THE LATE BLOWING DEFECT IN GRANA PADANO CHEESE: THE MECHANISMS OF MILK HEALING THROUGH NATURAL CREAMING AND THE EFFECTS OF CHEESE MAKING CONDITIONS IN INDUCING CLOSTRIDIUM SPORE GERMINATION

This thesis project was designed to explore the technological, chemical and microbiological factors involved in the late blowing defect (LBD) of Grana Padano (GP) with the aim of setting up every possible precaution to overcome this economically important flaw. LBD is a cheese defect caused by some Clostridium species, mostly Clostridium tyrobutyricum, and characterized by holes, cracks as well as an unpleasant flavour. Natural creaming of milk is the first and most critical step in GP making, during which C. tyrobutyricum spores are risen to the top of milk together with fat globules, thus being eliminated from partially skimmed milk. This step has been thoroughly investigated with particular reference to the interaction type between spores and fat globules. Transmission Electron Microscopy (TEM) revealed an electron-dense material sticking bacteria to fat globules. This material has been identified as immunoglobulins, mainly IgA and to a lesser extend IgM, by immunogold labelling. Many IgA molecules, but not IgM and IgG, were localized on C. tyrobutyricum cell wall suggesting the presence of anti-IgA receptors on it. Following this finding, immunoglobulins were purified from colostrum and their ability to enhance bacteria rising during natural creaming of milk investigated. Indeed, the addition of immunoglobulins led to a significant increase in spore rising with respect to untreated control. Specific natural creaming trials were also carried out to study the effect of creaming temperature on fat microstructure. Fat supramolecular organization, the unwanted fat globule structural rearrangement, was observed when creaming was carried out at 22 and 40 °C, as well as in sample tempered at 37 °C prior to the normal creaming at 8 °C. This suggests that the often-adopted thermal treatment before creaming is deleterious for fat microstructure. The behaviour of C. tyrobutyricum throughout the further steps of GP making and ripening was also investigated by an innovative approach. Vegetative bacteria cells and spores, independently sealed within dialysis tubes, were kept in the vat for the entire cheese making and then into the cheese over 6-month ripening. They were sampled and counted at milk renneting, curd cooking, curd extraction, in-mould acidification, brine salting, 3- and 6-month ripening. Furthermore, their morphological changes were monitored by TEM and the free amino acid utilization evaluated. Vegetative bacteria cells died during curd cooking up to 54 °C and then were no longer cultivable. However, 2 x 102 CFU/mL of these cells were sporulated in the curd at extraction and a higher number of spores was found at the end of 6-month ripening. Few spores germinated during curd acidification, when lactate was available. C. tyrobutyricum proved to convert arginine to citrulline and then to ornithine throughout the cheese ripening. This capability was confirmed in both milk and cheese model systems, indicating the vitality and metabolic activity of bacterial cells and, possibly, their ability of modifying GP sensorial properties. Lysozyme (LZ) is used in GP as well as in other cheese varieties to prevent the LBD. Thus, it has been considered important to verify its effects on lactic acid bacteria population and cheese free amino acid pattern. This study was carried out in 16 raw-milk hard cheeses produced in eight parallel cheese makings conducted at four different dairies using the same milk with (LZ+) or without (LZ-) addition. LZ- cheeses were characterized by higher numbers of cultivable microbial population and lower amount of DNA arising from lysed bacterial cells with respect to LZ+ cheeses. At both 9 and 16 months of ripening, Lactobacillus delbrueckii and L. fermentum proved to be the species mostly affected by LZ. The total content of free amino acids indicated that the proteolysis extent is characteristic of each dairy, regardless to the presence of LZ. Instead, in LZ+ cheeses, microbial degradation of arginine into citrulline and ornithine was always promoted. As, the amount of ornithine was quantitatively lower than citrulline it is likely that the arginine-deiminase pathway was only partially adopted. Working with C. tyrobutyricum spores gave rise to the need of a practical and effective staining method that could both facilitate spore detection and easily discriminate spore from vegetative bacterial cells. A fast and robust protocol for fluorescent staining of spores and vegetative cells of C. tyrobutyricum was set up, by using Hoechst 34580 and Propidium Iodide (HO/PI) stains that allowed to distinguish viable, dead and sporulated cells of C. tyrobutyricum. The HO/PI staining protocol proved to be suitable for other three Clostridia that can cause LBD: C. butyricum, C. sporogenes and C. beijerinkii. Furthermore, Hoechst 34580 dye was successfully used, together with Nile Red and Fast Green, to observe spores into the cheese matrix. In this case, spores were experimentally added to milk during cheese making and the triple staining was performed on slices of cheese after a month ripening. Finally, during the investigations related to this thesis project, it was possible to shed light on other inclusions present in GP matrix during and after ripening, such as specks, spots and microcrystals. Light microscopy revealed that the small, hard specks had the structure of crystalline tyrosine, as confirmed by amino acid analysis. Spots showed a complex structure, including several curd granules, cavities, and microcrystals, and were surrounded by a dense protein layer. Spots contained significantly less moisture and ash than the adjacent cheese area, and were significantly richer in protein, including significantly higher levels of valine, methionine, isoleucine, leucine, tyrosine and phenylalanine. Microcrystals were observed by light and electron microscopy and analysed by confocal micro Raman. Among others, calcium phosphate crystals appeared to consist of a central star-shaped structure immersed in a matrix of free fatty acids besides leucine and phenylalanine, in free form or in small peptides. A hypothetical mechanism for the formation of these structures was also formulated.