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Goals: To assess the effectiveness of Bifidobacterium breve B632 and BR03 association in the reduction of infants crying over time. The second endpoint was to observe the effect of the same strains on daily evacuations and on the number of regurgitations and vomits. Background: Infant colics represent a clinical condition in childhood, characterized by an uncontrollable crying that occurs without any apparent organic cause. An altered intestinal microbiota composition in the very first months may induce intestinal colics in infants. Thus far, no treatment is really effective for this problem, but recent literature shows an increasing attention toward probiotics. Study: A total of 83 subjects were enrolled, 60 breastfed infants and 23 bottle-fed infants. Sixty of them carried out the study: 29 infants were given probiotics, whereas 31 placebo. During the 90 days of the study, parents were asked to give 5 drops of active product (108 viable cells/strain) or placebo and to daily take note of: minutes of crying, number, color, and consistency of evacuations, and number of regurgitations or vomits. Results: No significant differences were detected in the infants treated with probiotics, compared with placebo group (P=0.75). The analysis of the 3 months of treatment demonstrated that during the third month, the probiotic group cried 12.14 minutes on average and the placebo cried 46.65 minutes. This difference is statistically significant (P=0.016). Conclusions: The evidence of the usefulness of some probiotic strains in the treatment and prevention of infant colics is growing, and therefore their use in clinical practice is spreading.

Coeliac disease (CD) is associated with alterations of the intestinal microbiota. Although several Bifidobacterium strains showed anti-inflammatory activity and prevention of toxic gliadin peptides generation in vitro, few data are available on their efficacy when administered to CD subjects. This study evaluated the effect of administration for three months of a food supplement based on two Bifidobacterium breve strains (B632 and BR03) to restore the gut microbial balance in coeliac children on a gluten free diet (GFD). Microbial DNA was extracted from faeces of 40 coeliac children before and after probiotic or placebo administration and 16 healthy children (Control group). Sequencing of the amplified V3-V4 hypervariable region of 16S rRNA gene as well as qPCR of Bidobacterium spp., Lactobacillus spp., Bacteroides fragilis group Clostridium sensu stricto and enterobacteria were performed. The comparison between CD subjects and Control group revealed an alteration in the intestinal microbial composition of coeliacs mainly characterized by a reduction of the Firmicutes/Bacteroidetes ratio, of Actinobacteria and Euryarchaeota. Regarding the effects of the probiotic, an increase of Actinobacteria was found as well as a re-establishment of the physiological Firmicutes/Bacteroidetes ratio. Therefore, a three-month administration of B. breve strains helps in restoring the healthy percentage of main microbial components.

Background: Knowledge about the interplay between diet, microbiota and short-chain fatty acids (SCFAs) so far exists. Moreover, raising evidence suggests their influence on the pathogenesis of the celiac disease (CD). Objective: Our aim was to study and evaluate differences in the composition of selected bacterial groups and SCFAs in faeces of healthy and CD children. Methods: The study included 41 children with CD, 8 newly discovered, not treated children (ND) and 33 children on gluten-free diet for more than 1 year (GFD) and 17 healthy children as a Control group. Bacterial communities and SCFAs in faecal samples were determined by real-time PCR and HPLC analysis, respectively. Results: There were no statistically significant differences between GFD and ND patients. GFD patients compared to Controls had significantly lower Lactobacillus spp. (p = 0.027) and Enterobacteriaceae family group (p = 0.003), but higher propionic acid (p = 0.034). Acetic (p = 0.027) and propionic acid (p = 0.014) were significantly higher in ND patients compared to Controls. Lactobacillus spp. negatively correlated with total SCFAs in the Control and the ND group. In ND and GFD patients, Lactobacillus spp. negatively correlated with Clostridium sensu stricto cluster I. A very strong positive correlation (p = 0.002) between Enterobacteriaceae family and Bacteroides fragilis was found in GFD patients. Conclusions: Changes in microbiota and SCFAs are clearly related to the pathogenesis of CD. As being potential pro-inflammatory agents in CD, acetic and propionic acid may serve as important disease-related markers. Their origin in relation to Lactobacillus and Bifidobacterium is debatable and still need to be further investigated. Enterobacteriaceae family might not be directly addressed to pathogenesis of CD.

Novel bio-based and biodegradable electrospun membranes, containing bactericidal ricinoleic acid (RA) units either by copolymerization with butylene succinate (BS) units or by blending PRA and PBS homopolymers, are characterized in their chemical, thermal, mechanical and surface properties. In the blends E. coli and S. aureus mortality rate increases with PRA amount, while the copolymer shows lower bactericidal activity compared to the blend with similar composition. XPS demonstrates that in the blends an enrichment of RA units at the fiber surface occurs during electrospinning due to phase separation, while in the copolymer RA units are homogeneously dispersed along fiber cross-section.

