Measuring phytic acid in complex and fermented food matrices: Modified protocol using enzymatic analysis

Abstract Background and Objective Quantifying phytic acid in complex or fermented foods is essential for assessing nutritional quality. Conventional enzymatic assays often overestimate phytic acid due to nonspecific hydrolysis of phosphorylated compounds. This study aimed to develop a modified enzymatic method with improved specificity by correcting for non‐phytate phosphorus interference. Results The modified protocol introduced a parallel alkaline phosphatase (ALP)‐only control to determine background phosphate release. Phytic acid content was then calculated as the difference between total (phytase + ALP) and background (ALP‐only) phosphorus. The method was validated using chicken, fungal biomass, fermented oats, and oat flour. It showed a detection limit of 11.29 mg P per 100 g (≈40 mg phytic acid per 100 g) and linearity from 0.5 to 7.5 μg P per assay. In interference‐prone samples, the apparent phytic acid content was reduced by 85%–99% compared with the standard Megazyme K‐PHYT assay (e.g., chicken: 0.45 → <0.04%), with results aligning closely with high‐performance ion chromatography (HPIC) data (e.g., fungal biomass: 0.07% vs. 0.15%). Conclusions The modified enzymatic assay improves analytical specificity and accuracy for phytic acid quantification in complex food matrices. It provides a robust, low‐cost, and high‐throughput alternative to chromatographic methods. This method introduces a simple background‐correction step for enzymatic phytic acid analysis, enhancing reliability in fermented or protein‐rich foods and facilitating integration into automated compositional analysis workflows.