The objectives of this PhD research were: i) to evaluate the use of bread making process to increase the content of β-glucans, resistant starch, fructans, dietary fibers and phenolic compounds of kamut khorasan and wheat breads made with flours obtained from kernels at different maturation stage (at milky stage and fully ripe) and ii) to study the impact of whole grains consumption in the human gut. The fermentation and the stages of kernel development or maturation had a great impact on the amount of resistant starch, fructans and β-glucans as well as their interactions resulted highly statistically significant. The amount of fructans was high in kamut bread (2.1g/100g) at the fully ripe stage compared to wheat during industrial fermentation (baker’s yeast). The sourdough increases the content of polyphenols more than industrial fermentation especially in bread made by flour at milky stage. From the analysis of volatile compounds it resulted that the sensors of electronic nose perceived more aromatic compound in kamut products, as well as the SPME-GC-MS, thus we can assume that kamut is more aromatic than wheat, so using it in sourdough process can be a successful approach to improve the bread taste and flavor. The determination of whole grain biormakers such as alkylresorcinols and others using FIE-MS AND GC-tof-MS is a valuable alternative for further metabolic investigations. The decrease of N-acetyl-glucosamine and 3-methyl-hexanedioic acid in kamut faecal samples suggests that kamut can have a role in modulating mucus production/degradation or even gut inflammation. This work gives a new approach to the innovation strategies in bakery functional foods, that can help to choose the right or best combination between stages of kernel maturation-fermentation process and baking temperature.

60 strains (belonging to the genera Lactobacillus, Bifidobacterium, Leuconostoc and Enterococcus) were tested for their capacity to inhibit the growth of 3 strains of Campylobacter jejuni: Lactobacilli and bifidobacteria were left to grow in MRS or TPY broth at 37°C overnight in anaerobic conditions; Campylobacter jejuni was inoculated in blood agar plates at 37°C for 24-48 hours in microaerophilic conditions. The inhibition experiments were carried out in vitro using ”Spot agar test” and “Well diffusion assay” techniques testing both cellular activity and that of the surnatant. 11 strains proved to inhibit the growth of Campylobacter jejuni. These strains were subsequently analised analised in order to evaluate the resistance to particular situations of stress which are found in the gastrointestinal tract and during the industrial transformation processes (Starvation stress, osmotic stress, heat stress, resistance to pH and to bile salts). Resistance to starvation stress: all strains seemed to resist the stress (except one strain). Resistance to osmotic stress: all strains were relatively resistant to the concentrations of 6% w/v of NaCl (except one strain). Resistance to heat stress: only one strain showed little resistance to the 55°C temperature. Resistance to pH: In the presence of a low pH (2.5), many strains rapidly lost their viability after approximately 1 hour. Resistance to bile salts: Except for one strain, all strains seemed to be relatively resistant to the 2% w/v concentration of bile salts. Afterward, strains were identified by using phenotipic and molecular techniques. Phenotipic identification was carried out by using API 50 CHL (bioMérieux) and API 20 STREP identification system (bioMérieux); molecular identification with species-specific PCR: the molecular techniques confirmed the results by phenotipic identification. For testing the antibiotic resistance profile, bacterial strains were subcultured in MRS or TPY broth and incubated for 18 h at 37°C under anaerobic conditions. Antibiotics tested (Tetracycline, Trimethoprim, Cefuroxime, Kanamycin, Chloramphenicol, Vancomycin, Ampycillin, Sterptomycin, Erythromycin) were diluted to the final concentrations of: 2,4,8,16,32,64,128,256 mg/ml. Then, 20 μl fresh bacterial culture (final concentration in the plates approximately 106 cfu/ml) were added to 160 μl MRS or TPY broth and 20 μl antibiotic solution. As positive control the bacterial culture (20 ul) was added to broth (160 ul) and water (20 ul). Test was performed on plates P96, that after the inoculum were incubated for 24 h at 37oC, then the antibiotic resistance was determined by measuring the Optical Density (OD) at 620 nm with Multiscan EX. All strains showed a similar behaviour: resistance to all antibiotic tested. Further studies are needed.