CONTENTS 16.1 Bifidobacteria and Their Use as Probiotics....................................................................................291 16.2 Dietary Flavonoid Bioconversion by Bifidobacteria.....................................................................293 16.2.1 Dietary Flavonoids...........................................................................................................293 16.2.2 Role of Bifidobacteria in Increasing the Biological Activity of Dietary Flavonoids.......295 16.3 Folate Production by Bifidobacteria..............................................................................................296 16.3.1 Structure of Folates..........................................................................................................296 16.3.2 Sources.............................................................................................................................298 16.3.3 Folate Production by Bifidobacteria.................................................................................298 16.4 Cancer Prevention Activities by Bifidobacteria............................................................................299 16.4.1 Cancer and Intestinal Microbiota.....................................................................................299 16.4.2 Bifidobacteria and Anticarcinogenic Activities...............................................................301 16.4.2.1 Binding/Absorption of Carcinogens.................................................................301 16.4.2.2 Modulation of Intestinal Bacterial Enzymes....................................................301 16.4.2.3 Effects on Oxidative Stress...............................................................................302 16.4.2.4 Production of Beneficial Compounds...............................................................302 References...............................................................................................................................................303

Characterization of strains for taxonomic purposes. The current methodologies used for the characterization of a strain for taxonomic purpose are a combination of those considered traditional and the new one that have been developed recently. In case of strains belonging to novel taxa the characterization needs to provide data for their allocation within the hierarchical framework laid down by the Bacteriological Code. The information on the strain should be as complete as possible and should include the location and the environment peculiarity from which the strain was isolated. Genetic and phenotypic methods are used for characterization. Genotypic criteria refers to sequence analysis of the 16S rRNA gene, DNA-DNA hybridization, DNA base composition, sequencing of the housekeeping genes. Phenotypic criteria typically comprises parameters such as cell shape, colony morphology, biochemical properties, pH and temperature optima etc. In addition, chemotaxonomy is in essence a part of the phenotypic characterization. Characterization of strains for probiotics applications. Functional characterization of probiotic strains have been performed in our laboratory regarding humans , animals and plants. Three examples are reported. The application to humans aimed at the characterization of Bifidobacterium strains capable of inhibiting pathogens typical of the infant gastrointestinal tract. After a preliminary screening regarding the antimicrobial properties against coliforms, Escherichia coli, Salmonella enteriditis, and Clostridium difficile strains, the most interesting Bifidobacterium strains were characterized for cytotoxic effects, adhesion to gut epithelium cell lines and for their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells. The examination of all these features allowed the identification of three Bifidobacterium breve strains and a Bifidobacterium longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The application to animals regarded the characterization of probiotic strains for the reduction of Campylobacter spp. levels in poultry aimed at increasing the safety of poultry meat. Within this work, a synbiotic mixture was developed, composed of a galactooligosaccharide combined with a probiotic Bifidobacterium strain (B. longum subsp. longum PCB133), characterized for its antimicrobial activity against C. jejuni. A significant reduction of C. jejuni in poultry feces was obseved in the treated chickens. The application to plants focused on the inoculation of microorganisms to improve plant growth and root development of perennial ryegrass. A microorganism-based commercial product was used to amend hydroponically grown Lolium perenne L. and results compared with the use of the same filtered product, a phytohormone solution and an untreated control. The main microbial groups present in the product were identified, evidencing bacterial and yeast species, including several Lactobacillus strains. The microbial composition of sand and root samples was investigated by PCR-DGGE, showing different profiles in the different treatments. Inoculated lactobacilli were found to be able to colonize plant roots. Plants treated with the product showed an increased resistance to tearing out with respect to other treatments and roots were longer with respect to the control.

Introduction. With the ban of dietary antimicrobial agents in animal farms, the use of probiotics, prebiotics or synbiotics to reduce pathogen load has attracted a great attention. In particular, Campylobacter jejuni has emerged as a leading bacterial cause of food-borne gastroenteritis in humans, mainly due to poultry food chain and poultry products. In a recent work a synbiotic mixture were administered for 15 days to young broilers and qPCR revealed an increase of the beneficial bacteria (i.e. bifidobacteria, lactobacilli) and a reduction of C. jejuni. In the current study the microbial gut community profiles and C. jejuni were evaluated in treated and untreated broilers by PCR-DGGE analysis to better understand the impact of the tested synbiotic product and support qPCR results. Methods. The synbiotic product was composed by the microencapsulated B. longum subsp. longum PCB133 (>109 UFC/g) and a galactooligosaccharide (3% w/w). It was administered to broilers for 15 days directly mixed to normal feed; faecal samples were collected three times along the trial (t0, t15, t20). DNA extracted from faecal samples was analyzed with DGGE technique after amplification with universal primers and with Campylobacter-specific primers (fla-DGGE). Obtained profiles analysed using the GelCompareII software package version 6.1 (Applied Maths, Kortrijk, Belgium). After normalization, a cluster analysis was performed using the Pearson similarity coefficient and unweighted pair group mathematical average (UPGMA) clustering algorithm. Results. DGGE profiles provided evidence that synbiotic consumption did not alter microbial profiles in the fecal microbiota of both untreated and treated animals; nevertheless a shift in the faecal microbiota of both groups was observed, due probably to animal growth and development. Moreover, DGGE results showed that the band corresponding to the administered probiotic strain PCB133 was detected in all treated animals and was persistent after the wash-out period. Preliminary fla-DGGE analysis underlined the effectiveness of the treatment on naturally infected animals. Discussion. The experimental study highlights the positive effect of the synbiotic approach in broiler chickens, confirming the efficacy of the probiotic PCB133 against C. jejuni without perturbation of gut microbial community.

