Continuous fluorescence-based quantitative antioxidant assay using vegetable oil as an oxidizable substrate.

Area of Science:

• Food Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Traditional antioxidant assays like DPPH and ORAC often lack real-world applicability for food systems.
• Direct autoxidation measurements are labor-intensive and require specialized equipment, limiting their widespread use.
• A need exists for accessible, relevant methods to assess antioxidant activity in actual food matrices.

Purpose of the Study:

• To develop a small-volume, microplate-based assay for measuring inhibited autoxidation in food substrates.
• To quantify the rate constant of antioxidant reactions with peroxyl radicals (k_inh) using a novel styrene-conjugated BODIPY (STY-BODIPY) probe.
• To evaluate the synergistic interactions of antioxidants within a food matrix.

Main Methods:

• Utilized stripped sunflower oil (SSO) as an oxidizable food substrate and STY-BODIPY as a reporter chromophore.
• Controlled the rate of initiation (R_i) using azobis(isobutyronitrile) (AIBN) at 30°C.
• Standardized the assay using 2,2,5,7,8-pentamethyl-6-chromanol (PMC) and assessed synergistic effects with other compounds.

Main Results:

• Determined the rate constant for peroxyl radical reaction with STY-BODIPY (k_ST = 890 ± 52 M⁻¹ s⁻¹).
• Quantified the k_inh for PMC in SSO as 1.5 × 10⁶ M⁻¹ s⁻¹.
• Demonstrated concentration-dependent increases in induction time (τ) due to synergistic interactions of PMC with γ-terpinene, quercetin, and caffeic acid.

Conclusions:

• The developed small-volume inhibited autoxidation assay is a reliable and accessible tool for evaluating antioxidant activity in food systems.
• The method accurately assesses antioxidant efficacy and synergistic effects, providing more relevant data than conventional assays.
• This approach offers a practical alternative for food quality and safety assessments related to oxidative stability.