A New Method to Determine Antioxidant Activities of Biofilms Using a pH Indicator (Resazurin) Model System

**Background:** Biopolymer films, particularly gelatin-based films, are of interest for active food packaging due to their biodegradability and ability to carry antioxidants. Traditional methods for measuring antioxidant activity of films (e.g., TBARS, peroxide value, DPPH) are time-consuming, require specialized skills, and are not easily applied to complex food systems. Resazurin (alamarBlue) is a redox-sensitive pH indicator that changes from blue to red in response to chemical reduction, but it had not previously been used to measure antioxidant activity or lipid oxidation in biopolymer film systems. This study aimed to develop a simple, rapid, low-cost colorimetric method using resazurin to determine antioxidant activity of gelatin biopolymer films (GBFs).

**Methods:** Type B limed bone gelatin (200 bloom) was used to prepare GBFs with glycerol as plasticizer. Three antioxidants—ascorbic acid, butylated hydroxyanisole (BHA), and phytic acid—were incorporated at 0.2%, 0.6%, and 1.0% (w/w of gelatin). Films were cast on Teflon-coated plates and dried overnight. Mechanical properties (tensile strength, elongation at break, energy to break) were measured using an Instron Universal Testing Machine per ASTM D882-01. Water vapor permeability (WVP) was determined gravimetrically at 25°C and 50% RH gradient using a modification of ASTM E96-00. Oxygen permeability (OP) was measured using an OX-TRAN 2/20 at 25°C and 50% RH per ASTM D3985. The model food system (AES-R) consisted of 5% agar, 30% (of agar weight) each of soybean oil and Tween 80 emulsifier, and 8 mM resazurin, adjusted to pH 7. For calibration, three systems were compared: agar only (A-R), agar + emulsifier (AE-R), and agar + emulsifier + soybean oil (AES-R). Color (brightness L and redness a) was measured every other day for 14 days using a Minolta Colorimeter. DPPH radical scavenging activity was measured at 517 nm after 2 h incubation. Statistical analysis used ANOVA with Duncan's Multiple Range Test (p < 0.05).

**Key Results:** Phytic acid incorporation increased tensile strength by up to 35% (37.7 MPa at 0.6% vs. 28.4 MPa for control) and energy to break by up to 40% (79.0 in-lbf/in³ at 0.2% vs. 56.5 for control), attributed to electrostatic cross-linking. BHA-based GBFs showed lower elongation (19.4–21.8%) compared to control (26.2%). Phytic acid at 0.6% showed the lowest WVP (1.29 ng m/m² s Pa), while ascorbic acid and BHA showed concentration-dependent increases. Oxygen permeability decreased with phytic acid and ascorbic acid but increased with BHA. In calibration experiments, the AES-R system showed the greatest redness increase (a-value 14.1 at day 14), 85% higher than A-R and 49% higher than AES-R-Cont. BHA-based GBFs showed the strongest antioxidant effect, with slowest color change from blue to red. Ascorbic acid at 1.0% showed prooxidant activity, with highest redness up to day 8 followed by a dramatic drop. Phytic acid showed no antioxidant effect in the AES-R system, consistent with DPPH results (phytic acid showed no radical scavenging; ascorbic acid 71.7% and BHA 41.7% at 1.0% concentration).

**Clinical Implications:** While this is a food science study without direct clinical application, the developed AES-R method provides a simple, rapid, low-cost, and instrument-free approach for screening antioxidant activity of active packaging films. This could improve food safety and shelf-life by enabling better design of antioxidant-releasing packaging, potentially reducing dietary exposure to oxidized lipids. The method's reliance on colorimetric pH/redox indicators may also have translational potential for biomedical applications such as wound dressing antioxidant activity testing or drug delivery film characterization.