Dissertations / Theses: 'AGR/16: MICROBIOLOGIA AGRARIA'

1

Aloisio, Irene <1980&gt. "Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4420/1/Tesi_Aloisio_Irene.pdf.

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Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.

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2

Aloisio, Irene <1980&gt. "Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4420/.

Full text

Abstract:

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.

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3

Babini, Valentina. "Formaggi tradizionali della regione Marche: caratterizzazione compositiva, microbiologica e sensoriale." Doctoral thesis, Università Politecnica delle Marche, 2011. http://hdl.handle.net/11566/242002.

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La tradizione casearia dell’Italia centrale comprende una grande varietà di formaggi prodotti con latte locale ed antiche tecniche di caseificazione. Tali formaggi sono parte integrante del patrimonio storico e culturale delle comunità locali, nonché risorse da conservare e valorizzare, anche a fronte del sempre più incalzante processo di globalizzazione. La regione Marche, in particolare, vanta un cospicuo numero di produzioni casearie tipiche, come riportato nell’”Elenco nazionale dei prodotti agro-alimentari tradizionali” (DM 18/07/2000). La valorizzazione di tali produzioni rappresenta un obiettivo primario delle politiche volte a promuovere la salvaguardia delle risorse e dello sviluppo socio-economico dei territori locali. Obiettivo della presente ricerca è stato la caratterizzazione compositiva, microbiologica e sensoriale di produzioni tradizionali di caciotta, pecorino e caprino della regione Marche, anche caratterizzate da tratti peculiari quali l’impiego di caglio d’origine vegetale (estratto acquoso di Cynara cardunculus), l’aggiunta di ingredienti caratteristici (erbe locali, olio d’oliva, buccia di limone grattugiata), la stagionatura in botte o all’interno di fosse tufacee. I dati ottenuti dalle analisi fisico-chimiche, microbiologiche e gas-cromatografiche sono stati elaborati statisticamente al fine di individuare correlazioni con le specifiche tecniche di caseificazione ed identificare marcatori oggettivi di qualità. A tale scopo, sono stati campionati complessivamente 29 formaggi, di cui 8 caprini, 12 pecorini e 9 caciotte, di cui 2 a latte vaccino e 7 a latte misto (bovino ed ovino); di questi, le produzioni a latte crudo (19 campioni) sono state prelevate presso caseifici artigianali della regione Marche, mentre i formaggi a latte pastorizzato (10 campioni) sono stati acquistati presso supermercati della provincia di Ancona. Tutti i campioni sono stati sottoposti a: (i) determinazione del contenuto di NaCl, proteina grezza e grassi totali; (ii) misurazione di pH, attività dell’acqua (aw), numero di perossidi della sostanza grassa; (iii) ricerca di Listeria monocytogenes, Salmonella spp. ed enterotossine stafilococciche; (iv) enumerazione di batteri lattici mesofili e termofili; (v) analisi PCR-DGGE della popolazione batterica; (vi) analisi della componente volatile mediante SPME-GC. L’insieme dei dati composizionali, microbiologici e del profilo aromatico è stato sottoposto ad analisi PCA (Principal Component Analysis) e PLS-DA (Partial Least Square Discriminant Analysis) utilizzando il software SIMCA-Pv 11.5 (UMETRICS). Tutte le produzioni in studio sono risultate conformi ai limiti legislativi per i parametri igienico-sanitari considerati. L’analisi multivariata mediante PCA dell’insieme dei dati ottenuti ha prodotto un modello a 2 componenti che spiega il 31.5% della varianza. Nello score plot t1/t2, le 3 tipologie di formaggio sono risultate ben separate; al contrario, non è stato possibile separare i formaggi sulla base del processo produttivo (da latte crudo o pastorizzato), indipendentemente dalla tipologia considerata (caciotta, pecorino o caprino). Il corrispondente loading plot ha permesso di evidenziare 3 gruppi di variabili; nello specifico, le variabili relative al profilo aromatico sono risultate responsabili della distinzione dei formaggi appartenenti alla tipologia caprino; quelle relative a conte microbiche, proprietà fisico-chimiche e composizionali hanno permesso di separare i formaggi appartenenti alla tipologia caciotta; infine, le variabili relative all’ecologia microbica sono risultate utili per discriminare i formaggi appartenenti alla tipologia pecorino. In particolare, conte più elevate di batteri lattici e valori più alti di aw hanno permesso di differenziare le caciotte dai pecorini e caprini, questi ultimi caratterizzati da concentrazioni più elevate di composti aromatici, mentre i pecorini si sono distinti per la presenza delle specie Lactobacillus coryniformis, Lb. rhamnosus e Lb. plantarum. L’analisi PLS-DA ha prodotto un modello a 2 componenti; più in dettaglio, lo score plot t1/t2 ha confermato la separazione delle 3 tipologie di formaggio. Dall’analisi del corrispondente loading plot, è emerso che tale separazione è principalmente dovuta a 10 descrittori legati alla definizione del profilo aromatico: acetone, etanolo, acido acetico, butirrico, caproico, enantico, caprilico, caprico, undecanoico ed undecenoico. L’approccio polifasico applicato nel presente studio ha permesso, nel complesso, di caratterizzare le produzioni casearie considerate, nonché di identificare marcatori utili per una discriminazione oggettiva delle stesse.
The dairy tradition of central Italy territories is characterized by a large variety of cheeses, manufactured with local milk according to ancient cheese-making techniques. These cheeses are an integral part of the historical and cultural heritage of local communities and resources to preserve and enhance, even in the face of increasingly insistent globalization process. The Marche region, in particular, boasts a large number of typical dairy products, as reported in the “Italian list of traditional agro-alimentary products” (DM 18/07/2000). The exploitation of these dairy products is a primary objective of politics to promote the conservation of resources and socio-economic development of local territories. The aim of this study was the compositional, microbiological and sensory characterization of traditional caciotta, caprino and pecorino cheeses produced in the Marche region, also characterized by peculiar traits such as the use of vegetable rennet (aqueous extract of Cynara cardunculus flowers), the addition of particular ingredients (herbs, olive oil, grated lemon peel) and ripening under unconventional conditions (in wooden barrels or pit ageing). All the data collected from physico-chemical, microbiological and chromatographic assays were statistically processed in order to assess the relationships between cheese-making techniques and compositional, microbiological and aromatic traits and identify objective markers of quality. To this aim, 29 cheeses manufactured in the Marche region, according to traditional techniques, were sampled; these included 8 goats’ (caprino), 12 ewes’ (pecorino) 2 cows’ and 9 mixed (ewes’ and cows’) milk (caciotta) cheeses. Raw milk cheeses (19 samples) were supplied by local artisan dairies, while pasteurized milk cheeses (10 samples) were bought from supermarkets. All the cheese samples were subjected to physico-chemical analyses for the determination of pH and aw values, NaCl, lipid and protein contents and peroxide index. Conventional microbiological analyses were carried out for the detection of pathogenic micro-organisms (Listeria monocytogenes and Salmonella spp.) and their metabolites (staphylococcal enterotoxins) as well as for viable counting of mesophilic and thermophilic lactic acid bacteria. The composition of the bacterial community was also evaluated by PCR-DGGE analysis of the bacterial DNA extracted directly from either the cheese samples or the bulk cells harvested from the MRS and M17 dilution plates. The cheese volatile profiles were determined by solid phase micro-extraction coupled with gas-chromatography (SPME-GC). All the data collected were subjected to Principal Component Analysis (PCA) and Partial Least Square (PLS) analysis by using SIMCA-P v 11.5 software (UMETRICS). The absence of bacterial hazard was demonstrated in both pasteurized and raw milk cheeses. Principal Component Analysis of the outcomes from physico-chemical, microbiological and chromatographic assays yielded a two components model, explaining the 31.5% of the variance. In the t1/t2 score plot, the three cheese types (pecorino, caciotta and caprino) were well separated; by contrast, no separation was seen between raw and pasteurized milk cheeses, irrespective of the cheese type considered. The corresponding loading plot revealed that variables clustered in three separated groups. In more detail, variables referring to aromatic profile are responsible for the grouping of caprino cheeses; variables referring to microbial loads, physico-chemical and compositional traits are responsible for the grouping of caciotta cheeses, while those referring to the PCR-DGGE results are responsible for the grouping of pecorino cheeses. Indeed, higher loads of mesophilic and thermophilic lactic acid bacteria and higher aw values differentiated caciotta from pecorino and caprino cheeses. This latter cheese type was characterized by higher amounts of volatile compounds, while pecorino cheeses were distinguished for the presence of distinctive bacterial species, namely Lactobacillus coryniformis, Lb. rhamnosus and Lb. plantarum. Partial Least Square Discriminant Analysis resulted in a 2 components model. The score plot of the first principal component vs the second principal component confirmed that the three cheese types were clearly separated. The analysis of the corresponding loading plot showed that such separation is mainly due to 10 descriptors related to the definition of the flavour profile: acetone, ethanol, acetic, butyric, caproic, enanthic, caprylic, capric, undecanoic, and undecenoic acids. The polyphasic approach used in this study allowed, in general, to characterize the dairy products concerned, and to identify useful markers for their discrimination.

