Understanding the bacterial communities of hard cheese with blowing defect

The environment of hard cheese encourages bacterial synergies and competitions along the ripening process, which might lead in defects such as clostridial blowing. In this study, Denaturing Gradient Gel Electrophoresis (DGGE), a quantitative Clostridium tyrobutyricum PCR and next-generation Illuminabased sequencing of 16S rRNA gene were applied to study 83 Grana Padano spoiled samples. The aimwas to investigate the community of clostridia involved in spoilage, the ecological relationships with the other members of the cheese microbiota, and the effect of lysozyme. Three main genera were dominant in the analysed cheeses, Lactobacillus, Streptococcus and Clostridium, and the assignment at the species level was of 94.3% of 4,477,326 high quality sequences. C. tyrobutyricum and C. butyricum were the most prevalent clostridia. Hierarchical clustering based on the abundance of bacterial genera, revealed three main clusters: one characterized by the highest proportion of Clostridium, a second where Lactobacillus was predominant and the last, dominated by Streptococcus thermophilus. Ecological relationships among species were found: cheeses characterized by an high abundance of S. thermophilus and L. rhamnosus were spoiled by C. tyrobutyricum while, when L. delbrueckii was the most abundant Lactobacillus, C. butyricum was the dominant spoiling species. Lysozyme also shaped the bacterial community, reducing C. tyrobutyricum in favour of C. butyricum. Moreover, this preservative increased the proportion of L. delbrueckii and obligate heterofermentative lactobacilli and lowered L. helveticus and non-starter species, such as L. rhamnosus and L. casei.