Use of fluorescent CTP1L endolysin cell wall-binding domain to study the evolution of Clostridium tyrobutyricum during cheese ripening

Clostridium tyrobutyricum is a bacteria of concern in the cheese industry, capable of surviving the manufacturing process and causing butyric acid fermentation and late blowing defect of cheese. In this work, we implement a method based on the cell wall-binding domain (CBD) of endolysin CTP1L, which detects C. tyrobutyricum, to monitor its evolution in cheeses challenged with clostridial spores and in the presence or absence of reuterin, an anti-clostridial agent. For this purpose, total bacteria were extracted from cheese samples and C. tyrobutyricum cells were specifically labelled with the CBD of CTP1L attached to green fluorescent protein (GFP), and detected by fluorescence microscopy. By using this GFP-CBD, germinated spores were visualized on day 1 in all cheeses inoculated with clostridial spores. Vegetative cells of C. tyrobutyricum, responsible for butyric acid fermentation, were detected in cheeses without reuterin from 30 d onwards, when LBD symptoms also became evident. The number of fluorescent Clostridium cells increased during ripening in the blowing cheeses. However, vegetative cells of C. tyrobutyricum were not detected in cheese containing the antimicrobial reuterin, which also did not show LBD throughout ripening. This simple and fast method provides a helpful tool to study the evolution of C. tyrobutyricum during cheese ripening.