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4

D'Aimmo, Maria Rosaria <1975&gt. "Uso di simbiotici e di nitrati come alternativa agli antibiotici in allevamenti zootecnici." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/142/1/Tesi_pdf.pdf.

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5

D'Aimmo, Maria Rosaria <1975&gt. "Uso di simbiotici e di nitrati come alternativa agli antibiotici in allevamenti zootecnici." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/142/.

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6

Stefanini, Ilaria <1972&gt. "Uso di probiotici e prebiotici quale barriera a patogeni enterici in suinetti in svezzamento." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/144/1/Tesi_Ilaria_Stefanini.pdf.

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7

Stefanini, Ilaria <1972&gt. "Uso di probiotici e prebiotici quale barriera a patogeni enterici in suinetti in svezzamento." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/144/.

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8

Sado, Kamdem Sylvain Leroy <1973&gt. "Effect of diet supplementation in unsaturated fatty acids on meat keeping qualities: study of selected fatty acids antimicrobial properties and inhibition mechanism on Staphylococcus aureus." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/418/1/Manuscript_Tesi_di_Dottorato_Sado_Sylvain.pdf.

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9

Sado, Kamdem Sylvain Leroy <1973&gt. "Effect of diet supplementation in unsaturated fatty acids on meat keeping qualities: study of selected fatty acids antimicrobial properties and inhibition mechanism on Staphylococcus aureus." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/418/.

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10

Saracino, Pasquale <1977&gt. "New signalling molecules in some foodborne bacteria." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/421/1/Saracino_Pasquale_tesi_dottorato.pdf.

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11

Saracino, Pasquale <1977&gt. "New signalling molecules in some foodborne bacteria." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/421/.

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12

Carri, Simone <1980&gt. "Attività antagonistica di batteri lattici isolati da salami verso muffe e lieviti." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/697/1/Tesi_Carri_Simone.pdf.

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13

Carri, Simone <1980&gt. "Attività antagonistica di batteri lattici isolati da salami verso muffe e lieviti." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/697/.

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14

Nissen, Lorenzo <1977&gt. "Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/790/1/Tesi_Nissen_Lorenzo.pdf.

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The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

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15

Nissen, Lorenzo <1977&gt. "Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/790/.

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Abstract:

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

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16

Caffarri, Elena <1979&gt. "Studio della maturazione di formaggi pecorino stagionati in stabilimento e in grotta." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/1759/1/Caffarri_Elena_tesi.pdf.

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Aims: Ripening evaluation of two different Pecorino cheese varieties ripened according either to a traditional method in plant and in cave. Different ripening features have been analyzed in order to evaluate the cave as possible ripening environment with the aim of obtaining a peculiar product which could also establish an added value to the cultural heritage of the local place in which it has been originally manufactured. Methods and Results: Chemical-physical features of Pecorino cheese have been initially analyzed into two different ripening environments and experimentations, among which: pH, weight reduction and subsequent water activity. Furthermore, the microbial composition has been characterized in relationship with the two different ripening environments, undertaking a variety of microbial groups, such as: lactic bacteria, staphylococci, yeasts, lactococci, enterobacteria, enterococci. Besides, an additional analysis for the in-cave adaptability evaluation has been the identification of biogenic amines inside the Pecorino cheese (2-phenilethylamine, putrescine, cadaverine, hystidine, tyramine, spermine and spermidine). Further analysis were undertaken in order to track the lipid profile evolution, reporting the concentration of the cheese free fatty acids in object, in relation with ripening time, environment and production. In order to analyse the flavour compounds present in Pecorino cheese, the SPME-GC-MS technique has been widely employed. As a result, it is confirmed the trend showed by the short-chain free fatty acids, that is to say the fatty acids which are mostly involved in conveying a stronger flavor to the cheese. With the purpose of assessing the protheolytic patterns of the above-mentioned Pecorino cheese in the two different ripening environments and testing methods, the technique SDS-PAGE has been employed into the cheese insoluble fraction, whereas the SDS-PAGE technique has been carried out into the cheese soluble portion. Furthermore, different isolated belonging to various microbial groups have been genotypically characterized though the ITS-PCR technique with the aim to identify the membership species. With reference to lactic bacillus the characterized species are: Lactobacillus brevis, Lactobacillus curvatus and Lactobacillus paraplantarum. With reference to lactococci the predominant species is Lactococcus lactis, coming from the employed starter used in the cheese manufacturing. With reference to enterococcus, the predominant species are Enterococcus faecium and Enterococcus faecalis. Moreover, Streptococcus termophilus and Streptococcus macedonicus have been identified too. For staphylococci the identified species are Staphyilococcus equorum, Staphylococcus saprophyfiticus and Staphylococcus xylosus. Finally, a sensorial analysis has been undertaken through on one side a consumer test made by inexperienced consumers, and on the other side through a panel test achieved by expert consumers. From such test Pecorino cheese ripened in cave were found to be more pleasant in comparison with Pecorino cheese ripened in plant. Conclusions: The proposed approach and the undertaken analysis showed the cave as preferential ripening environment for Pecorino cheese and for the development of a more palatable product and safer for consumers’ health.