The normal gut microbiota has several important functions in host physiology and metabolism, and plays a key role in health and disease. Bifidobacteria, which are indigenous components of gastrointestinal microbiota, may play an important role in maintaining the well-being of the host although its precise function is very difficult to study. Its physiological and biochemical activities are controlled by many factors, particularly diet and environment. Adherence and colonization capacity are considered as contributing factors for immune modulation, pathogen exclusion, and enhanced contact with the mucosa. In this way, bifidobacteria would fortify the microbiota that forms an integral part of the mucosal barrier and colonization resistance against pathogens. Bifidobacteria are not only subjected to stressful conditions in industrial processes, but also in nature, where the ability to respond quickly to stress is essential for survival. Bifidobacteria, like other microorganisms, have evolved sensing systems for/and defences against stress that allow them to withstand harsh conditions and sudden environmental changes. Bacterial stress responses rely on the coordinated expression of genes that alter various cellular processes and structures (e.g. DNA metabolism, housekeeping genes, cell-wall proteins, membrane composition) and act in concert to improve bacterial stress tolerance. The integration of these stress responses is accomplished by regulatory networks that allow the cell to react rapidly to various and sometimes complex environmental changes. This work examined the effect of important stressful conditions, such as changing pH and osmolarity, on the biosynthesis of cell wall proteins in B. pseudolongum subsp. globosum. These environmental factors all influence heavily the expression of BIFOP (BIFidobacterial Outer Proteins) in the cell-wall and can have an impact in the interaction with host. Also evidence has been collected linking the low concentration of sugar in the culture medium with the presence or absence of extracromosomal DNA.

L’insorgenza e la diffusione dell’antibiotico resistenza sta diventando un problema a livello mondiale. Molti sono gli ambienti in cui può avvenire tale diffusione, ma una delle principali vie di trasmissione passa attraverso la catena alimentare. Infatti, l’utilizzo di sostanze antimicrobiche è largamente diffuso negli allevamenti di animali ad uso alimentare e in agricoltura. In particolare, negli allevamenti gli antibiotici non solo vengono usati per trattare eventuali patologie, ma anche come profilassi e come promotori di crescita. Di conseguenza, questo uso a volte sconsiderato ha portato all’insorgenza di batteri resistenti a tali sostanze. Un ruolo fondamentale nella trasmissione e diffusione di tali resistenze a livello alimentare è svolto da batteri non patogeni che sono parte del naturale microbiota degli alimenti. Questi microorganismi infatti, pur non essendo essi stessi nocivi per l’uomo, possono fungere da reservoir di antibiotico resistenze per eventuali batteri patogeni. I batteri che generalmente svolgono questo ruolo sono i batteri lattici. Per questo motivo molto importante è stato identificare e studiare l’antibiotico resistenza anche di tali microorganismi. Negli ultimi anni, tuttavia, c’è stato un crescente interesse per un’altra classe di microorganismi, chiamata Haloarchaea o alobatteri o archaea alofili, poiché la loro presenza è stata rilevata in alimenti particolarmente salati. Dal momento che in letteratura ci sono pochi lavori che studiano i profili di antibiotico resistenza di tali microorganismi e, comunque, tali profili non sono stati studiati su un numero significativo di microorganismi appartenenti alla stessa specie, il presente lavoro di tesi è volto a definire il profilo di antibiotico resistenza del capostipite degli archaea alofili, che è l’Halobacterium salinarum, verificare se ci sono ceppi che presentano antibiotico resistenze e controllare se tali resistenze possono essere trasferite a batteri patogeni. / Antimicrobial resistance is now widely acknowledged as a major global public health challenge. There are many environments through which the transmission and diffusion of antibiotic resistance could happen, but one of the main routes of transmission is the food chain. As a matter of fact, antibiotic use is widely spread in animal husbandry and in agriculture. In particular, in animal husbandry antimicrobials have been used both for therapeutic reasons and as growth promoters. As a consequence, a selective pressure on pathogenic and commensal bacteria of animal origin has been exerted during the time, leading to the onset of microorganisms resistant to such compounds. A pivotal role in the spread in the food chain of antibiotic resistance has been played by non-pathogenic bacteria present in food. These microorganisms are not harmful for humans, but they could represent a reservoir of antibiotic resistance for foodborne pathogenic bacteria. Usually lactic acid bacteria play this role, since they are present in all fermented food. For this reason, the antibiotic resistance profile of lactic acid bacteria has been assessed. In recent years, another class of microorganisms called halophilic archaea have raised an increasing scientific interest, since they have been found in the human intestinal mucosa as well as in foods such as salted codfish and fermented Asiatic seafood. As a few papers have studied the antibiotic resistance profiles of halophilic archaea, and the only present do not consider a statistically significant number of microorganisms belonging to the same species, the aim of the present work is to define the antibiotic resistance profile of the major exponent of halophilic archaea, named Halobacterium salinarum, and consequently to verify if some strains present antibiotic resistances and if they can transfer these resistances to bacteria present in the food chain.

