Immunoassay-Based Identification of Dairy Thermal Processing Technology and Mechanism Analysis Using Lactoferrin as a Model

Accurate differentiation between dairy processing technologies such as pasteurization and ultra-high temperature sterilization is crucial for ensuring product quality, protecting consumer rights, and maintaining market order. This study identified antigen structure-sensitive antibodies and developed a high-performance sELISA detection method. By detecting antigenic changes in lactoferrin (LF) during processing, it enables the identification of milk thermal processing conditions. This antibody pair not only exhibits excellent binding affinity for native LF but also possesses processing-dependent recognition ability for heat-treated LF. While mild denaturation at 75°C reduces its recognition efficiency, it promotes the formation of stable lactose-LF complexes, thereby preserving conformation-dependent antigenicity. In contrast, combined high-temperature and pressure processing overrides the protective effect of lactose, rendering sELISA detection ineffective. Based on experimental and molecular docking results, the positive effect of lactose on LF antigenicity during thermal processing provides key support for distinguishing between pasteurization and ultra-high temperature (UHT) processing. Furthermore, validation with real samples confirms that this method can effectively differentiate pasteurized milk from UHT milk/reconstituted milk, offering technical support for industrial quality control and product authenticity verification.