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17

Caffarri, Elena <1979&gt. "Studio della maturazione di formaggi pecorino stagionati in stabilimento e in grotta." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/1759/.

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Aims: Ripening evaluation of two different Pecorino cheese varieties ripened according either to a traditional method in plant and in cave. Different ripening features have been analyzed in order to evaluate the cave as possible ripening environment with the aim of obtaining a peculiar product which could also establish an added value to the cultural heritage of the local place in which it has been originally manufactured. Methods and Results: Chemical-physical features of Pecorino cheese have been initially analyzed into two different ripening environments and experimentations, among which: pH, weight reduction and subsequent water activity. Furthermore, the microbial composition has been characterized in relationship with the two different ripening environments, undertaking a variety of microbial groups, such as: lactic bacteria, staphylococci, yeasts, lactococci, enterobacteria, enterococci. Besides, an additional analysis for the in-cave adaptability evaluation has been the identification of biogenic amines inside the Pecorino cheese (2-phenilethylamine, putrescine, cadaverine, hystidine, tyramine, spermine and spermidine). Further analysis were undertaken in order to track the lipid profile evolution, reporting the concentration of the cheese free fatty acids in object, in relation with ripening time, environment and production. In order to analyse the flavour compounds present in Pecorino cheese, the SPME-GC-MS technique has been widely employed. As a result, it is confirmed the trend showed by the short-chain free fatty acids, that is to say the fatty acids which are mostly involved in conveying a stronger flavor to the cheese. With the purpose of assessing the protheolytic patterns of the above-mentioned Pecorino cheese in the two different ripening environments and testing methods, the technique SDS-PAGE has been employed into the cheese insoluble fraction, whereas the SDS-PAGE technique has been carried out into the cheese soluble portion. Furthermore, different isolated belonging to various microbial groups have been genotypically characterized though the ITS-PCR technique with the aim to identify the membership species. With reference to lactic bacillus the characterized species are: Lactobacillus brevis, Lactobacillus curvatus and Lactobacillus paraplantarum. With reference to lactococci the predominant species is Lactococcus lactis, coming from the employed starter used in the cheese manufacturing. With reference to enterococcus, the predominant species are Enterococcus faecium and Enterococcus faecalis. Moreover, Streptococcus termophilus and Streptococcus macedonicus have been identified too. For staphylococci the identified species are Staphyilococcus equorum, Staphylococcus saprophyfiticus and Staphylococcus xylosus. Finally, a sensorial analysis has been undertaken through on one side a consumer test made by inexperienced consumers, and on the other side through a panel test achieved by expert consumers. From such test Pecorino cheese ripened in cave were found to be more pleasant in comparison with Pecorino cheese ripened in plant. Conclusions: The proposed approach and the undertaken analysis showed the cave as preferential ripening environment for Pecorino cheese and for the development of a more palatable product and safer for consumers’ health.

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18

Di, Biase Letizia <1974&gt. "Effetto dell'alta pressione di omogeneizzazione e dei trattamenti termici sulla risposta e sull'espressione genica in Listeria monocytogenes." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/1801/1/Di_Biase_Letizia_Tesi.pdf.

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19

Di, Biase Letizia <1974&gt. "Effetto dell'alta pressione di omogeneizzazione e dei trattamenti termici sulla risposta e sull'espressione genica in Listeria monocytogenes." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/1801/.

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20

Santini, Cecilia <1977&gt. "Characterisation of probiotic strains for the control and prevention of enteropathogens in the food chain." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2040/1/Santini_Cecilia_tesi.pdf.

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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.

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21

Santini, Cecilia <1977&gt. "Characterisation of probiotic strains for the control and prevention of enteropathogens in the food chain." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2040/.

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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.

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22

Tacconi, Stefano <1961&gt. "Rapporti fra fattori ambientali e proteine di parete in Bifidobacterium." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2041/1/tacconi_stefano_tesi.pdf.

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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.

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23

Tacconi, Stefano <1961&gt. "Rapporti fra fattori ambientali e proteine di parete in Bifidobacterium." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2041/.

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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.

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24

Baruffa, Elisa <1979&gt. "Microrganismi probiotici per le piante: una strategia di riduzione degli input nella coltura del pomodoro col metodo biologico." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2078/1/baruffa_elisa_tesi.pdf.

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In the recent years, consumers became more aware and sensible in respect to environment and food safety matters. They are more and more interested in organic agriculture and markets and tend to prefer ‘organic’ products more than their traditional counterparts. To increase the quality and reduce the cost of production in organic and low-input agriculture, the 6FP-European “QLIF” project investigated the use of natural products such as bio-inoculants. They are mostly composed by arbuscular mycorrhizal fungi and other microorganisms, so-called “plant probiotic” microorganisms (PPM), because they help keeping an high yield, even under abiotic and biotic stressful conditions. Italian laws (DLgs 217, 2006) have recently included them as “special fertilizers”. This thesis focuses on the use of special fertilizers when growing tomatoes with organic methods in open field conditions, and the effects they induce on yield, quality and microbial rhizospheric communities. The primary objective was to achieve a better understanding of how plant-probiotic micro-flora management could buffer future reduction of external inputs, while keeping tomato fruit yield, quality and system sustainability. We studied microbial rhizospheric communities with statistical, molecular and histological methods. This work have demonstrated that long-lasting introduction of inoculum positively affected micorrhizal colonization and resistance against pathogens. Instead repeated introduction of compost negatively affected tomato quality, likely because it destabilized the ripening process, leading to over-ripening and increasing the amount of not-marketable product. Instead. After two years without any significant difference, the third year extreme combinations of inoculum and compost inputs (low inoculum with high amounts of compost, or vice versa) increased mycorrhizal colonization. As a result, in order to reduce production costs, we recommend using only inoculum rather than compost. Secondly, this thesis analyses how mycorrhizal colonization varies in respect to different tomato cultivars and experimental field locations. We found statistically significant differences between locations and between arbuscular colonization patterns per variety. To confirm these histological findings, we started a set of molecular experiments. The thesis discusses preliminary results and recommends their continuation and refinement to gather the complete results.

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25

Baruffa, Elisa <1979&gt. "Microrganismi probiotici per le piante: una strategia di riduzione degli input nella coltura del pomodoro col metodo biologico." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2009. http://amsdottorato.unibo.it/2078/.