Il tratto gastro intestinale infantile al momento del parto è considerato virtualmente sterile e viene rapidamente colonizzato da microrganismi di origine materna e/o ambientale nei primi giorni di vita. Studi accoppiati di microbiologia classica e molecolare hanno dimostrato come la cavità amniotica sia popolata da microrganismi alcuni dei quali appartenenti a taxa non ancora coltivati e caratterizzati. Lo scopo del presente lavoro di tesi è stato quello di valutare la composizione del microbiota infantile durante i primi due anni di vita, in particolare, di popolazioni parzialmente “trascurate”. La tesi è suddivisibile in tre tematiche principali: presenza di popolazioni idrogenotrofiche, la distribuzione della famiglia delle Lachnospiraceae in soggetti sani prima del secondo anno di vita, e la possibile correlazione tra gli archaea metanogeni e la dieta in modello animale. Le tecniche impiegate nel presente lavoro di tesi sono state la PCR-DGGE e la PCR quantitativa (qPCR) e il sequenziamento Illumina. Le principali conclusioni derivabili dai tre studi sono correlate alla necessità di sviluppare nuove coppie di primers che meglio possano descrivere la complessa ecologia delle comunità microbiche intestinali. L’ambizioso obiettivo potrà considerarsi raggiunto quando si potranno identificare e stimare in modo preciso e corretto anche le popolazioni batteriche poco abbondanti nel microbiota intestinale infantile. / At birth, the gastrointestinal tract is virtually sterile, but is rapidly colonized during the first days of life until a relatively stable state is reached. Several studies using both bacterial culture techniques and bio-molecular methods revealed that the amniotic cavity harbors microorganisms and, among them, uncultivated and uncharacterized taxa. The aim of the present PhD thesis was to evaluate the composition of some neglected populations inhabiting the infant gut microbiota until the second year of life. The PhD thesis is composed of three main chapters related to the presence of hydrogenotrophic populations, the occurrence of the Lachnospiraceae family in healthy subjects before the second year and the possible linkage between methanogens and diet in a piglet’s model. PCR-DGGE, the quantitative PCR (qPCR) and the Illumina deep sequencing have been the prevalent molecular techniques used in this work. Main conclusions obtained from these studies were mainly linked to the need of new primer sets that better describe the ecological complexity of the gut microbial community. This ambitious objective will be reached when it is possible to properly identify and quantify less represented bacterial populations within the infant gut ecosystem.