Introduction: The infant gut microbiota composition is established in a relatively short time during the first two years of life. In particular birth represents a sensitive checkpoint to the future microbiota development which will exert a strong influence on human health. Several factors are known to influence the early microbial colonization of the gut in newborns as the mode of delivery, maternal microbiota of intestine, vagina and epidermis, type of infant feeding, gestational age at birth, hospitalization after birth and the use of antibiotics during the first few months of life. Conversely, the use of antibiotics on the mother during labour, referred to as intrapartum antibiotic prophylaxis (IAP), has been scarcely investigated, although this practice is routinely used in the group B Streptococcus (GBS) positive women. GBS infection remains one of the major causes of neonatal morbidity and mortality although deaths have declined significantly in the last decade (2001-2011) due to the introduction of IAP. This work is therefore aimed at verifying whether IAP can influence the main microbial groups of the newborn gut microbiota at an early stage of microbiota formation. Methods: 26 infants whose mothers gave positive results to GBS and subjected to IAP with ampicillin (IAP group) were compared to 26 infants whose mothers gave negative results to GBS (control group) were enrolled. Faecal samples were obtained at the age of 1 week. Real-time PCR on DNA extracted from faeces was used to quantify selected microbial groups (Lactobacillus spp., Bidobacterium spp., Bacteroides fragilis group, C. difficile and E.coli). In order to investigate the potential perturbations within the Bidobacterium population that were observed with the real time PCR, PCR-DGGE analyses using genus-specific primers targeted to bifidobacteria were carried out both on the IAP samples and on the controls. Results: The real-time PCR analyses showed that the gut microbiota of the IAP group had a significantly lower counts of bifidobacteria (P= 0.001) with respect to the control group. The other microbial genera and species analysed were not significantly affected by the IAP. Furthermore PCR-DGGE analysis revealed that bifidobacteria were affected by IAP not only quantitatively but also qualitatively. In fact, IAP determined a decrement in the frequency of B. breve (P= 0.003, 25% reduction), B. bifidum (P= 0.003, 25% reduction) and B. dentium (P= 0.001, 25% reduction) with respect to the control group. On the other hand, B. pseudocatenulatum, B. pseudolongum and B. longum seemed to be less influenced by the treatment. Discussion: This study has shown for the first time that IAP has a significant influence on the early bifidobacterial pattern of newborns, both quantitatively and qualitatively. However, the obtained results on a larger number of IAP newborns and the possibility of microbial restoration over time should be considered in future studies.

Development of antibacterial electrospun non-woven meshes is of critical importance in several areas such as healthcare products, food packaging, water and air filtration. In this work a novel biocidal polymer, which results active against Escherichia coli and Staphylococcus aureus, was synthesized starting from the biobased ricinoleic acid monomer (RA) and processed through electrospinning in combination with a biodegradable polymer poly(butylene succinate) (PBS), through a blending approach. The optimization of electrospinning process parameters allowed fabrication of defect-free nanofibers with uniform fiber diameter distribution. The chemical, physical and mechanical properties of the obtained non woven meshes were characterized and related to mesh structure and composition. The electrospun constructs were microbiologically evaluated and antibacterials tests confirmed their high biocidal efficacy. Mortality rate of E.Coli and S.Aureus was very high and it increased with the amount of PRA in the blends. Notably, the antimicrobial performances of the meshes were retained after 3 cycles of incubation with cell bacterial suspension. The obtained biobased and biodegradable antibacterial electrospun meshes can find useful application in several sectors.

Next-Generation DNA sequencing data from the human intestinal microbiome provides new instruments to explore the correlation between changes in microbiome composition and the host physiologic status. Some studies show that newborns have a highly unstable microbiome composition, but few information are available in literature on the potential effect due to intrapartum antibiotic prophylaxis (IAP) administrated to the mothers positive to the infection by group B Streptococcus, which affects around 10% of pregnant woman. According to this, the aim of the project is to evaluate the main effects of the mentioned IAP on microbiome composition of newborns at seven days after birth, by a massive parallel sequencing of seven different regions of 16S gene. The analysis was performed with Ion PGM platform, taking advantage from “Ion 16s Metagenomics Sequencing Technology Access Program” (LifeTechnologies). Our first results show an evident difference in the microbial composition of treated newborns, resulting in a general decrease of microbial biodiversity and an over representation of Proteobacteria, suggesting that these infants are potentially more exposed to gastrointestinal diseases.

Next-Generation DNA sequencing data from the human intestinal microbiome provides new mechanism to explore the correlation between changes in microbiome composition and the host physiological status. Previous studies show that newborns have a highly unstable microbiome composition, but little information are available in literature on the potential effect due to intrapartum antibiotic prophylaxis (IAP) administrated to the mothers positive for the infection by group B Streptococcus, which affects around 10% of pregnant woman. According to this, the aim of the research project is to evaluate the main effects of the mentioned IAP on microbiome composition using the retrospective samples of newborns at seven days after birth, by massive parallel sequencing of seven different regions of 16S gene. The analysis was performed with Ion PGM platform, taking advantage from “Ion 16S Metagenomics Sequencing Technology Acces Program” (Life Technologies), which provides us both an Ion Torrent Metagenomics 16S kit (for research use only, not for use in diagnostic procedures) and the support of the Ion ReporterTM Software specific for 16S results’ analysis. Our first results show an evident difference in the microbial composition of treated newborns, resulting in a general decrease of microbial biodiversity and an overrepresentation of Proteobacteria, suggesting that these infants are potentially more exposed to gastrointestinal disease.