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Abstract:

In the recent years, consumers became more aware and sensible in respect to environment and food safety matters. They are more and more interested in organic agriculture and markets and tend to prefer ‘organic’ products more than their traditional counterparts. To increase the quality and reduce the cost of production in organic and low-input agriculture, the 6FP-European “QLIF” project investigated the use of natural products such as bio-inoculants. They are mostly composed by arbuscular mycorrhizal fungi and other microorganisms, so-called “plant probiotic” microorganisms (PPM), because they help keeping an high yield, even under abiotic and biotic stressful conditions. Italian laws (DLgs 217, 2006) have recently included them as “special fertilizers”. This thesis focuses on the use of special fertilizers when growing tomatoes with organic methods in open field conditions, and the effects they induce on yield, quality and microbial rhizospheric communities. The primary objective was to achieve a better understanding of how plant-probiotic micro-flora management could buffer future reduction of external inputs, while keeping tomato fruit yield, quality and system sustainability. We studied microbial rhizospheric communities with statistical, molecular and histological methods. This work have demonstrated that long-lasting introduction of inoculum positively affected micorrhizal colonization and resistance against pathogens. Instead repeated introduction of compost negatively affected tomato quality, likely because it destabilized the ripening process, leading to over-ripening and increasing the amount of not-marketable product. Instead. After two years without any significant difference, the third year extreme combinations of inoculum and compost inputs (low inoculum with high amounts of compost, or vice versa) increased mycorrhizal colonization. As a result, in order to reduce production costs, we recommend using only inoculum rather than compost. Secondly, this thesis analyses how mycorrhizal colonization varies in respect to different tomato cultivars and experimental field locations. We found statistically significant differences between locations and between arbuscular colonization patterns per variety. To confirm these histological findings, we started a set of molecular experiments. The thesis discusses preliminary results and recommends their continuation and refinement to gather the complete results.

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26

Baffoni, Loredana <1979&gt. "Use of probiotics and prebiotics: a strategy to modulate the intestinal microbiota of poultry and control C. jejuni colonization." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2010. http://amsdottorato.unibo.it/3062/2/Baffoni_Loredana_Tesi.pdf.

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27

Baffoni, Loredana <1979&gt. "Use of probiotics and prebiotics: a strategy to modulate the intestinal microbiota of poultry and control C. jejuni colonization." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2010. http://amsdottorato.unibo.it/3062/.

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28

Tabanelli, Giulia <1982&gt. "Use of sub-lethal high pressure homogenization (HPH) treatments to enhance functional properties of lactic acid bacteria probiotic strains." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3598/1/Tabanelli_Giulia_tesi.pdf.

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The aim of this PhD thesis was to evaluate the effect of a sub-lethal HPH treatment on some probiotic properties and on cell response mechanisms of already-known functional strains, isolated from Argentinean dairy products. The results achieved showed that HPH treatments, performed at a sub-lethal level of 50 MPa, increased some important functional and technological characteristics of the considered non intestinal probiotic strains. In particular, HPH could modify cell hydrophobicity, autoaggregation and resistance to acid gastric conditions (tested in in vitro model), cell viability and cell production of positive aroma compounds, during a refrigerate storage in a simulated dairy product. In addition, HPH process was able to increase also some probiotic properties exerted in vivo and tested for two of the considered strains. In fact, HPH-treated cells were able to enhance the number of IgA+ cells more than other not treated cells, although this capacity was time dependent. On the other hand, HPH treatment was able to modify some important characteristics that are linked to the cell wall and, consequently, could alter the adhesion capacity in vivo and the interaction with the intestinal cells. These modifications, involving cell outermost structures, were highlighted also by Trasmission Electron Microscopy (TEM) analysis. In fact, the micrographs obtained showed a significant effect of the pressure treatment on the cell morphology and particularly on the cell wall. Moreover, the results achieved showed that composition of plasma membranes and their level of unsaturation are involved in response mechanisms adopted by cells exposed to the sub-lethal HPH treatment. Although the response to the treatment varied according to the characteristics of individual strains, time of storage and suspension media employed, the results of present study, could be exploited to enhance the quality of functional products and to improve their organoleptic properties.

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29

Tabanelli, Giulia <1982&gt. "Use of sub-lethal high pressure homogenization (HPH) treatments to enhance functional properties of lactic acid bacteria probiotic strains." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3598/.

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Abstract:

The aim of this PhD thesis was to evaluate the effect of a sub-lethal HPH treatment on some probiotic properties and on cell response mechanisms of already-known functional strains, isolated from Argentinean dairy products. The results achieved showed that HPH treatments, performed at a sub-lethal level of 50 MPa, increased some important functional and technological characteristics of the considered non intestinal probiotic strains. In particular, HPH could modify cell hydrophobicity, autoaggregation and resistance to acid gastric conditions (tested in in vitro model), cell viability and cell production of positive aroma compounds, during a refrigerate storage in a simulated dairy product. In addition, HPH process was able to increase also some probiotic properties exerted in vivo and tested for two of the considered strains. In fact, HPH-treated cells were able to enhance the number of IgA+ cells more than other not treated cells, although this capacity was time dependent. On the other hand, HPH treatment was able to modify some important characteristics that are linked to the cell wall and, consequently, could alter the adhesion capacity in vivo and the interaction with the intestinal cells. These modifications, involving cell outermost structures, were highlighted also by Trasmission Electron Microscopy (TEM) analysis. In fact, the micrographs obtained showed a significant effect of the pressure treatment on the cell morphology and particularly on the cell wall. Moreover, the results achieved showed that composition of plasma membranes and their level of unsaturation are involved in response mechanisms adopted by cells exposed to the sub-lethal HPH treatment. Although the response to the treatment varied according to the characteristics of individual strains, time of storage and suspension media employed, the results of present study, could be exploited to enhance the quality of functional products and to improve their organoleptic properties.

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30

Montanari, Chiara <1983&gt. "Oxylipins biosynthesis in Lactobacillus helveticus in the presence of oxidative stress." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3599/1/Montanari_Chiara_tesi.pdf.