I lattobacilli probiotici devono sopravvivere al passaggio attraverso il tratto intestinale, che significa principalmente al pH acido nello stomaco e alla concentrazione di bile nell'intestino tenue. Questi tratti richiedono un adattamento specifico, suggerendo che gli isolati intestinali hanno più probabilità di sopravvivere dopo l'ingestione. L. helveticus CNBL 1254, un ceppo isolato dall'ambiente lattiero-caseario, mostra - in vitro - una gamma completa di fenotipi correlati all'adattamento intestinale: è, infatti, in grado di sopravvivere al transito gastrointestinale chimicamente simulato e ai sali biliari, anche se il suo gene bsh ha una delezione che lo rende non funzionale. L'analisi di RNA-Seq condotta in presenza dello 0,1% di sali biliari evidenzia una sovraespressione della proteina precursore del S-layer e del aggregation promoting factor. Inoltre, nelle proteine selezionate dall'analisi VIP della statistica multivariata OPLS-DA condotta per i dati proteomici, abbiamo riscontrato un aumento globale delle proteine ribosomiali e del S-layer con un VIP-score di 1,33 e un fold change di 2,41 che suggerisce la presenza, in questo ceppo di L. helveticus, di un meccanismo di resistenza non ancora descritto per l'ambiente intestinale. / Probiotic lactobacilli have to survive to the passage through the intestinal tract, mainly acid pH in the stomach and bile concentration in the small intestine. These traits require a specific niche adaptation, suggesting that intestinal isolates are most likely to survive after ingestion. However L. helveticus CNBL 1254, a strain isolated from dairy environment, shows in vitro a full range of phenotypes related to gut adaptation. It is able to survival to the chemically simulated gastrointestinal transit and to bile salts, even if its bsh gene has a deletion that make it non-functional. RNA-Seq analysis conducted in the presence of 0.1% of bile salts highlights an over expression of S-layer protein precursor and aggregation promoting factor. Furthermore in the proteins selected by VIP analysis of OPLS-DA multivariate statistic conducted for proteomic data ,we have found global increase in the ribosomal proteins and a S-layer protein with a VIP-score of 1.33 and a fold change of 2.41 suggesting the presence in this strain of L. helveticus of a not yet described mechanism of resistance to the gut environment.

Obiettivo di questo lavoro è stato valutare la diffusione di AR in differenti ceppi di S. thermophilus isolati tra il 1947 e il 2004 e provenienti da differenti ambienti, in modo da avere un chiaro andamento del fenomeno; questo è stato possibile analizzando un numero significativo di ceppi isolati in un periodo di tempo che va da prima dell'utilizzo degli antibiotici fino ai giorni nostri. L'espressione fenotipica è stata valutata con tre differenti metodi (microdiluizioni in brodo, E-test e Disk Diffusion), in accordo con gli standard NCCLS, per la determinazione delle MICs (Minimum Inhibitory Concentration, ovvero Concentrazioni Minime Inibenti). Per la valutazione genetica è stata impiegata la tecnica dei microarrays a DNA utilizzando oligonucleotidi da 50 e 60-mer, per un totale di 300, appartenenti a 10 diverse classi di antibiotici. La conferma dei risultati è stata ottenuta mediante PCR e sequenziamento. In 9 ceppi di S. thermophilus è stato possibile mettere in evidenza la presenza di almeno uno dei geni tetS ed ermB responsabili della resistenza agli antibiotici Tetraciclina e Eritromicina rispettivamente. / The aim of the present work was to assess the AR diffusion in a total of 70 different strains of Streptococcus thermophilus, collected between 1950 and 2004 and from different environments; in this way we had the possibility to obtain a clear overview of the response of these bacteria to a large variety of antibiotics, having been able to analyze a significant number of different strains, originated from different areas and distributed over a wide time period, since before the use of antibiotics up to the present day. The phenotypic expression has been evaluated by using three different methods: microdilution, E-test and disk diffusion. The genetic analysis was performed using 50 and 60-mer oligonucleotides DNA based micro array for the identification of AR genes; the AR genes represented by the oligonucleotides on the micro array belong to: Aminoglycoside, Extended Spectrum ?-lactamase (ESBL), Chloramphenicol, Macrolide Lincosamides and Streptogramin (MLS) group, Sulfonamide, Tetracycline, Trimethoprim and Vancomycin. tetS and ermB genes were found and sequenced in 4 out of the total of the S. thermophilus investigated. Furthermore we have wanted to establish the genetic location of above-mentioned genes and assess their transfer intra and inter species adopting the conjugation technique in plate.