Introduction: Food safety and preservation are major priorities for consumers and associated industry. Ancient methods of food preservation are still employed in the production of meat products, such as curing with nitrite or nitrate. Nitrite deriving from nitrate conversion is the real preservative agent. It has several toxic effects to humans, therefore its elimination or reduction is envisaged. Objective: to explore the possibility of using lactic acid bacteria (LAB) as protective cultures to prevent Clostridium spp. growth. Material and Methods: Lactobacillus plantarum PCS20 and Lactobacillus delbruecki DSM20074 were used as protective cultures; Clostridium botulinum DSM1975 and Clostridium perfringens DSM756 were the target pathogens. In-vitro studies (co-culture experiments and spot agar texts) were performed to evaluate the capability of the Lactobacillus strains of inhibiting the Clostridium strains. The survival of both LAB in pork meat batter supplied with NaCl at 2% was checked by using traditional plate counts and RAPD-PCR. Challenge texts were then performed aimed at studying in vivo the antimicrobial activity of the selected LAB inoculated in pork meat batter challenged with Clostridium strains in the presence and absence of 150 mg/kg NaNO3. Results: Both the spot agar texts and the co-culture inhibition assays showed an important antimicrobial activity of the two LAB against both clostridia. L.plantarum PCS20 could survive well in pork meat batter, as shown by RAPD-PCR analyses on colonies isolated from MRS plates. When pork meat batter was inoculated with C.botulinum DSM1975 or with Clostridium perfringens DSM 756, an important reduction of both pathogens was observed both in the presence and in the absence of nitrite. On the contrary, L.delbrueckii DSM20074 strain did not show extensive in vivo antimicrobial activity. Conclusion: The strain L.plantarum PCS20 has the potential of being used as a protective culture against Clostridium spp. in pork meat batter used for sausage production.

Lactic Acid Bacteria [LAB] are worldwide known as probiotic microorganisms. One of the most important probiotic feature is their strong activity against a wide range of harmful bacteria/fungi (Gaggìa et al., 2011). Production of organic acids and production of antagonistic compounds are the main mechanisms described nowadays. This work focuses on the in vitro selection of LAB strains isolated from environmental sources against Fusarium genus. Fusarium is a well distributed genus of filamentous fungi affecting plant, animal and human health. In the agricultural field, Fusarium is one of the most important plant pathogenic genera, with a record of devastating infections in various economically important plants. In this study, in vitro antagonistic effects of 18 isolates of Lactobacillus spp. were evaluated against F. oxysporum f. sp. lactucae, agent of tracheofusariosis in Lactuca sativa, F. culmorum and F. graminearum, agents of severe diseases in cereals. Fungal inhibition was performed using spot agar test in a triplicate assay (Magnusson et al., 2003). Overnight cultures of LAB were inoculated as spot on MRS agar plates and allowed to grow at 30°C for 24 h. The plates were then overlaid with 10 ml of PDA containing 104 mould spores/ml. After 48-72 h of aerobic incubation at 25°C, the zone of inhibition was measured. All LAB strains (except one) showed antifungal activity against all Fusarium species (inhibition halo between 23.7- 65.3 mm). The highest halos were obtained against F. graminearum on which 50% of the LAB strains showed a total growth inhibition of the colony. These results are probably related to the high amount of organic acids produced by LAB strains but it can also be hypothesized the presence of antifungal compounds. In vivo application of the most successful LAB strains is foreseen towards wheat and lettuce to confirm in vitro results.

Use of biological control agents, such as plant growth promoting rhizobacteria (PGPR), and Lactic Acid Bacteria (LAB) can be a suitable approach in control of disease in vegetable crops to reduce and limit the widespread use of chemicals. PGPR, such as Pseudomonas and Bacillus strains, are the major root colonizers that can elicit plant defense and compete with pathogens. Lactic Acid Bacteria [LAB] are worldwide known as probiotic microorganisms and one of the most important probiotic feature is their activity against a wide range of harmful bacteria/fungi. Their use as “effective microorganisms” is increasing as active components of soil amendments for sustainable agriculture. This work focuses on the in vitro screening of 32 PGPR and LAB strains isolated from environmental sources against Fusarium spp, Sclerotinia spp. and Rhizoctonia spp which are widely distributed fungi affecting plant, animal and human health. In the agricultural field, they are considered important plant pathogenic fungi, with a record of devastating infections in various economically important crop plants. Fungal inhibition was performed using spot agar test in a triplicate assay with different conditions for LAB and PGPR. All LAB strains (except one) showed an antifungal activity against all Fusarium species tested (inhibition halo between 23.7- 65.3mm); the highest halos were obtained against F. graminearum on which 50% of the strains showed a total growth inhibition of the colony. Among PGPR, preliminary results showed a strong inhibition by some B. subtilis strains against all fungi tested. These results are promising for the formulation of a commercial product that could be used for a wide spectrum of plant crops. In vivo application with the most successful strains is foreseen towards wheat and lettuce to confirm in vitro results.

