Integrated Culture-Dependent and qPCR Monitoring Reveals Persistent Penicillium Contamination Throughout Smear-Ripened Cheese Production

Research background. Smear-ripened cheeses rely on surface microbiota dominated by Brevibacterium, coryneform bacteria, and yeasts to develop their characteristic sensory properties. However, open-surface production increases the risk of airborne mold contamination, particularly by Penicillium spp., which may compromise product quality and shelf life. Identifying contamination sources and critical control points is essential for effective HACCP-based risk management. Experimental approach. Environmental and product-associated microbiota were monitored in an industrial smear-ripened cheese facility using cultivation, microscopy, and real-time PCR. Environmental contamination was assessed at 24 locations during two sampling periods before and after ethylene oxide fumigation. In parallel, microbiological succession was monitored in two cheese batches throughout ten technological stages of production. Results and conclusions. Penicillium spp. represented the predominant contamination risk within the production environment and on cheese surfaces. Real-time PCR detected fungal DNA more frequently than cultivation, demonstrating the value of combining molecular and culture-dependent approaches. Ethylene oxide fumigation reduced fungal contamination associated with cheese production, although environmental recolonization was observed at facility entry points and cold-storage areas. Key contamination hotspots were identified during forming, drying, washing, and packaging operations. The results indicate that integrated environmental and product monitoring can effectively support contamination tracking and targeted hygiene interventions in smear-ripened cheese production. Novelty and scientific contribution. This study demonstrates the practical value of combining environmental surveillance with product microbiota monitoring to identify contamination sources and verify hygiene measures in industrial cheese production. The integrated use of cultivation and molecular methods provides a robust framework for HACCP verification and contamination management in smear-ripened cheese facilities.