Non Proteolytic Aminoacyl Derivatives (NPADs) in cheeses: Synthesis, Origin, Properties

La presente tesi di dottorato riguarda la caratterizzazione di derivati amminoacidici (gamma-glutamil amminoacidi, lattoil-amminoacidi, piroglutamil-amminoacidi), denominati Non Proteolyltic Aminoacyl Derivatives (NPADs) presenti nel Parmigiano Reggiano, considerando in particolare la caratterizzazione strutturale, l’origine e le proprietà biologico-funzionali. Dopo un’introduzione generale riguardante lo studio e l’evoluzione peptidica nel formaggio durante la stagionatura, i peptidi bioattivi, la sintesi/analisi peptidica ed i risultati riportarti in letteratura riguardanti gli NPADs, la prima parte della tesi riguarda l’uso di tali molecole come marker di stagionatura avvalendosi dell’analisi statistica. La produzione e la degradazione enzimatica degli NPADs sono esaminate nel terzo capitolo, indicando i potenziali enzimi responsabili della loro produzione ed eventuale degradazione. Inoltre, la bioaccessibilità di tali composti è stata studiata nel medesimo capitolo dimostrando la resistenza degli NPADs alla digestione gastrointestinale simulata ed al siero umano. La biodisponibilità della gamma-glutamil-fenilalanina, molecola maggiormente rappresentativa degli NPADs, è stata studiata utilizzando un sistema che impiega cellule Caco-2. L’ultima parte, infine, riporta i primi risultati riguardanti le proprietà immunomodulatorie ed antiossidanti degli NPADs.The present Ph. D. thesis is focused on the characterization of unusual aminoacidic derivatives (gamma-glutamyl amino acids, lactoyl amino acids, pyroglutamyl amino acids), collectively named Non Proteolytic Aminoacyl Derivatives (NPADs), present in Parmigiano Reggiano cheese, considering in particular their structural characterization, the origin and biological-functional properties. After a general introduction concerning cheese peptidomics approaches, bioactive peptides, peptides synthesis/analysis and previous results about NPADs, the first part deals with the use of NPADs as a marker of cheese ripening, through appropriate statistical analysis. The enzymatic production and degradation of NPADs are then examined in depth in the third chapter, indicating which enzymes in cheese might be responsible for their production and eventual degradation. Also bioaccessibility was evaluated , demonstrating the resistance of NPADs to gastrointestinal digestion and blood serum. Also the bioavailability of gamma-Glutamyl-Phenylalanine, the most important representative of NPADs, was studied using Caco-2 model. The last part reports the first studies about the immunomodulatory and antioxidant properties of NPADs

The Interrelationship Between Microbiota and Peptides During Ripening as a Driver for Parmigiano Reggiano Cheese Quality

Cheese microbiota contribute significantly to the final characteristics of cheeses due to the growth and interaction between cheese microorganisms during processing and ripening. For raw milk cheeses, such as Parmigiano Reggiano (PR), the microbiota derive from the raw milk itself, the dairy environment, and the starter. The process of cheese making and time of ripening shape this complex ecosystem through the selection of different species and biotypes that will drive the quality of the final product by performing functions of their metabolism such as proteolysis. The diversity in the final peptide and amino acid composition of the cheese is thus mostly linked to the diversity of this microbiota. The purpose of this study was to get more insight into the factors affecting PR cheese diversity and, more specifically, to evaluate whether the composition of the bacterial community of cheeses along with the specific peptide composition are more affected by the ripening times or by the cheese making process. To this end, the microbiota and the peptide fractions of 69 cheese samples (from curd to cheese ripened 24 months) were analyzed during 6 complete PR production cycles, which were performed in six different dairies located in the PR production area. The relation among microbial dynamics, peptide evolution, and ripening times were investigated in this unique and tightly controlled production and sampling set up. The study of microbial and peptide moieties in products from different dairies – from curd to at least 12 months, the earliest time from which the cheese can be sold, and up to a maximum of 24 months of ripening – highlighted the presence of differences between samples coming from different dairies, probably due to small differences in the cheese making process. Besides these differences, however, ripening time had by far the greatest impact on microbial dynamics and, consequently, on peptide composition