Il progetto “Impact of Nanoparticles in Environmental Sustainability and Ecotoxicity” (INESE), finanziato dall’Istituto Italiano di Tecnologia (IIT), ha studiato gli effetti delle nanoparticelle (NPs) ingegnerizzate su diversi ecosistemi (acquatici e terrestri), proponendo differenti sistemi biologici di studio (piante acquatiche, ricci di mare, organismi del suolo, pomodoro-impollinatore, ecc). In particolare l’UO dell’Università di Bologna ha focalizzato l’attenzione sullo studio dei modelli di interazione tra particelle di suolo, microorganismi (biomassa microbica), invertebrati (Lumbricus rubellus) e pianta (Solanum lycopersicum). In questa sede verranno riportati i risultati ottenuti dall’impatto delle nanoparticelle di argento (Ag-NPs) nell’ecosistema suolo. La crescita esponenziale dell’uso di materiali contenenti Ag-NPs è dovuta alla loro importante azione antimicrobica, sempre più sfruttata in diversi campi: dalla medicina (bendaggi) all’abbigliamento (magliette, calzini etc.), ai prodotti di igiene personale (spazzolini da denti), dalla sanificazione delle acque reflue con conseguente produzione di biosolidi impiegati come ammendanti agricoli, all’utilizzo in agricoltura di prodotti dedicati ("Nano-Argentum 10", Fa Nanosys, Svizzera). Studi basati su monitoraggio registrano l’aumento di Ag-NPs nei diversi comparti ambientali (aria-acqua e suolo) e dimostrano che il suolo è destinato ad esserne il maggior ricettore. Il C contenuto nella biomassa microbica del suolo (Cmic) è significativamente diminuita dopo l'esposizione ad Ag-NPs rispetto al controllo, e ciò viene anche confermato dalla diminuzione del DNA microbico estraibile e dall’analisi PCR-DGGE. Quest’ultima ha mostrato la capacità battericida dell’Ag-NPs, riducendo la comunità microbica di un suolo forestale a solo 5 ceppi di batteri resistenti. Riguardo all’impatto sugli invertebrati, il maggior contenuto di Ag rispetto al controllo trovato nei tessuti dei lombrichi è stato riconfermato dall’analisi ESEM che ha mostrato la presenza di Ag-NPs nell’intestino dei lombrichi, dopo depurazione. Inoltre l’esposizione di questi organismi ad Ag-NPs ha causato significativi cambiamenti nella composizione lipidica dei tessuti, rilevando quindi condizioni di stress. La somministrazione di Ag-NPs alle piante di pomodoro tramite l’acqua di irrigazione ha messo in evidenza come le NPs possano essere traslocate dalle radici alle foglie e infine al frutto edule. I potenziali rischi per la salute umana ed ambientale dovuti all’incontrollata diffusione delle NPs costituiscono motivi di grave preoccupazione per la comunità scientifica internazionale.

The production of engineered nanoparticles (NPs) is growing day by day; thanks to their physical and chemical properties, NPs are used in different sectors. However, manufacture, use and potential release of these particles have preceded the evaluation of the environmental risks. The aim of this work was to assess the impact of Ag, CeO2, Fe3O4, SnO2 NPs on the soil microbial community. To evaluate if NPs can affect the soil quality, 500 mg of NPs / kg of soil were mixed to an A1 horizon of Epileptic Cambisols (sandy clay loam texture; subacid pH) and incubated at constant temperature and controlled moisture (e.g. 25°C and 60% WHC). Aliquots of soil samples were taken at 30, 60 and 90 days and transferred to the laboratory for the following analysis: viable bacterial and moulds/yeasts counts , total soil microbial biomass, estimation of soil respiration, DNA extraction and PCR-DGGE. Preliminary results of the biological assays shows an increment of the basal respiration and a decrease in the amount of carbon soil microbial biomass. The relation between these parameters determine a higher metabolic quotient (qCO2) compared to the control test, that identifies a stressful situation, most evident in the thesis with Ag-NPs. Regarding microbial plate counts, no significant differences among the four treatments and the soil control was observed, except for the Ag at 60 days of incubation (Ag-60), where 2 log decrease was evidenced for the viable bacterial count. Conversely, the quantification of the extracted genomic DNA showed a gradual and evident decrease in Ag treatment as incubation proceeded. PCR-DGGE, that was performed to evaluate NPs impact on microbial soil community, clearly highlighted a different microbial profile in Ag treatment between control and Ag-30. Moreover, the profile gave rise to a different banding in Ag-60 with no variation in Ag-90, but some bands showed an increased intensity as the incubation with Ag NPs proceeded. On the other hand, the general banding profile corresponding to the control was mainly conserved in CeO2, Fe3O4 and SnO2 treated soil. Therefore, results obtained so far show an important influence of the Ag NPs on the microbial community in soil by decreasing richness and diversity. Studies are in progress to characterize the member of the microbial community that are positively/negatively selected in the presence of Ag NPs.