La DGGE ha messo in evidenza le modificazioni indotte da un prebiotico sul genere Bacteroides mentre non ha evidenziato variazioni a carico dei bifidobatteri. Ha messo in risalto come, durante lo svezzamento, i generi batterici dominanti subiscano variazioni correlate alla variabilità individuale mentre, in soggetti affetti dal morbo di Crohn, sia la TGGE che la DGGE evidenziano i drastici cambiamenti prodotti dalla nutrizione enterale. La quantificazione mediante Real-time PCR non ha messo in risalto variazioni statisticamente significative per Bifidobacterium, Bacteroides e C. difficile tra i soggetti trattati e quelli non trattati con prebiotico. Per i soggetti in svezzamento Enterococcus, Bifidobacterium, C. perfringens, E. coli e i membri della famiglia delle Enterobacteriaceae non subiscono variazioni statisticamente significative. / DGGE analysis put in evidence that prebiotic treatment induces modification of Bacteroides population while any modifications have not been underlined for Bifidobacterium. During weaning modifications of the dominant bacterial genera are correlated to the individual variability and not to dietary changes. TGGE and DGGE techniques also underlined important changes of the intestinal microflora composition due to the enterale nutrition. Quantification of the most important genera and species shows that Bifidobacterium, Bacteoides and C. difficile are not characterised by statistically significant modifications during prebiotic treatment. More over Enterococcus, Bifidobacterium, Enterobacteriaceae, E. coli and C. perfringens did not show any statistically significant changes during weaning.

Per varie ragioni, tra cui le loro specifiche condizioni di crescita, la diagnosi di infezione e di contaminazione alimentare da clostridi presenta ancora numerose difficoltà sia a livello clinico-batteriologico che a livello molecolare. In questo lavoro di tesi si è cercato di ampliare lo spettro di conoscenze riguardo i clostridi e la loro diffusione; durante il primo anno di ricerca è stato studiato, applicando nuove tecniche di microscopia, il processo di germinazione di Clostridium tyrobutyricum, uno dei batteri maggiormente responsabili del gonfiore tardivo dei formaggi a pasta dura; l’applicazione di tecniche di Real-Time PCR ha nel contempo reso possibile una determinazione quantitativa dello stesso in latte. Successivamente, è stata condotta un’analisi di tipizzazione molecolare di clostridi nell’ambito di una filiera agro-zoo-casearia finalizzata alle matrici di processo al fine di individuare le possibili vie di diffusione dei microrganismi. La parte finale del lavoro è stata dedicata allo studio di espressione genica di un altro Clostridium responsabile di gonfiore ma scelto perché geneticamente indistinguibile da Clostridium botulinum, ovvero il Clostridium sporogenes; l’analisi trascrizionale dei suoi geni durante le fasi vegetativa, di sporulazione, germinazione ed esocrescita ha permesso di assegnare diverse funzioni a geni singoli o a gruppi di geni allo scopo di utilizzare queste informazioni per formulare ipotesi future anche su altre specie di clostridi patogeni. / For several reasons, including their specific growth requirements, the diagnosis of infections and food contamination caused by clostridia still presents much difficulty at the clinical, bacteriological and molecular levels. The main purpose of this work is to learn more about clostridia and their interactions with environment. First, new microscopy techniques have been used to study the germination process in Clostridium tyrobutyricum, an anaerobic bacterium responsible for late blowing defects during cheese ripening; meanwhile, the application of real-time PCR methods have been employed to enumerate C. tyrobutyricum cells and spores in milk. Then, a molecular genotyping has been set in order to identify the most common clostridia in a agro-dairy production aimed to detect the possible ways of diffusion of these microbial species. The last part concerns the study of expression patterns of Clostridium sporogenes, an apathogenic gram positive clostridium usually involved in food damage and frequently isolated from late bowled cheese; Clostridium sporogenes is genetically indistinguishable from Clostridium botulinum and is often used as a model for the toxic subtypes. The objective of this study is to use an array-based large-scale transcriptional analysis in order to study gene expression in four different steps of Clostridium sporogenes life cycle: vegetative cells, sporulating cells, dormant spores and germinating ones. Our aims includes being able to relate gene-expression patterns to specific phenotypes and to discover gene expression divergences between the different phases of living, germination and outgrowth of spore-forming bacteria. An important aim is to assign functions to groups of or individual C. sporogenes genes and use this information to formulate specific hypotheses for further testing also on pathogenic clostridia types.

