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<p>Accurate determination of total phenolic content in protein-rich foods remains challenging due to methodological interferences. This study systematically investigated the effects of protein properties (concentration, molecular weight, and isoelectric point) on three common total phenolic content determination methods (Folin-Ciocalteu, Ferrous tartrate, and Prussian blue assays). Using bovine serum albumin (BSA, 66&#xa0;kDa, pI 4.7), casein (23.6&#xa0;kDa, pI 4.6), and cytochrome C (12.4&#xa0;kDa, pI 10.2) as model proteins, with gallic acid as the phenolic standard. Results demonstrated that the synergistic effect of protein concentration and molecular weight significantly influenced the measurements. The Folin-Ciocalteu assay exhibited the most pronounced interference from high protein concentrations (≥ 1&#xa0;mg/mL) and large molecular weights, primarily due to protein-copper chelation and competitive chromogenic reactions involving aromatic amino acids. The Ferrous tartrate method was sensitive to phosphorylated proteins (e.g., casein) owing to competitive Fe<sup>2+</sup> complexation by phosphate groups. In contrast, the Prussian blue method showed superior anti-interference capability (recovery &gt; 92%), though protein precipitation at high concentrations (≥ 2&#xa0;mg/mL) could lead to underestimation. A multiple linear regression model was established to quantify the relationship between protein characteristics and interference intensity. These findings provide quantitative relationships between protein properties and assay performance, offering practical guidance for selecting appropriate total phenolic content methods in complex food matrices. The study highlights the need for protein-specific method optimization to ensure accurate phenolic quantification in nutritional and functional food analysis.</p>

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Effects of protein characteristics on three methods for determination of total phenolic content in food systems

  • 期刊论文

  • 发表日期：

    2025年12月12日

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  • Qiu-Yue Ma,
  • Qian-Da Xu,
  • Nan Chen,
  • Wei-Cai Zeng

摘要

Accurate determination of total phenolic content in protein-rich foods remains challenging due to methodological interferences. This study systematically investigated the effects of protein properties (concentration, molecular weight, and isoelectric point) on three common total phenolic content determination methods (Folin-Ciocalteu, Ferrous tartrate, and Prussian blue assays). Using bovine serum albumin (BSA, 66 kDa, pI 4.7), casein (23.6 kDa, pI 4.6), and cytochrome C (12.4 kDa, pI 10.2) as model proteins, with gallic acid as the phenolic standard. Results demonstrated that the synergistic effect of protein concentration and molecular weight significantly influenced the measurements. The Folin-Ciocalteu assay exhibited the most pronounced interference from high protein concentrations (≥ 1 mg/mL) and large molecular weights, primarily due to protein-copper chelation and competitive chromogenic reactions involving aromatic amino acids. The Ferrous tartrate method was sensitive to phosphorylated proteins (e.g., casein) owing to competitive Fe2+ complexation by phosphate groups. In contrast, the Prussian blue method showed superior anti-interference capability (recovery > 92%), though protein precipitation at high concentrations (≥ 2 mg/mL) could lead to underestimation. A multiple linear regression model was established to quantify the relationship between protein characteristics and interference intensity. These findings provide quantitative relationships between protein properties and assay performance, offering practical guidance for selecting appropriate total phenolic content methods in complex food matrices. The study highlights the need for protein-specific method optimization to ensure accurate phenolic quantification in nutritional and functional food analysis.