The postnatal period is the critical phase for the formation of the microbiota of the human gut. Bifidobacteria are among the first colonizers in breast-fed infants, whereas the microbiota of bottle-fed infants is different and contains higher counts of Bacteroides, Clostridium and Enterobaceriaceae that are the main responsible for bacterial infant gastrointestinal disorders like diarrhea (Penders et al., 2006). Moreover, recent evidences have indicated gas-forming coliform bacteria and C. difficile as a possible cause of infantile colics, which are widely diffused among newborns (Savino et al., 2009). Several studies have focused on the role of bifidobacteria as antagonist of pathogens in the gut. Therefore, there is a great interest in selecting Bifidobacterium strains to be added to formula-fed infants to improve the intestinal microbial balance. The aim of this work is to use in vitro assays to select bifidobacteria to be used as probiotics in newborns for the treatment of enteric disease. 46 strains of bifidobacteria, isolated from infant faeces, were screened to determine their capability of inhibiting potential pathogenic bacteria in infants by using the spot agar test, which evidenced the inhibition of indicator bacteria by the Bifidobacterium strains. Furthermore, inhibitory potency of bifidobacteria neutralized supernatants was investigated. The 16 strains showing the highest antimicrobial activity were subjected to further investigations, such as the susceptibility to 13 antibiotics to determine MIC breakpoints. Additionally, bacterial adhesion to intestinal epithelium was studied in different experimental in vitro models involving human intestinal epithelial and macrophage cell lines. The strains were also compared for their cytotoxic effects, their ability to stimulate the non specific immune response in the gut as well as the metabolic activity in the human functional cell model. The spot agar test of the selected Bifidobacterium strains against Escherichia coli, C. difficile, Salmonella enteriditis and two gas-forming coliforms isolated from colicky infants, identified as Klebsiella pneumoniae and Enterobacter cloacae , evidenced that a number of the assayed strains were capable of inhibiting a large array of pathogens. Ampicillin, kanamicym and amoxicillin resistances were found to be widely diffused among Bifidobacterium strains, whereas a number of strains possessing sensitivity to the other antibiotics assayed could be evidenced. All bacterial strains examined showed a good ability to adhere to polarized human epithelial cells and macrophages but some of them appeared slightly cytotoxic towards the cell monolayers at the highest bacterial concentration used (107 CFU/mL). In particular the ability to stimulate NO production in eukaryotic cells does not seem to be a common ability of member of the Bifidobacterium genus but rather of few strains, while increased H2O2 production was observed for cells stimulated with all tested strains. The results obtained allowed to identify four strains possessing strong antimicrobial activity against selected pathogens, sensitivity to the majority of the antibiotics tested and a good adhesion on human epithelial cells and no cytotoxic effects on them. These strains, belonging to the species B. breve and B. longum , are therefore potential candidates to be used for in vivo trials in newborns.

Bifidobacteria are well known for their health promoting properties and are widely used as probiotics for therapeutic purposes (Sanders et al., 2010). A wide literature documentation reports clinical benefits with treatment of infant gastrointestinal disease with probiotics. In addition, the supplementation of infant formula with oligosaccharides stimulates the growth of bifidobacteria in the intestine resembling the effect of breast-feeding (Boehm and Moro, 2008). A recent study in our laboratory has allowed the selection of four Bifidobacterium strains (B. breve B632, B2274 and B7840 and B. longum subsp. longum B1975) as potential probiotics for the treatment of enteric disorders in newborns such as infant colics (Aloisio et al., 2012). This work was therefore aimed at the formulation of a synbiotic product with an appropriate prebiotic fibre capable of supporting the growth of the previously selected Bifidobacterium strains. The bifidogenic potential of eleven commercial fibres was determined. The polysaccharides used included galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulins, glucooligosaccharide, an arabinogalactan and a partially hydrolised guar gum. The growth of the previously selected em>Bifidobacterium strains was evaluated by using the fibres (1% w/v) as the sole carbon source. The assay was performed incubating the bifidobacteria cultures at 37 °C under anaerobic conditions and the bacterial growth was determined by measuring A620 nm at regular intervals. Significant differences were obtained in the growth profiles of the four Bifidobacterium strains on the different fibres, which is not surprising considering that early studies on prebiotics reported that carbohydrate utilization pattern differs greatly among Bifidobacterium species and strains. A common feature was that the 4 strains could grow well on two GOS (Vivinal®GOS and Cup-Oligo®), on the FOS Actilight® and the low molecular weight inulin Orafti®HSI. In addition, B632 could also grow well on the glucooligosaccharide BioEcolians®. The efficacy of a prebiotic fibre has to be evaluated not only by studying its capability of sustaining the growth of a probiotic strain but also taking into consideration the growth stimulation of potentially harmful bacteria (Huebner et al., 2007). A prebiotic activity score was therefore calculated for the four strains on each fibre by comparing the growth of bifidobacteria with that of a mixture of coliform strains isolated from colicky infants and potentially involved in enteric diseases in newborns (E.coli, K.pneumonia, E. cloacae). The results obtained evidenced a significant prebiotic*strain interaction. The highest prebiotic scores were obtained with Orafti®HSI*B632, Orafti®HSI*B2274, Orafti®HSI*B7840 and Actilight®*B7840. These results provide a basis for the formulation of a synbiotic product for the treatment of enteric disorders in newborns. In addition, considering the high prebiotic scores obtained with Vivinal, a mixture of GOS and FOS can be assayed in further studies.

