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Sattin, Eleonora (2016) 16S amplicon Next Generation Sequencing approach evaluation and its application to food microbial communities characterization. [Tesi di dottorato]

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Abstract (inglese)

Foods are complex ecosystems, due to the influence of either intrinsic and extrinsic factors such as the chemical composition, microbial communities and the environment that can have an effect on the final product. In cheese microbes are the most influencing factor because they can effect both shelf-life and quality flavor/taste of the food. In addition microbiome establishes complex interactions within the bacterial community itself and between the food environment, such that nowadays these dynamics have been only partially highlighted with traditional culture-dependent technique. Thus, culture-independent methods such as Next Generation Sequencing (NGS) approach have become a powerful tool to study in depth microbial communities contained in a variety of ecological niches, including food. In fact they should be increasingly considered as a complementary technique to culture-dependent methods in microbiology.
In this comprehensive study we have evaluated and applied a 16S Next Generation Sequencing approach to the study of Ricotta cheese shelf life, its raw materials and to food technology studies in soft or ripened cheeses using adjunct cultures. In all these studies NGS method have always been supported with culture-dependent approaches, other molecular methods and biochemical analysis. The different studies are separated in the subsequent chapters of this dissertation.
First of all using a mock community we evaluated the impact of 16S NGS pipeline on the final community structure and identification, showing that bioinformatic analysis, and above all the OTU-picking strategy, can affect the alpha-diversity (within the sample) of the community but not its beta-diversity (between samples). To this extent both strategies can be used in order to compare community structures. Moreover the protocol was showed as robust and repeatable.
Secondly we evaluated the raw materials (wheys and cream) used for ricotta production showing that the microbiological quality of these materials must be improved. As they showed a very biodiverse micro flora (mainly Streptococcaceae and Pseudomonadaceae) and a high concentration of aerobic spores (10 to 104 CFU/ml, not present in cream) their usage for Ricotta-making could damage the final product. Moreover NGS combined with Gas Cromatography approach on organoleptic non satisfactory frozen wheys showed that under-hygienic storage of the raw materials drove to the production of fermentation or lipid oxidation compounds influenced by particular bacteria such as Acinetobacter, Lactococcus and Pseudomonas.
In the third chapter we evaluated Ricotta shelf life during winter and summer season, showing that Ricotta microbiome was mainly composed of spore-forming bacteria, Bacillus, Paenibacillus and Clostridium (more than 80% of relative abundance). Moreover we found a summer ricotta with a pink discoloration and, applying a molecular Multi-locus Sequence Typing approach, we demonstrated that the bacterium causing the defect belonged to the Bacillus cereus group, in particular it was a B. mycoides/wehienstephanensis strain. This work has been published in Food Microbiology journal.
In the last chapter we applied NGS analysis to the technological use of adjunct cultures on soft or ripened cheeses in order to evaluate their anti-spoilers effect. We evaluated the anti-gammaproteobacteria effect of some strains such as L. rhamnosus, L. sakei and Carnobacterium maltoaromaticum in fresh industrial cheeses prone to package swelling and premature spoiling. From a NGS point of view we found that C. maltoaromaticum managed to eliminate almost all contaminants at different storage temperatures.
In conclusion with NGS approach we could improve food microbiological quality assessment and support food technology research. In fact the results obtained in the present studies demonstrate how the application of NGS technologies, describing in-depth microbial communities, could in the next future become a scalable, cost-effective and suitable tool in the food industry for improving the quality and the safety of products.

Abstract (italiano)