I lactobacilli sono considerati dei microorganismi non-patogeni. Molti di loro appartengono al gruppo batterico GRAS e/o sono nell’elenco QPS. Dal momento che i lactobacilli intenzionalmente aggiunti agli alimenti possono agire come reservoir di geni di resistenza, la valutazione del rischio deve essere continuamente aggiornata. Lo scopo di questa tesi era la valutazione di alcuni metodi usati per testare e caratterizzare le specie del genere Lactobacillus per quanto riguarda la sicurezza e la potenziale attività probiotica. Nella prima parte due metodi di micro diluzione, il metodo ISO e CLSI, soni stati comparati testando la resistenza agli antibiotici di 54 ceppi L. plantarum. Sulla base di risultati ottenuti il metodo ISO era più adatto per valutare la resistenza di questa specie. Il test del limite di sensibilità della PCR per 8 paia di primers specifici per il rilevamento dei lactobacilli e bifido batteri da feci ha confermato i loro diversi livelli di efficacia. La seconda parte della tesi descrive un progetto di ricerca mirato sulla identificazione di nuovi ceppi probiotici fra diversi ceppi di Lactobacillus paracasei e Lactobacillus rhamnosus identificando dei geni o loci responsabili della interazione con l’ospite, immunomodulazione, e l’inibizione della crescita dei patogeni. Le analisi fenotipiche dei 40 ceppi hanno confemato una grande variabilità fra di loro, che può servire per associare delle caratteristiche fenotipiche a quelle genotipiche. Tra i ceppi dello stesso progetto è stato individuato un ceppo di L. mucosae. Dal momento che questa è una specie relativamente nuova, le sue caratteristiche sono state analizzate comparandole con altri 3 ceppi appartenenti alla stessa specie. In questo modo sono state confermate alcune informazioni su L. mucosae, ma soprattutto sono stati forniti dei dati nuovi sulle proprietà di questa specie. / The species of the Lactobacillus genus are generally believed to be microorganisms with no pathogenic potential. Many of them have granted GRAS and QPS status. Non-pathogenic bacteria as lactobacilli-intentionally added or accidentally present in food-are under evaluation, as they could act as reservoir of resistant genes. This thesis was aimed to evaluate some methods used for testing and to characterize some Lactobacillus species, as regards their safety and potential probiotic activity. The first part of the research focused on the comparison of two broth microdilution methods: ISO and CLSI, in order to assess the resistance of 54 L. plantarum strains to antimicrobial agents. The results suggest better performances of the phenotypic assay developed by ISO, at least for strains belonging to L. plantarum species.Then the assessment of the PCR detection limit for 8 sets of primers for the detection of lactobacilli and bifidobacteria from infant faeces confirmed different levels of effectiveness for the primers. Next part of the thesis was the research project aimed at identifying genes or genetic loci of different strains of two Lactobacillus species (i.e. Lactobacillus paracasei and Lactobacillus rhamnosus) involved in the interaction with the host, immune-modulation of host cells and pathogen growth inhibition in order to find new probiotic strains. The phenotypic analysis of 40 selected strains demonstrated large variability between strains of these species, which could serve to the association of phenotypic differences to genome specificities. A strain of Lactobacillus mucosae was found within the framework of the same project. As it is a relatively new species, it was chosen to further investigate its properties, comparing it with three other L. mucosae strains. This study led to confirm some information but first and foremost it has provided new data on the examined species.