Recently, basing on consumer´s requirements, particular attention has been devoted to the development of functional food. Such is the case of food that contains probiotic microorganisms and/or prebiotic compounds that promote the growth of these microorganisms. “Espina Corona” gum (EC) is obtained from the seeds of Gleditsia amorphoides, growing in the north of Argentina. Due to its thickener ability, it is utilized together with gelatin, as additive in the manufacture of dairy products. The interaction between microorganisms and other components of the food matrix may be necessary to understand the inhibition or benefit in the growth of such microorganisms. Other gums (like guar gum) have proven prebiotic activity over different species of bifidobacteria and the great composition similarity between guar gum and EC motivates the study of the latter as a potential prebiotic in food matrices. The aim of this work was to study the ability to ferment commercial prebiotics such as Cup-Oligo® (a galactooligosaccharide, GOS), Actilight®950P (a fructooligosaccharide, FOS), Orafti®Synergy (a high solubility inulin, HSI), Orafti®Raftiline (a high polymerization inulin, HP), EC, and Gelatin (G) by different species of bifidobacteria and lactobacilli isolated from commercial dairy products and others present in the culture collection BUSCOB at Bologna University. The strains were identified according to molecular techniques (species-specific PCR and ERIC-PCR). FOS, GOS and HSI were the substrates fermented by the largest number of species. Lactobacillus casei Shirota and L. rhamnosus isolated from Yakult™ and Yomo™ products, respectively, only grew in the presence of GOS. L. rhamnosus SP1 did not use any substrates. EC and HP did not show significant effects on the growth of lactobacilli species and on the 4 strains of Bifidobacterium animalis ssp. lactis isolated from commercial products. While EC was fermented by B. animalis ssp. lactis DSM 10140T and P23 , B. longum ssp. longum B1835 (deriving from BUSCOB) and DSM 20219T, the gelatin inhibited the growth of all the strains tested. These results point out that EC can be used not only as a thickener additive but that it also has a potential prebiotic activity for some bifidobacteria strains

Aquaculture is one of the fastest growing sector of food production in the world. Despite the encouraging trends, several constraints have had a negative impact on the growth of aquaculture. Diseases are the primary limiting factor in large scale production facilities, where aquatic animals are exposed to stressful conditions. Bacterial diseases are responsible for heavy mortality in both wild and cultured fish and result in serious economic lossess. Prevention and control of diseases have led during recent decades to a substantial increase in the use of veterinary medicines. However, the utility of anti-microbial agents as a preventive measure has been questioned, given the extensive documentation on the development and spread of antimicrobial resistance genes among bacteria. Several mechanisms can be responsible for the acquirement of antibiotic resistances, the main common of which are chromosomal mutation or acquisition of plasmids. Chromosomal mutations cannot be transferred to other bacteria but plasmids can transfer resistance rapidly. Several bacterial pathogens can develop plasmid-mediated resistances: plasmids carrying genes for resistance to antibiotics have been found in marine Vibrio species and they could be laterally exchanged. The concept of biological disease control using probiotics has received widespread attention during the last decade. When looking at probiotics intended for an aquatic usage, it is important to take into account the complex relationship that an aquatic organism has with its direct environment, compared to terrestrial animals. Gram-negative facultative anaerobic bacteria are dominant in fish and shellfish digestive tract, but the intestinal microbiota of aquatic animals may change very rapidly with the ingestion of microorganisms coming from water and food. This is probably the reason why a large number of probiotics developed in aquaculture are bacteria directly originating from the aquatic environment. However, more ‘traditional’ bacterial or yeast species marketed for animal nutrition (Lactobacillus spp., Pediococcus spp., Bacillus spp., and S. cerevisiae) are also used. They can target fish eggs and larvae, fish juveniles and adults, crustaceans, bivalve molluscs and also live food such as rotifers, artemia, or unicellular algae (Kesarcodi-Watson et al., 2008). Growth-promoting effects, through better feed utilisation and digestion, as well as biological control of pathogen colonisation are the most important expected benefits of probiotic applications. Disease outbreaks caused by Vibrio spp. or Aeromonas spp. have been recognised as the most significant constraint on aquaculture production, particularly in the shrimp subsector, where vibriosis is currently one of the main diseases identified. Whereas in vitro, antagonism to pathogens has been clearly demonstrated for a wide range of probiotic strains, in vivo evidence of efficacy is still very scarce. Most probiotics proposed as biological control agents in aquaculture belong to the lactic acid bacteria (Lactobacillus spp. and Carnobacterium spp.), to the genus Vibrio (V. alginolyticus), to the genus Bacillus, or to the genus Pseudomonas, although other genera or species have also been mentioned (Aeromonas and Flavobacterium). Few multinational pharmaceutical companies have introduced commercial preparations into the market as probiotics feed/food supplement in various commercial names as Aqualact, Spilac, Protexin (Verschuere et al., 2000). This chapter will give some examples on how probiotics can be applied to reduce the incidence of diseases usually affecting aquatic animals, including fish, crustaceans and mollusks. Moreover, a brief consideration on the recent approach of prebiotic administration will also be provided.

