Understanding the bacterial communities of hard cheese with blowing defect

Hard cheese production require long ripening time creating the optimal conditions for spoiling microorganisms able to survive in the food matrix. Late blowing in cheese caused by butyric clostridia is a well-fitting example (Ingham et al., 1998; Klijn et al., 1995; Vissers, 2007). Different clostridial species alone or in association, have been related during time to the blowing problem, but few data are available about their dynamic changes in the cheese shape and their relationships all along the ripening period. In Italy, Grana Padano (GP) hard cheese is produced from raw cow's milk added with natural whey starter cultures and protected from clostridia spoilage by lysozyme addition (www.granapadano.com). The aim of the present work was to assess the microbial communities of spoiled hard cheese using new NGS technologies associated to quantitative and qualitative cultivation-independent techniques. These approaches have not yet been applied for the study of microbial communities involved in late blowing spoilage. Respectively, a PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) approach to obtain a qualitative characterization of clostridia heterogeneity, a TaqMan qPCR on single C. tyrobutyricum species and an NGS approach based on Illumina MiSeq sequencing of total bacteria were applied to 83 Grana Padano cheese samples from nine production facilities, with or without the addition of lysozyme and with blowing defects appearance at different ripening times. Information gathered from this study could be useful to assess the effect of lysozyme as a preservative and to measure the effects of ripening time on clostridial population and their relationships with other bacterial species present in the cheese paste