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Food fortification is a current practise to increase the bioavailability of certain nutrients. In this study, 5% (w/w) pistachio powder was added to tender wheat flour or durum wheat semolina to evaluate its contribution to augment the amount of lysine in bread. Bread production was carried out by sourdough technology using a selected 3-species lactic acid bacterial (LAB) starter culture (Lactobacillus sanfranciscensis PON100336, Leuconostoc citreum PON10079 and Weissella cibaria PON10030). In order to determine the influence of pistachio powder on the evolution of LAB, sourdough bread productions were compared with control trials obtained from flour or semolina and evaluated for the development of the main microbial groups by plate counts. All sourdoughs were subjected to a long-time fermentation (21 h) and showed levels of LAB around 109 CFU/g, indicating the suitability of pistachio powder for lactic fermentation. Yeasts were also detected, in particular in semolina trials. MySeq Illumina technology was applied to investigate the bacterial composition of sourdoughs evidencing a different distribution of LAB species among the trials with Lactobacillus as being the major bacterial group in almost all sourdoughs, except that produced from flour supplemented with pistachio powder which displayed Weissella as dominant group. Physicochemical parameters were comparable among the trials. After baking, pistachio powder was found not to influence the height of the breads, but pistachio breads were more firm than control breads. Colour of the breads, as well as void fraction and cell density, were influenced by pistachio powder. The amount of lysine increased consistently thanks to pistachio supplementation which also determined a higher presence of o-xylene, p-cymene and limonene and the appearance of alpha-pinene and 1-octen-3-ol in the final breads. Sensory tests showed a higher appreciation of the judges towards the breads produced from flour and semolina supplemented with pistachio powder rather than control breads.

This work was carried out to pursue a double objective: to improve the hygienic safety of cheeses produced from raw ewes’ milk; and to produce a new typology of cheese through the application of “Grana” technology. With this in mind, raw milk from an individual farm was transformed under controlled condition at a pilot dairy plant. The production technology included the partial skimming of the evening and morning milk mixture by cream surfacing, the addition of a natural whey starter cultures (NWSC) prepared with four selected Streptococcus thermophilus strains (PON6, PON244, PON261 e PON413) isolated from raw ewes’ cheese productions (Gaglio et al., 2014), milk clotting at 38°C, curd cooking at 56°C, cheese moulding at room temperature, immersion salting and ripening for 9 months. In this study, 10 microbial groups were investigated from raw milk until ripened cheeses. Lactic acid bacteria (LAB) were in the range 104 - 105 CFU/ml before NWSC addition. After curdling, these bacteria increased to 3 Log cycles and were counted at 106 CFU/ml after curd cooking. A rapid pH drop (to 6.05) was registered after almost 3 h from NWSC addition. The levels of members of the Enterobacteriaceae family were at about 103 CFU/ml in raw milk and decreased after curd cooking to 1 Log cycle. A similar behavior was shown by the other undesired microbial group till the complete disappearance of staphylococci. The microbiological counts of 9-month ripened cheeses showed the dominance of LAB and undetectable levels of undesired bacteria. Next Generation Sequence analyses are in progress to evaluate the total microbial composition of the final cheeses. On the basis of the results, the main hypothesis of the work was accepted since the undesired microbial groups were reduced in number in comparison with uncooked hard pressed technology.

Phytothermotherapy ("grass baths") is a traditional phytotherapy for rheumatism consisting of taking baths in hot fermenting grass. Scientific studies have demonstrated its efficiency in treating several rheumatic diseases. However the efficiency and repeatability of the therapy is dependent on the wild fermentations, determining sometimes the appearance of unpleasant conditions leading to the early abandonment of the therapy. The metabolism undergoing in the grass baths is unknown and there is not an established method to evaluate and predict grass baths quality. The aim of this study is to establish a simple VOCs profiling method able to evaluate the grass baths, predicting their evolution, through the identification of marker volatiles related to the best conditions and/or the spoilage. After replicating in real scale the traditional grass baths, the volatile profiles were measured using passive diffusion samplers injected in a thermal desorption-comprehensive GC × GC-TOF-MS. The high dimensionality of the data coupled with the limited number of time points, required a rigorous method development for the analysis of the data, achieved through the development of a novel R package for variable selection in GC × GC data matrices. The further application of a fuzzy clustering approach demonstrated to be a useful tool dealing with short time series, allowing to discard un-trending volatiles and giving a clear snapshot of the main trends in the data. A broad coverage of the volatolome was provided, thus suitable to describe the main metabolic changes ongoing in the grass baths. Coupling this data with the temperature and pH, and comparing it to the data from similar processes, like silage and compost, we demonstrated that the established method can be helpful to evaluate short time series, allowing us to obtain a list of volatiles as candidate markers for the quality of the grass baths. The established method gave a list of markers applicable to real scale grass baths to predict spoilage; furthermore it provides a list of volatiles where to search for candidate markers with reported health-related effects and can be used to generate hypothesis on the mechanisms of action of the treatment.