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This PhD thesis is aimed at studying the possible pathways and the mechanisms that can trigger oxylipins biosynthesis, and particularly that of short chain aldehydes and alcohols, in Lactobacillus helveticus, also in the presence of oxidative stress, using a totally labelled linoleic acid as precursor. In plants and fungi these molecules, involved in defence mechanisms against pathogens and in communication systems, derive from the oxidation of cellular unsaturated fatty acids (UFAs) and their accumulation is associated with stress exposure. Since some oxylipins are produced also by lactobacilli, it is possible to hypothesize that a metabolic pathway from UFAs to oxylipins, similar to what happens in plants and fungi, is present also in lactic acid bacteria. The results obtained pointed out that some volatile molecules are the result of UFAs catabolism, since they appear only when cells are incubated in their presence. Labelled linoleic acid is integrated in the membrane and subsequently transformed into aldehydes and alcohols, whose extent and carbon atoms number depend on stress exposure. The enzymes responsible for this metabolic pathway in plants and fungi (e.g. lipoxygenase, dioxygenase) seem to be absent in Lactobacillus helveticus and in other lactobacilli. Proteomic analyses show the over expression of many proteins, including thioredoxin reductase (part of the bacterial oxidative defence system), mainly in cells grown with linoleic acid without oxidative stress exposure, confirming that linoleic acid itself induces oxidative stress. 6 general oxidoreductases (class including dioxygenases and peroxidase) were found and therefore a deeper investigation on them could be productive in elucidating all steps involved in oxylipins biosynthesis in bacteria. Due to the multiple role of oxylipins (flavouring agents, antimicrobial compounds and interspecific signalling molecules) the identification of genes involved and regulating factors should have an important biotechnological impact, also allowing the overproduction of selected bioactive molecules.

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31

Montanari, Chiara <1983&gt. "Oxylipins biosynthesis in Lactobacillus helveticus in the presence of oxidative stress." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3599/.

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Abstract:

This PhD thesis is aimed at studying the possible pathways and the mechanisms that can trigger oxylipins biosynthesis, and particularly that of short chain aldehydes and alcohols, in Lactobacillus helveticus, also in the presence of oxidative stress, using a totally labelled linoleic acid as precursor. In plants and fungi these molecules, involved in defence mechanisms against pathogens and in communication systems, derive from the oxidation of cellular unsaturated fatty acids (UFAs) and their accumulation is associated with stress exposure. Since some oxylipins are produced also by lactobacilli, it is possible to hypothesize that a metabolic pathway from UFAs to oxylipins, similar to what happens in plants and fungi, is present also in lactic acid bacteria. The results obtained pointed out that some volatile molecules are the result of UFAs catabolism, since they appear only when cells are incubated in their presence. Labelled linoleic acid is integrated in the membrane and subsequently transformed into aldehydes and alcohols, whose extent and carbon atoms number depend on stress exposure. The enzymes responsible for this metabolic pathway in plants and fungi (e.g. lipoxygenase, dioxygenase) seem to be absent in Lactobacillus helveticus and in other lactobacilli. Proteomic analyses show the over expression of many proteins, including thioredoxin reductase (part of the bacterial oxidative defence system), mainly in cells grown with linoleic acid without oxidative stress exposure, confirming that linoleic acid itself induces oxidative stress. 6 general oxidoreductases (class including dioxygenases and peroxidase) were found and therefore a deeper investigation on them could be productive in elucidating all steps involved in oxylipins biosynthesis in bacteria. Due to the multiple role of oxylipins (flavouring agents, antimicrobial compounds and interspecific signalling molecules) the identification of genes involved and regulating factors should have an important biotechnological impact, also allowing the overproduction of selected bioactive molecules.

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32

Dei, Più Lucilla <1981&gt. "Characterization and exploiment of Microbial Strains to be used in Meat Processing and Fermentation." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4523/1/deipiu_lucilla_tesi.pdf.

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The principal aim of this research project has been the evaluation of the specific role of yeasts in ripening processes of dry-cured meat products, i.e. speck and in salami produced by adding Lactobacilli starter cultures, i.e. L. sakei, L. casei, L. fermentum, L. rhamnosus, L.sakei + S.xylosus. In particular the contribution of the predominant yeasts to the hydrolytic patterns of meat proteins has been studied both in model system and in real products. In fact, although several papers have been published on the microbial, enzymatic, aromatic and chemical characterization of dry-cured meat e.g. ham over ripening, the specific role of yeasts has been often underestimated. Therefore this research work has been focused on the following aspects: 1. Characterization of the yeasts and lactic acid bacteria in samples of speck produced by different farms and analyzed during the various production and ripening phases 2. Characterization of the superficial or internal yeasts population in salami produced with or without the use of lactobacilli as starter cultures 3. Molecular characterization of different strains of yeasts and detection of the dominant biotypes able to survive despite environmental stress factors (such as smoke, salt) 4. Study of the proteolytic profiles of speck and salami during the ripening process and comparison with the proteolytic profiles produced in meat model systems by a relevant number of yeasts isolated from speck and salami 5. Study of the proteolytic profiles of Lactobacilli starter cultures in meat model systems 6. Comparative statistical analysis of the proteolytic profiles to find possible relationships between specific bands and peptides and specific microorganisms 7. Evaluation of the aromatic characteristics of speck and salami to assess relationships among the metabolites released by the starter cultures or the dominant microflora

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33

Dei, Più Lucilla <1981&gt. "Characterization and exploiment of Microbial Strains to be used in Meat Processing and Fermentation." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4523/.

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The principal aim of this research project has been the evaluation of the specific role of yeasts in ripening processes of dry-cured meat products, i.e. speck and in salami produced by adding Lactobacilli starter cultures, i.e. L. sakei, L. casei, L. fermentum, L. rhamnosus, L.sakei + S.xylosus. In particular the contribution of the predominant yeasts to the hydrolytic patterns of meat proteins has been studied both in model system and in real products. In fact, although several papers have been published on the microbial, enzymatic, aromatic and chemical characterization of dry-cured meat e.g. ham over ripening, the specific role of yeasts has been often underestimated. Therefore this research work has been focused on the following aspects: 1. Characterization of the yeasts and lactic acid bacteria in samples of speck produced by different farms and analyzed during the various production and ripening phases 2. Characterization of the superficial or internal yeasts population in salami produced with or without the use of lactobacilli as starter cultures 3. Molecular characterization of different strains of yeasts and detection of the dominant biotypes able to survive despite environmental stress factors (such as smoke, salt) 4. Study of the proteolytic profiles of speck and salami during the ripening process and comparison with the proteolytic profiles produced in meat model systems by a relevant number of yeasts isolated from speck and salami 5. Study of the proteolytic profiles of Lactobacilli starter cultures in meat model systems 6. Comparative statistical analysis of the proteolytic profiles to find possible relationships between specific bands and peptides and specific microorganisms 7. Evaluation of the aromatic characteristics of speck and salami to assess relationships among the metabolites released by the starter cultures or the dominant microflora

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34

Gottardi, Davide <1983&gt. "Production of bioactive peptides through sequencial action of Yarrowia lipolytica proteases and chemical glycation." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5718/1/gottardi_davide_tesi.pdf.

