Metaproteomics applied to milk and cheese: Methodological advances and perspectives in dairy microbiome characterization

Abstract

Metaproteomics has emerged as a key tool for understanding the molecular complexity of dairy products by integrating the simultaneous identification and quantification of both animal- and microbe-derived proteins with their biological functions within the dairy ecosystem. Supported by high-resolution mass spectrometry and advanced bioinformatics, this approach has provided deeper insights into the dynamics of the microbiome during milk and cheese fermentation, ripening, and preservation. This review discusses the methodological foundations of metaproteomics, recent advances in bottom-up, shotgun, and label-free approaches, and its integration with multi-omics strategies. In addition, it examines the main applications in microbial characterization, authenticity, safety, and biopreservation of dairy products, highlighting recent studies on protein biomarkers, fraud detection, and the discovery of bacteriocins and enzymes of biotechnological interest. Finally, current challenges are discussed, including protocol standardization, database curation, and the incorporation of artificial intelligence into metaproteomic data interpretation. Recent evidence indicates that metaproteomics is becoming a cornerstone for scientific and technological innovation toward a safer, more traceable, and sustainable dairy industry.