Obiettivo di questo lavoro era lo studio dell´evoluzione della carica microbica totale e psicrotrofa indesiderata nel latte per produzione di Grana Trentino al variare del sistema di raccolta del latte. Il conferimento due volte al giorno del latte raccolto in bidoni senza refrigerazione o in cisterna refrigerata a 18°C è stato confrontato con il conferimento una volta al giorno del latte raffreddato alla stalla a 8 o 12°C. I latti sono stati seguiti dalla stalla alla caldaia, i campioni di latte e panna sono stati analizzati tramite tecniche classiche e molecolari per la definizione del quadro microbico. La composizione microbica del latte intero non differiva nelle 4 tesi: in tutti i campioni sono stati trovati S. thermophilus, L. helveticus, L. delbrückii, L. rhamnosus, Ln. mesenteroides, E. coli, specie appartenenti alla famiglia delle Pseudomonadaceae. Dopo l´affioramento, il latte a 12 o 8°C alla stalla aveva un quadro microbiologico sovrapponibile a quello del latte refrigerato a 18°C. Nel latte non refrigerato trasportato con bidoni le cariche batteriche totali in caldaia erano più alte rispetto ai latti refrigerati. La refrigerazione del latte a 12 o 8°C non ha comportato una dominanza dei batteri psicrotrofi sui lattici nel latte in caldaia. Durante l’affioramento notturno i microrganismi psicrotrofi aumentano solo in latti non refrigerati alla stalla, e le specie microbiche si distribuiscono differentemente nelle frazioni che si separano: Pseudomonas e Flavobacteriaceae maggiormente concentrati nella panna, Enterobacteriaceae e Streptococcaceae nella fase meno grassa.

La composizione microbica del Grana Trentino e del suo siero-innesto è stata studiata non solo in relazione alla differente tecnologia di raccolta e conservazione del latte ma anche in relazione al tempo di stagionatura e alla zona di campionamento. Per una serie di lavorazioni si è determinata la composizione microbica di latte, siero e di due forme gemelle, aperte dopo 9 e 18 mesi. Queste sono state tagliate lungo l’asse verticale e sono state campionate tre diverse zone lungo la diagonale: sotto la crosta, al centro e in posizione mediana. I campioni sono stati analizzati tramite metodi coltura-dipendenti e coltura-indipendenti (DGGE e 454 pyrosequencing) e i risultati sono stati messi in relazione con le analisi chimiche degli acidi grassi a corta catena. Batteri termofili sono stati trovati, come atteso, in grandi quantità sia nel siero cotto che nel siero-innesto. Dopo 9 mesi le conte erano più alte che dopo 18, e nel centro erano più basse che nelle altre due zone campionate. Le principali specie isolate da formaggio, all’analisi fenotipica, erano diverse da quelle isolate da latte e siero. Il microbiota nel sotto crosta e nella zona intermedia era dominato da Lactobacillus paracasei e Lactobacillus rhamnosus, mentre al centro vi era una maggiore presenza di Lb. rhamnosus. Dopo 18 mesi, i batteri lattici (LAB) erano assenti. L’analisi tramite pyrosequencing 454 ha permesso di individuare la dominanza di Streptococcaceae nella parte centrale delle forme di 18 mesi.

La tecnologia di lavorazione del Grana Trentino che prevede la raccolta doppia giornaliera del latte è stata messa a confronto con un’altra che prevede la conservazione a bassa temperatura, in stalla, del latte munto la mattina, e la sua unione con il latte munto alla sera. Il conferimento al caseificio del latte di un’intera giornata avviene una sola volta, la sera. Si sono impostate due temperature di conservazione del latte in stalla, 8°C e 12°C, per il sistema di raccolta unica, che è stato messo a confronto con il latte conferito due volte al giorno, che è stato conservato a 18 °C (il cosiddetto “raffrescato”), o trasportato senza refrigerazione in bidoni da 50 litri. I confronti sono avvenuti in tre caseifici ed in due periodi dell’anno (estate ed inverno). Come prevedibile, si sono osservate differenze nella stagionalità del latte, e tra le tecnologie adottate nei diversi caseifici. Le due modalità di conferimento si sono rivelate differenti per alcune caratteristiche, quali il tempo di affioramento, il contenuto in grasso della panna dopo la scrematura, il contenuto in cellule somatiche, ed anche, indirettamente, il contenuto in microrganismi.