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This PhD thesis is aimed at studying the suitability of proteases realised by Yarrowia lipolytica to hydrolyse proteins of different origins available as industrial food by-products. Several strains of Y. lipolytica have been screened for the production of extracellular proteases by zymography. On the basis of the results some strains released only a protease having a MW of 37 kDa, which corresponds to the already reported acidic protease, while other produced prevalently or only a protease with a MW higher than 200 kDa. The proteases have been screened for their "cold attitude" on gelatin, gluten and skim milk. This property can be relevant from a biotechnological point of view in order to save energy consumption during industrial processes. Most of the strains used were endowed with proteolytic activity at 6 °C on all the three proteins. The proteolytic breakdown profiles of the proteins, detected at 27 °C, were different related to the specific strains of Y. lipolytica. The time course of the hydrolysis, tested on gelatin, affected the final bioactivities of the peptide mixtures produced. In particular, an increase in both the antioxidant and antimicrobial activities was detected when the protease of the strain Y. lipolytica 1IIYL4A was used. The final part of this work was focused on the improvement of the peptides bioactivities through a novel process based on the production of glycopeptides. Firstly, the main reaction parameters were optimized in a model system, secondly a more complex system, based on gluten hydrolysates, was taken into consideration to produce glycopeptides. The presence of the sugar moiety reduced the hydrophobicity of the glycopeptides, thus affecting the final antimicrobial activity which was significantly improved. The use of this procedure could be highly effective to modify peptides and can be employed to create innovative functional peptides using a mild temperature process.

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35

Gottardi, Davide <1983&gt. "Production of bioactive peptides through sequencial action of Yarrowia lipolytica proteases and chemical glycation." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5718/.

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Abstract:

This PhD thesis is aimed at studying the suitability of proteases realised by Yarrowia lipolytica to hydrolyse proteins of different origins available as industrial food by-products. Several strains of Y. lipolytica have been screened for the production of extracellular proteases by zymography. On the basis of the results some strains released only a protease having a MW of 37 kDa, which corresponds to the already reported acidic protease, while other produced prevalently or only a protease with a MW higher than 200 kDa. The proteases have been screened for their "cold attitude" on gelatin, gluten and skim milk. This property can be relevant from a biotechnological point of view in order to save energy consumption during industrial processes. Most of the strains used were endowed with proteolytic activity at 6 °C on all the three proteins. The proteolytic breakdown profiles of the proteins, detected at 27 °C, were different related to the specific strains of Y. lipolytica. The time course of the hydrolysis, tested on gelatin, affected the final bioactivities of the peptide mixtures produced. In particular, an increase in both the antioxidant and antimicrobial activities was detected when the protease of the strain Y. lipolytica 1IIYL4A was used. The final part of this work was focused on the improvement of the peptides bioactivities through a novel process based on the production of glycopeptides. Firstly, the main reaction parameters were optimized in a model system, secondly a more complex system, based on gluten hydrolysates, was taken into consideration to produce glycopeptides. The presence of the sugar moiety reduced the hydrophobicity of the glycopeptides, thus affecting the final antimicrobial activity which was significantly improved. The use of this procedure could be highly effective to modify peptides and can be employed to create innovative functional peptides using a mild temperature process.

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36

Siroli, Lorenzo <1984&gt. "Use of essential oils and biocontrol cultures for the improvement of shelf-life of fresh cut products." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6278/1/final_thesis.pdf.

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The demand of minimally processed fruits and vegetables has increased in the last years. However, their intrinsic characteristics may favor the growth of pathogens and spoilage microbiota. The negative effects on human health reported for some traditional chemical sanitizers have justified the search for substitutes to guarantee food safety and quality. In this work we have evaluate the potential of some essential oils and their components to improve the safety and the shelf life of Lamb’s lettuce (Valerianella locusta) and apples (Golden delicious). Moreover, the effects of selected lactic acid bacteria alone or in combination with essential oils or their components, on the shelf-life and safety as well as organoleptic properties of minimally processed products, were evaluated. Since the lack of knowledge of microbial cell targets of essential oils represent one of the most important limit to the use of these molecules at industrial level, another aim of this thesis was the study of the action mechanisms of essential oils and their components. The results obtained showed the beneficial effects of the natural antimicrobials as well as the selected lactic acid bacteria on minimally processed fruit and vegetable safety and shelf-life, without detrimental effects on the quality parameters. The beneficial effects obtained by the use of the selected biocontrol agents were further increased combining them with selected natural antimicrobials. The natural antimicrobial employed induced noticeable modifications of membrane fatty acid profiles and volatile compounds produced by microbial cells during the growth. The modification of the expression in genes involved in fatty acid biosynthesis suggesting that the cytoplasmic membrane of microbial cells is one of the major cellular target of essential oils and their components. The comprehension of microbial stress response mechanisms can contribute to the scaling up of natural antimicrobials and bio-control agents at industrial level.

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37

Siroli, Lorenzo <1984&gt. "Use of essential oils and biocontrol cultures for the improvement of shelf-life of fresh cut products." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6278/.

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Abstract:

The demand of minimally processed fruits and vegetables has increased in the last years. However, their intrinsic characteristics may favor the growth of pathogens and spoilage microbiota. The negative effects on human health reported for some traditional chemical sanitizers have justified the search for substitutes to guarantee food safety and quality. In this work we have evaluate the potential of some essential oils and their components to improve the safety and the shelf life of Lamb’s lettuce (Valerianella locusta) and apples (Golden delicious). Moreover, the effects of selected lactic acid bacteria alone or in combination with essential oils or their components, on the shelf-life and safety as well as organoleptic properties of minimally processed products, were evaluated. Since the lack of knowledge of microbial cell targets of essential oils represent one of the most important limit to the use of these molecules at industrial level, another aim of this thesis was the study of the action mechanisms of essential oils and their components. The results obtained showed the beneficial effects of the natural antimicrobials as well as the selected lactic acid bacteria on minimally processed fruit and vegetable safety and shelf-life, without detrimental effects on the quality parameters. The beneficial effects obtained by the use of the selected biocontrol agents were further increased combining them with selected natural antimicrobials. The natural antimicrobial employed induced noticeable modifications of membrane fatty acid profiles and volatile compounds produced by microbial cells during the growth. The modification of the expression in genes involved in fatty acid biosynthesis suggesting that the cytoplasmic membrane of microbial cells is one of the major cellular target of essential oils and their components. The comprehension of microbial stress response mechanisms can contribute to the scaling up of natural antimicrobials and bio-control agents at industrial level.

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38

Taneyo, Saa Danielle Laure <1984&gt. "Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6433/1/Final_dissertationTANEYO13.03.pdf.

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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.

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39

Taneyo, Saa Danielle Laure <1984&gt. "Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6433/.

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Abstract:

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.

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40

Stenico, Verena <1983&gt. "Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6604/1/Stenico_Verena_tesi.pdf.

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Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid.

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41

Stenico, Verena <1983&gt. "Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amsdottorato.unibo.it/6604/.

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Abstract:

Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid.

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42

Mazzola, Giuseppe <1985&gt. "The role of bifidobacteria in newborn health and the intestinal microbial balance." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6832/1/mazzola_giuseppe_tesi.pdf.

