Ensuring fish freshness is critical for food safety, yet conventional assessment methods are destructive and unsuitable for real-time monitoring. This study develops a pH-responsive colorimetric film based on a chitosan–polyvinyl alcohol (Cs/PVA) matrix incorporated with red amaranth (Amaranthus tricolor L.) leaves extract (RAE), a highly betacyanin-rich natural pigment source. LC-HRMS analysis identified betacyanin (C₃₀H₃₄N₂O₁₉) as the dominant metabolite with the highest ion area (1.15 ×10 ¹⁰), alongside phenolics, carotenoids, and alkaloids that may enhance stability and responsiveness. UV–Vis characterization revealed a strong absorption peak at 535 nm and a total betacyanin content of 261 mg/L, with marked spectral shifts and color degradation above pH 9. Five film formulations were produced by varying extract volume of 1–5 mL (RAE1–RAE5), corresponding to 0.261–1.305 mg/L betacyanin. Film thickness increased with pigment loading (0.085 ± 0.01–0.138 ± 0.02 mm). FESEM revealed improved uniformity in RAE4, while FTIR confirmed strong hydrogen-bonding interactions between betacyanin and the Cs/PVA matrix. The films displayed distinct color transitions across pH 5–9, with ΔE values exceeding 50 for RAE4 and RAE5. Ammonia-vapor assays demonstrated high sensitivity, achieving > 50 % response within 180 min at 1 % NH₃. Application to tilapia fillets stored under refrigerated (6 ± 1 °C, 11 days) and ambient (23 ± 1 °C, 24 h) conditions showed strong correlation between film responses, pH elevation, and sensory quality decline. These findings establish red amaranth as a potent natural pigment and identify Cs/PVA-RAE4 and Cs/PVA-RAE5 films as sensitive, stable, and scalable freshness indicators suitable for real-time food monitoring.
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