Gli alimenti sono ecosistemi complessi in quanto vengono influenzati sia da fattori intrinseci che da fattori estrinseci, come la composizione chimica, il profilo microbiologico e l'ambiente, che possono avere forte impatto sul prodotto finale. In particolare nei formaggi i microbi sono il fattore più influente perché possono modificare sia la vita commerciale che il sapore ed il gusto del prodotto. Inoltre il microbiota stabilisce delle complesse interazioni internamente alla comunità batterica e con l'ambiente, a tal punto che al giorno d'oggi queste dinamiche sono state solo in parte evidenziate con metodi tradizionali coltura-dipendenti. Di conseguenza i metodi coltura-indipendenti, come il Sequenziamento di Nuova Generazione (NGS), stanno diventando un potente strumento per studiare in profondità le comunità microbiche presenti in una varietà di nicchie ecologiche, compresi gli alimenti. È probabile che ormai tali tecniche debbano essere considerate complementari ai metodi di microbiologia coltura-dipendenti.
In questo ampio progetto abbiamo valutato e applicato un approccio NGS sul gene 16S rRNA allo studio della vita commerciale della Ricotta, delle sue materie prime e ad alcuni studi di tecnologia degli alimenti su formaggi molli o stagionati addizionati con colture batteriche antagoniste. In tutti questi studi i metodi NGS sono sempre stati supportati da approcci cultura-dipendenti, altri metodi molecolari e analisi biochimiche. I vari studi sono suddivisi tra i successivi capitoli di questa tesi.
Per prima cosa utilizzando una comunità batterica artificiale abbiamo valutato l'impatto del workflow di processo del protocollo 16S-NGS sulla struttura e sull'identificazione della comunità reale. Con questo approccio abbiamo dimostrato che l'analisi bioinformatica, e principalmente la strategia di raggruppamento delle sequenze in OTU (Operational Taxonomic Unit), può influenzare la diversità della comunità di tipo alfa (intra-campione), ma non la sua beta-diversità (inter-campioni). In questo senso entrambe le strategie possono essere utilizzate al fine di confrontare le strutture delle comunità microbiche. Inoltre il protocollo si è dimostrato robusto e ripetibile.
In secondo luogo abbiamo valutato le materie prime (siero e panna) utilizzate per la produzione di ricotta, dimostrando che la qualità microbiologica di queste materie deve essere migliorata. Poiché queste hanno mostrato una forte biodiversità (principalmente di Streptococcaceae e Pseudomonadaceae) e un'alta concentrazione di spore aerobie (da 10 a 104 UFC/ml, non presenti nella panna), il loro utilizzo per la produzione della ricotta potrebbe danneggiare il prodotto finale. Non di meno l'approccio combinato NGS con Gas Cromatografia su sieri congelati con difetti organolettici ha mostrato che lo stoccaggio non igienico delle materie prime conduce alla produzione di composti di fermentazione o di ossidazione lipidica, guidati anche da particolari batteri come Acinetobacter, Lactococcus e Pseudomonas.
Nel terzo capitolo abbiamo valutato la vita commerciale della Ricotta durante l'inverno e l'estate, dimostrando che il microbioma della Ricotta era composto principalmente da batteri sporigeni, Bacillus, Paenibacillus e Clostridium (oltre l'80% di abbondanza relativa). Inoltre abbiamo trovato una ricotta estiva con colorazione rosa e, applicando l'approccio molecolare Multi-locus Sequence Typing, abbiamo dimostrato che il batterio che causava il difetto apparteneva al gruppo del Bacillus cereus, in particolare si trattava di un ceppo di B. mycoides/wehienstephanensis. Tale lavoro è stato pubblicato nella rivista Food Microbiology.
Nell'ultimo capitolo abbiamo applicato l'analisi NGS a studi di culture ausiliari aggiunte a formaggi a pasta molle o stagionati, al fine di evidenziare il loro effetto antibatterico. Abbiamo così valutato l'effetto anti-Gammaproteobacteria di alcuni ceppi come L. rhamnosus, L. sakei e Carnobacterium maltoaromaticum in formaggi freschi industriali con problematiche di rigonfiamento della confezione e deterioramento prematuro. Dal punto di vista NGS abbiamo evidenziato che C. maltoaromaticum riusciva ad eliminare quasi tutti i contaminanti a temperature di conservazione differenti.
In conclusione, con l'approccio NGS, è stato possibile migliorare la valutazione della qualità microbiologica alimentare e sostenere la ricerca tecnologica alimentare. Infatti i risultati ottenuti in questo studio dimostrano come l'applicazione di tecnologie NGS, descrivendo approfonditamente le comunità microbiche, potrebbe diventare nel prossimo futuro uno strumento scalabile ed efficace, adatto per migliorare la qualità e la sicurezza alimentare, nonché favorire la produzione di nuovi alimenti.

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Tipo di EPrint:Tesi di dottorato
Relatore:Squartini, Andrea
Correlatore:Simionati, Barbara
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > BIOSCIENZE E BIOTECNOLOGIE > BIOTECNOLOGIE
Data di deposito della tesi:28 Gennaio 2016
Anno di Pubblicazione:Gennaio 2016
Parole chiave (italiano / inglese):alimenti/food, microbiologia/microbiology, Sequenziamento di nuova generazione/Next Generation Sequencing, Qiime, colture antagoniste/adjunct culture, Miseq Illumina, Bacillus cereus, pink discoloration, ricotta, cheese/formaggio siero/whey.
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:9325
Depositato il:24 Ott 2016 16:17
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