Profiling antimicrobial and resistance proteins in milk from diverse cow breeds

Milk is a complex fluid containing nutritional and bioactive compounds, including antimicrobial peptides (AMPs) and resistome proteins. AMPs, crucial for innate immunity, protect against pathogens in the mammary gland and infant gut. Resistome proteins, linked to antimicrobial resistance (AMR), pose a global health concern. Understanding these proteins in milk from different bovine breeds, such as the Podolica breed, can shed light on antimicrobial properties and bioactive potential. Monitoring resistome proteins is crucial due to the spread of AMR genes in animal production. This study used LAP-MALDI MS, proteomics, and metaproteomics to classify Podolica milk, identify AMPs, and investigate resistome proteins across various breeds and environments.

Eight farms in Calabria (Italy) were studied: four Podolica farms and four with other cattle breeds. Milk samples were collected twice from each farm, 7 days apart, using a sanitized dipper after 5–10 minutes of agitation. Each 250 mL sample was partially used for somatic cell count (SCC). Samples were transported at 4°C to the Centro Interdipartimentale di Servizi Veterinari, Magna Graecia University of Catanzaro, Italy, and stored at -80°C.

LAP-MALDI MS profiling differentiated Podolica milk from other breeds. Milk samples were processed via centrifugation and TCA precipitation, followed by hybrid quadrupole-time of flight mass spectrometer analysis. Protein concentrations were measured, and samples were digested with trypsin. LC-MS/MS analysis was performed using a ThermoFisher Scientific Orbitrap Fusion Tribrid® mass spectrometer. Data analysis involved PEAKS XPpro and MaxQuant software for label-free quantification (LFQ), using Bos taurus, bacteria, and resistome databases.

LAP-MALDI MS profiling coupled with LDA analysis yielded 86% accuracy in classifying Podolica versus control milk samples in the training set. The test set achieved 98.4% accuracy. Significant ion species for classification were identified, showing notable abundance differences.

Bottom-up proteomics identified about 220 proteins from LC-MS/MS searches. Cathelicidins and annexins were more abundant in control cow milk. Bacterial taxonomy analysis showed different bacterial presences between groups. The CARD database analysis detected beta-lactamases and tetracycline resistance proteins in both sample groups, with an isoform of beta-lactamase more abundant in Podolica milk, highlighting the importance of monitoring AMR.

In conclusion, this study analyzed milk composition from various bovine breeds, focusing on AMPs and resistome proteins. Using LAP-MALDI MS profiling and bottom-up proteomics, we distinguished Podolica milk, identifying around 220 proteins. Both control and Podolica samples contained beta-lactamases and tetracycline resistance proteins, emphasizing the need for routine AMR monitorin