Dual-indicator approach for real-time milk freshness detection using butterfly pea anthocyanins and riboflavin fluorescence

Abstract

Milk is highly prone to spoilage due to its rich nutrient and moisture content. This study presents a dual-indicator system for real-time milk freshness assessment by integrating a natural pH-sensitive dye from Clitoria ternatea (butterfly pea) with riboflavin-based fluorescence spectroscopy. Anthocyanins were extracted in aqueous media and characterized via UV–vis (400–700 nm) and FTIR spectroscopy, confirming pH-sensitive transitions (λ_max shift from 548 nm to 628 nm) and relevant functional groups. A chromogenic assay showed progressive color change during storage at 37 °C, with CIE-LAB values shifting from L* = 64.61 to 13.42, a* from −2.24 to 27.90, and ΔE from 23.26 to 60.27 over 24 hours. Simultaneously, riboflavin fluorescence (λ_ex = 450 nm, λ_em = 520–525 nm) quenched from ∼8100 to 4300 RFU, correlating with pH drop (from 6.67 to 4.81), titratable acidity rise (0.125% to 0.456% lactic acid), and SPC increase (5.00 × 10⁴ to 7.85 × 10⁶ cfu mL⁻¹). The study successfully demonstrates a dual-indicator platform combining visual anthocyanin-based chromogenic strips and riboflavin fluorescence spectroscopy for non-invasive, real-time milk spoilage detection. Both these indicators are extremely potent and mainly work on pH.