VOCs Profiling and Quality Assessment of Milk Employing Odorant-Binding Proteins-Based Fluorescence Biosensor

The quality of cow's milk is critical for human nutrition; thus, it is important to develop rapid, sensitive, and cost-effective methods to monitor milk quality. Volatile Organic Compounds (VOCs) from milk are odorant molecules that can be used as key indicators of milk quality, since their presence is influenced by important factors such as animal metabolism, animal diet, and farming practices. In this work, we used the porcine odorant-binding protein (pOBP) and the bovine odorant-binding protein (bOBP) as molecular recognition elements (MREs) of an innovative fluorescence biosensor to detect the presence of odorant molecules in (a) milk produced by intensive livestock farming and (b) milk produced by extensive livestock farming. For biosensors, it is important to use proteins that are stable under operative conditions; therefore, we used fluorescence spectroscopy for a biophysical characterization of the pOBP and of the bOBP at different temperatures. The proposed biosensor employs a system to capture the odorant molecules from milk, which are then transferred to a liquid phase for quantitative and qualitative analyses. The binding of the odorant molecules to the OBPs triggers a Förster Resonance Energy Transfer (FRET) signal, allowing for real-time VOC quantification. The performance of the assays was evaluated by Headspace Solid-Phase Microextraction coupled with Gas Chromatography-Mass Spectrometry (HS-SPME/GC-MS) experiments. The experimental approach used for the development of the biosensor demonstrated high sensitivity and specificity, enabling the differentiation of milk from intensive and extensive farming systems. The results indicate the potential of this method for the real-time monitoring of VOCs in milk samples for food traceability and quality control.