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Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

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43

Mazzola, Giuseppe <1985&gt. "The role of bifidobacteria in newborn health and the intestinal microbial balance." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/6832/.

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Abstract:

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

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44

Gozzi, Giorgia <1987&gt. "Atmospheric plasma processes for microbial inactivation: food applications and stress response in Listeria monocytogenes." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/7171/1/Gozzi_Giorgia_tesi.pdf.

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This PhD thesis is focused on cold atmospheric plasma treatments (GP) for microbial inactivation in food applications. In fact GP represents a promising emerging technology alternative to the traditional methods for the decontamination of foods. The objectives of this work were to evaluate: - the effects of GP treatments on microbial inactivation in model systems and in real foods; - the stress response in L. monocytogenes following exposure to different GP treatments. As far as the first aspect, inactivation curves were obtained for some target pathogens, i.e. Listeria monocytogenes and Escherichia coli, by exposing microbial cells to GP generated with two different DBD equipments and processing conditions (exposure time, material of the electrodes). Concerning food applications, the effects of different GP treatments on the inactivation of natural microflora and Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli on the surface of Fuji apples, soya sprouts and black pepper were evaluated. In particular the efficacy of the exposure to gas plasma was assessed immediately after treatments and during storage. Moreover, also possible changes in quality parameters such as colour, pH, Aw, moisture content, oxidation, polyphenol-oxidase activity, antioxidant activity were investigated. Since the lack of knowledge of cell targets of GP may limit its application, the possible mechanism of action of GP was studied against 2 strains of Listeria monocytogenes by evaluating modifications in the fatty acids of the cytoplasmic membrane (through GC/MS analysis) and metabolites detected by SPME-GC/MS and 1H-NMR analyses. Moreover, changes induced by different treatments on the expression of selected genes related to general stress response, virulence or to the metabolism were detected with Reverse Transcription-qPCR. In collaboration with the Scripps Research Institute (La Jolla, CA, USA) also proteomic profiles following gas plasma exposure were analysed through Multidimensional Protein Identification Technology (MudPIT) to evaluate possible changes in metabolic processes.

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45

Gozzi, Giorgia <1987&gt. "Atmospheric plasma processes for microbial inactivation: food applications and stress response in Listeria monocytogenes." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2015. http://amsdottorato.unibo.it/7171/.

Full text

Abstract:

This PhD thesis is focused on cold atmospheric plasma treatments (GP) for microbial inactivation in food applications. In fact GP represents a promising emerging technology alternative to the traditional methods for the decontamination of foods. The objectives of this work were to evaluate: - the effects of GP treatments on microbial inactivation in model systems and in real foods; - the stress response in L. monocytogenes following exposure to different GP treatments. As far as the first aspect, inactivation curves were obtained for some target pathogens, i.e. Listeria monocytogenes and Escherichia coli, by exposing microbial cells to GP generated with two different DBD equipments and processing conditions (exposure time, material of the electrodes). Concerning food applications, the effects of different GP treatments on the inactivation of natural microflora and Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli on the surface of Fuji apples, soya sprouts and black pepper were evaluated. In particular the efficacy of the exposure to gas plasma was assessed immediately after treatments and during storage. Moreover, also possible changes in quality parameters such as colour, pH, Aw, moisture content, oxidation, polyphenol-oxidase activity, antioxidant activity were investigated. Since the lack of knowledge of cell targets of GP may limit its application, the possible mechanism of action of GP was studied against 2 strains of Listeria monocytogenes by evaluating modifications in the fatty acids of the cytoplasmic membrane (through GC/MS analysis) and metabolites detected by SPME-GC/MS and 1H-NMR analyses. Moreover, changes induced by different treatments on the expression of selected genes related to general stress response, virulence or to the metabolism were detected with Reverse Transcription-qPCR. In collaboration with the Scripps Research Institute (La Jolla, CA, USA) also proteomic profiles following gas plasma exposure were analysed through Multidimensional Protein Identification Technology (MudPIT) to evaluate possible changes in metabolic processes.

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46

Bargossi, Eleonora <1988&gt. "Characterization of Tyrosine Decarboxylase (tyrDC) Activity in Genus Enterococcus." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7460/1/Bargossi_Eleonora_Tesi.pdf.

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The presence of biogenic amines in foods and the risks associated to their consumption (especially considered the increase of susceptible consumers) are well known since long time, but systematic studies about their production have been carried out only in the last decades. Fermented foods, in particular, are often characterized by the presence of relevant concentrations of biogenic amines due to the activity of the microbiota responsible for the secondary fermentation. Within this microbiota, enterococci play a controversial role, in fact they contribute to the definition of typical organoleptic profile of some fermented foods, but at the same time they are characterized by different virulence factors and by the ability to produce high concentrations of biogenic amines, in particular tyramine and 2-phenylethylamine. In this perspective, this PhD thesis represents a contribution for a deeper insight of factors, biological mechanisms and genetic characteristics that influence the activity of tyrosine decarboxylase enzymes in strains belonging to the species Enterococcus faecalis, Enterococcus faecium and Enterococcus mundtii. The results of these studies indicated that qualitatively and quantitatively kinetics of tyramine and 2-phenylethylamine production can vary within species, but also strains. These differences can be related to the effects of the specific composition of substrates, the technological variables (temperature, NaCl concentrations, pH) and the specific genetic characteristics of tyrosine decarboxylase cluster and its transcription. Moreover this thesis highlighted the differences in the responses to environmental factors of a pure enzyme respect microbial cells, to better understand the relationships between decarboxylating activity and the integrity of microbial cells. The data obtained indicating that the decarboxylation activity has to be viewed in the light of the overall cell metabolism. Finally, the use of bioprotective cultures producing bacteriocins as antagonists against biogenic amine producing microorganisms has been exploited to reduce the risks of biogenic amine accumulation in fermented foods.

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47

Michelini, Samanta <1987&gt. "Bifidobacteria Ecology of non-Human Primates: Characterization of Novel Species with Unexpected Functionalities for Probiotic Applications and a Co-Evolutionary Host-Microbe Analysis." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7513/1/michelini_samanta_tesi.pdf.

