposed in vitro assay provides a controlled approach for screening protease efficacy and investigating enzyme interactions in feed ingredients. The method may serve as a useful tool for evaluating enzymatic strategies aimed at improving protein solubility and, potentially, protein utilization in animal feed.