CHARACTERISATION OF PDO CHEESE SAMPLES THROUGH THE IDENTIFICATION OF BIOCHEMICAL MARKERS

Bitto and Valtellina Casera are two PDO cheeses whose origin and production is strongly rooted in the Alpine area, since cows are fed using only local forage and pastures and milk is processed on place. They both are middle-hard, middle-ripened, but different production and storage conditions lead to different physical, chemical and microbiological changes during cheese ripening. In particular, the cooking temperature (48-52°Cx30min for Bitto and 40-45°Cx30min for Valtellina Casera) can influence the microflora growing and the associated biochemical events. Bitto cheese is produced in summer, exclusively in the mountain, in itinerant huts, within 1 hour from milking, using raw full-fat cow milk, and a maximum of 10% of goat milk can be added. Ripening starts in the mountain to conclude in the valley, following natural climatic conditions. Valtellina Casera is a middle-fat cheese produced all year long in social dairy firms, collecting milk from one or two milking. Spontaneous acidification or the addition of starter cultures is used. Cheese ripening is made under controlled conditions. The two products are ripened at least 70 days before labelling and trading. The aim of this PhD work was to improve knowledge on these two products, considering the evolving dairy techniques applied during their production and the poorness of data about their composition. The challenge was to improve and update information in order to identify a set of specific biochemical markers related to the process/product which could be used to classify and assess quality of products for authentication purposes. In this perspective, Bitto and Valtellina Casera cheese samples representative of the current productive reality, were analysed to assess their chemical composition (moisture, protein, fat and ash contents). Particular attention was paid to determine nitrogen fractions and their quantification, since proteolysis is one of the most important biochemical events taking place during cheese ripening. Chemical data showed a high variability due to the traditional technologies adopted during cheese-making and to the different ripening time of samples. Anyway, the presence of significant differences between the two kinds of cheese, although belonging to the same variety, with regard to chemical parameters has been demonstrated. Significant differences (p<0.001) were observed in their moisture, proteins, ash, moisture/solids non-fat, proteins/DM and MFFS (moisture in the fat-free substance) contents, indicating that the interaction between the main cheese constituents in Bitto and Valtellina Casera is different. Valtellina Casera showed significantly higher values (p<0.001) of NCN and NPN, which indicate a more extensive degradation of caseins if compared to Bitto cheese. Protein pattern was evaluated in detail by Capillary Zone Electrophoresis (CZE): an extensive hydrolysis of beta-caseins into the gamma-caseins by means of plasmin was evidenced. Also alpha s1-casein was highly degraded and a peak at the same migration time as alpha s1-I-casein appeared, suggesting some residual activity of chymosin or cathepsin D. Protein profile was a useful fingerprint for product characterization and a valuable tool to differentiate the two products: some peptides characterised the two cheese profiles either for presence/absence or for variation in their concentration and contributed to the partial distribution of samples in a PCA plot. Free amino acids (FAA) quantification by HPLC analysis evidenced strong differences in the proteolysis behaviour, which was by far deeper in Valtellina Casera, confirming results obtained for nitrogen fractions. The two varieties showed very different concentration values of single amino acids but the same relative percentage distribution: Glu, Val, Phe, Leu, Orn and Lys confirmed to be the most relevant ones in both cheese productions, representing 67.6% of total FAA in Bitto and 64% in Valtellina Casera. Anyway, amino acids which allowed samples separation in PCA plot were those which showed most significantly different values in the two products. Organic acids content was investigated. Results showed a very high variability and PCA simultaneously applied to all the variables could not differentiate between the two cheese productions. Anyway, through pair-wise comparison, Bitto cheese showed higher butyrate concentrations, while Valtellina Casera had higher lactate, pyruvate and pyroglutamate contents. In conclusion, since the final aim of this work was Bitto and Valtellina Casera cheese characterisation and differentiation, PCA was applied to all parameters found to be highly significant (p<0.001) in order to test the whole data set for cheese classification. PCA explained 72% of the total variance and correctly grouped samples into two classes.