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Bifidobacterium spp. are known as probiotic strains and recently new features emphasize their importance for human health, as EPSs and folate production. The relationship between Bifidobacterium spp. and their hosts is unknown, but probably links to peculiarities in the bifidobacterial cell-wall structures or to bifidobacterial ability to metabolize substrates from the host diet. Recently, a richness and diversity of bifidobacteria was observed in Callithrix jacchus and Saguinus midas, introducing the existence of a storehouse in primate guts. Several techniques were developed to deepen the microbial diversity, mainly based on the PCR. The RFLP-PCR of 16S rRNA gene represents a fast tool to distinguish human or animal origin bifidobacteria, useful in “Microbial Source Tracking” and probiotic selection. The project aim was the exploration of the bifidobacterial occurrence and diversity in evolutionary primate hosts to improve the knowledge about bifidobacteria distribution in non-human primates, and to identify bifidobacteria with new probiotic features (EPSs and folate production). 17 subjects from Strepsirrhini, Eulemur macaco, Eulemur rubriventer, Hapalemur alaotrensis and Lemur catta, and from Simiiformes, the New World Monkeys Callithrix jaccus, Pithecia pithecia, Saguinus oedipus and Saguinus imperator, and the Old World Monkeys, Chlorocebo aethiops and Macaca Sylvanus, were studied. Strains tested for probiotics traits, acid and bile tolerance, revealed B. aesculapii, B. myosotis and B. spp. MRM_8.19 strains as the most resistance. The folate production on strains from ring-tailed lemur and common marmoset revealed autotrophy only in strains from common marmoset. The distribution of microbial communities in non-human primates from 8 babies of common marmosets, golden faced saki and Barbary macaques and 11 adults of ring-tail lemurs, black lemurs, red-bellied lemur, Alaotran bamboo lemur, Barbary macaques, grivet, cotton top-tamarin and emperor tamarin, was carried out using ARDRA and rep-PCR. Results revealed a richness in both abundance and diversity of Bifidobacterium in primates.

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48

Michelini, Samanta <1987&gt. "Bifidobacteria Ecology of non-Human Primates: Characterization of Novel Species with Unexpected Functionalities for Probiotic Applications and a Co-Evolutionary Host-Microbe Analysis." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7513/.

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Abstract:

Bifidobacterium spp. are known as probiotic strains and recently new features emphasize their importance for human health, as EPSs and folate production. The relationship between Bifidobacterium spp. and their hosts is unknown, but probably links to peculiarities in the bifidobacterial cell-wall structures or to bifidobacterial ability to metabolize substrates from the host diet. Recently, a richness and diversity of bifidobacteria was observed in Callithrix jacchus and Saguinus midas, introducing the existence of a storehouse in primate guts. Several techniques were developed to deepen the microbial diversity, mainly based on the PCR. The RFLP-PCR of 16S rRNA gene represents a fast tool to distinguish human or animal origin bifidobacteria, useful in “Microbial Source Tracking” and probiotic selection. The project aim was the exploration of the bifidobacterial occurrence and diversity in evolutionary primate hosts to improve the knowledge about bifidobacteria distribution in non-human primates, and to identify bifidobacteria with new probiotic features (EPSs and folate production). 17 subjects from Strepsirrhini, Eulemur macaco, Eulemur rubriventer, Hapalemur alaotrensis and Lemur catta, and from Simiiformes, the New World Monkeys Callithrix jaccus, Pithecia pithecia, Saguinus oedipus and Saguinus imperator, and the Old World Monkeys, Chlorocebo aethiops and Macaca Sylvanus, were studied. Strains tested for probiotics traits, acid and bile tolerance, revealed B. aesculapii, B. myosotis and B. spp. MRM_8.19 strains as the most resistance. The folate production on strains from ring-tailed lemur and common marmoset revealed autotrophy only in strains from common marmoset. The distribution of microbial communities in non-human primates from 8 babies of common marmosets, golden faced saki and Barbary macaques and 11 adults of ring-tail lemurs, black lemurs, red-bellied lemur, Alaotran bamboo lemur, Barbary macaques, grivet, cotton top-tamarin and emperor tamarin, was carried out using ARDRA and rep-PCR. Results revealed a richness in both abundance and diversity of Bifidobacterium in primates.

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49

Braschi, Giacomo <1988&gt. "Action mechanisms of natural antimicrobials against food-borne pathogenes." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2018. http://amsdottorato.unibo.it/8479/1/Action%20mechanisms%20of%20natural%20antimicrobials%20against%20food-borne%20pathogenes.pdf.

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Essential oils (EOs) or their components represent one of the most promising natural feasible alternatives to improve food safety, shelf-life and quality. Although their antimicrobial properties are well documented few and fragmented are the information about their mechanisms of action, cellular targets and on the stress response strategies of microorganisms after the exposure to such compounds. In this framework, the main aim of the PhD project was to investigate on the effects of one hour exposure to sublethal concentrations of selected natural antimicrobials, such as citral, carvacrol, (E)-2-hexenal and thyme EO, on the food-borne pathogens Listeria monocytogenes Scott A and Escherichia coli K12 MG1655. The action mechanisms of the natural antimicrobials and the cellular targets were studied through multiple approaches able to give information on cell morphological, physiological, transcriptome and proteome changes. The results obtained allowed to define for each strain and each antimicrobial the cell targets and the response mechanism, respectively. The use of the different multi-parametric approaches provided useful information on citral, carvacrol, (E)-2-hexenal and thyme EO action mechanisms on microbial cell targets as well as to elucidate the behavior and the stress response strategies used by Listeria monocytogenes Scott A and Escherichia coli K12 MG1655 after the one hour exposure to such natural antimicrobials. The validation in apple juice allowed to understand the real potential of one of the antimicrobials (chosen on the basis of its sensory compatibility with the food matrix) to improve food safety and shelf life. The data obtained can speed up the exploitation at industrial level of natural antimicrobials as alternative food preservatives.

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Alberoni, Daniele <1990&gt. "Beneficial microorganisms for honey bees health." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2018. http://amsdottorato.unibo.it/8626/1/Ph.D_Thesis_Alberoni_D.pdf.

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Honeybees (Apis mellifera and other species) are considered as the most economically important insect species for humans and the ecosystems, not only as honey producers but also and especially as pollinators of agricultural, horticultural crops and wild plants, contributing at the pollination of 35% of the global food production. Unfortunately, honeybee decline started about 30 years ago, with the arrival from Asia of the bee mite Varroa destructor. Since then, honeybees have been damaged by different kinds of biotic and abiotic stressor factors, cumulating any kind of damages, and posing a serious threat to the agricultural field. Many scientists agree that bee decline is a multifactorial process in which a mechanism seems to be more important in a given period of the year than in another, and different mechanisms may predominate in another period or in other environments. Of those multifactorial processes, leading factors are the new emergent pathogens, such as Nosema ceranae a gut pathogen causing serious threat to bees and the consequent death of the colony; Pesticides and other environmental stress factors are furthering enhancing the high pathogenicity on bees, weakening more and more the delicate beehive superorganism balance. The major science concern about the bees usually regards the study of the bee pathogens and their interaction with an increasingly anthropized environment (e.g.: pollution and sub lethal poisonings). Only few research projects (of high scientific importance) have been carried out using an approach aimed to fix the problems linked with it. Even less are the researches investigating probiotic microorganisms as growth promoter, in order to obtain a better wealth and wellbeing of the bees. In the light of these possibilities the aim of my research is the development of -environmental friendly- microbial technologies aimed to increase the health of the bees.

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Dissertations / Theses on the topic 'AGR/16: MICROBIOLOGIA AGRARIA'

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