Application of culture dependent and cultural-independent techniques to investigate the dynamics of microorganisms during industrial cheese making of a Gouda type cheese
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- Master's theses (KBM) 
The aim of this work was to study the microbial dynamic in a Gouda type cheese applying a comparison between culture-dependent technique, plate count, and culture-independent technique as PCR-DGGE. The study was also focus on the analyse of the efficiency of the pasteurizer, and its cleaning steps. The study was conducted analysing 4 batches for each of the two days of production, Monday and Friday. Between batch 16 and 17 there was a quick cleaning, and between batch 32 and batch 1, of the following day, there was a complete cleaning. For each batch samples of raw milk, pasteurized milk, starter, milk before rennet, cheese after pressing, cheese after brine were collected to understand the microbial development during the cheese making-process. Plate count was conducted with selective media, M17, for lactococci; MRSV, (MRS with vancomycin), to select Leuconostoc; LBS, for lactobacilli. The PCR-DGGE was conducted both with universal primer for V1 and V3 of 16S DNA, and with specific primer for lactobacilli, Leuconostoc/Pediococci, and selective for Lactococci/Enterococci and Streptococci. The gradient 35-55% of urea-formamide was used during DGGE (100% denaturant corresponding to 7M urea and 40% [v/v] formamide was used). Identification of the DGGE bands was possible by the comparison among different migration distance of pure strains, loaded in the gel, like a marker and by the sequencing of the band. The monitoring of total lactic acid bacteria (LAB) and non-starter lactic acid bacteria (NSLAB), from the dairy samples and starter cultures the presence of Lactococcus (Lc.) lactis subsp. lactis and Lc. lactis subsp. cremoris, as the dominant species of LAB was shown, while Lc. Raffinolactis, Lactobacillus (Lb.) casei and Lb. paracasei could be considered NSLAB. Studying the pasteurization step, Anoxybacillus flavithermus, Aeribacillus pallidus/ Geobacillus Pallidus were identified, which may produce, heat resistant spores and these may also create biofilm on the steel pasteuriser surface. It was supposed that the cleaning in at middle of the production day was not sufficient to eliminate this bacillus. At the other hand it was possible to conclude that the pasteurized treatment was effective. Streptococcus dysgalactiae subsp. dysgalactiae, may underline the presence of mastitis in the milk.However the combination of temperature and time in the pasteurizer, was able to kill this species which was not found after pasteurisation. The technique, which was applied in this study, could not permit the quantification of the amount of microorganism with precision. In fact thanks to the comparison between culture dependent and culture independent techniques, it was possible to understand the proportion and the balance between the species of LAB, indicating which were predominant.