Nonylphenol ethoxylates (NPnEOs, n is the number of ethoxylic units in the molecule) are non-ionic surfactants widely used in several industrial applications, such as textile and leather processing, paper industry, formulation of pesticides, paints and washing cleaners. 4-Nonylphenol (4-NP), the main product of NPnEO biodegradation, is a toxic xenobiotic compound classified as endocrine disrupter. While numerous studies reported the toxicity and estrogenic activity of nonylphenols, little is known about the mutagenicity of these compounds. In this paper, the genotoxicity of 4-NP and NPnEO mixtures was evaluated by using the D7 strain of Saccharomyces cerevisiae as experimental model. The same tests were applied to effluents deriving from laboratory scale packed-bed bioreactors, developed for the treatment of NPnEO contaminated wastewater, in order to evaluate the residual genotoxic potential with respect to the influent waste. The target compounds fed to the bioreactors were 4-NP and NPnEO mixtures possessing an average of 5 or 1.5 ethoxylic units (Igepal CO-520 and Igepal CO-210, respectively). The results showed that 4-NP induced significant cytotoxic effect on S. cerevisiae cells at 60 mg/L, as well as mutagenic effects at 15 and 30 mg/L. 4-NP was the most genotoxic compound among those assayed, followed by Igepal CO-210, whereas Igepal CO-520 did not induce genotoxicity at any of the assayed concentrations. The genotoxic effects evidenced on yeast cells treated with 4-NP disappeared after the treatment in the bioreactor. This indicates that the biological treatment is capable of removing not only the pollutant, but also the toxicity associated to the compound and its degradation metabolites. This study represents, to the best of our knowledge, the first report that evaluates the genotoxicity of both 4-NP, NPnEOs and their potential aerobic degradation products on an eukaryotic organism. The obtained results suggest that the S. cerevisiae D7 strain is a very effective model to study the induction of genotoxic damage by the compounds under study. In addition, this method is much more simpler and easier to be applied than other tests described in the literature to study genotoxic effects. Moreover, the test described in this work has also proven to be effective in evaluating the toxicity of effluents deriving from laboratory scale biotreatment processes. It would be really interesting to apply the described tests on real wastewater treatment system in order to estimate the detoxification potential of the process applied, which is often not directly correlated with the removal of the target pollutant.

Bifidobacteria and lactobacilli due to their health promoting activity are commonly used as probiotic in dairy products for human consumption. Their suggested effective concentration is up to 106CFU/g of product. The correct identification and the monitoring of viable probiotics in the final product is an indispensable prerequisite for applications in industrial processes and in safety and quality control applications. The aim of this study was the optimization of routine laboratory methods for the enumeration and identification of probiotic species commercialized in dairy products. Nine fermented milk products of different marketed brands, purchased in Italian supermarkets, were tested. In these products, with or without starter cultures, Bifidobacterium animalis subsp.lactis and/or L. acidophilus, L. casei, L. johnsonii, L. paracasei, L. rhamnosus have been declared in the product label. The shelflife for all the products was about 20 days. Bifidobacteria were counted in TPY-mupirocin medium while lactobacilli were all counted using MRS-bile both in aerobiosis and anaerobiosis; in anaerobiosis MRS-pH 5.4 for L. acidophilus, L. johnsonii and L. paracasei, and MRS-vancomycin for L. casei and L. rhamnosus were also used. The obtained isolates have been catalogued basing on cell morphology and identified by species-specific PCR and ERIC-PCR. TPY-mupirocin has been confirmed as an optimal selective media for bifidobacteria; for L. rhamnosus and L. paracasei all the media tested were suitable while for L. acidophilus and L. johnsonii MRS-pH5.4 gave better results. All the probiotic dairy products contained the recommended levels of 106–107 CFU/g probiotic bacteria except one brand with 105 CFU/g. Morphology cannot help in distinguishing between the species L. casei, L. paracasei and L. johnsonii while L. acidophilus and L. rhamnosus show peculiar morphology; B. animalis subsp.lactis show the typical "bone" morphology. The present work confirm that ERIC-PCR is a valuable method to rapidly discriminate between lactobacilli closely related species while species-specific PCR does not discriminate between L. casei and L. paracasei. All the products declaring the presence of L. casei in the label actually contain L. paracasei.

Lactobacilli, bifidobacteria and various yeasts are worldwide known as probiotic microorganisms. They are part of the natural gut microbiota of both humans and animals, they have a long history of consumption and they are present in almost all fermented foods. Recently they have been also exploited as “Effective Microorganisms” (EM) for the production of bio-fertilizers to promote plant growth and control diseases in sustainable agriculture systems. This work focuses on the effect of inoculation of a commercial product, containing lactobacilli, bifidobacteria, yeasts and aerobic bacteria on perennial ryegrass (Lolium perenne L.), hydroponically grown for five weeks. Perennial ryegrass is a cool-season turfgrass species that is widely used on home lawns, sports fields, and golf courses due to its rapid establishment and superior traffic tolerance. Following an activation process to revitalize the microorganisms, the inoculation was performed via the irrigation system, combined or not with seeds imbibitions with the same product. Six different treatments were compared: 1- EM combined with seed imbibition: 2- EM without seed imbibition ; 3- filtered EM combined with seed imbibition; 4- filtered EM without seeds imbibition; 5- control (mineral Hoagland solution), 6- commercial plant hormones rich solution. Data on aboveground and underground biomass were collected with special regard to root development representing a critical issue for the maintenance of lawns and sport fields. Soil and root microbiota have been studied by DGGE analysis. qPCR has been performed to assess the Lactobacillus colonization of the radical tissues. In all experimental conditions no significant effect of different treatments was observed at the leaf level, while EM and phytohormone treatments induced significant effects on root development, with an average increase of total root length equal to 35% and 70%, respectively. Among treatments significant differences in root fresh weight were observed, while the effects of treatments on root dry weight were negligible. As regards microbiological data, DGGE evidenced different microbial profiles in EM treated plants compared to control and phytohormone treatment, which were comparable. EM seed imbibition induced the greatest microbial diversity at the root level. qPCR revealed a significant increase of Lactobacillus spp. in EM treated roots. A potential colonization of this microbial group at root level could be an important feature to improve plant resistance against pathogens. In conclusion, considering these preliminary results, a potential use of EM as bio-fertilizer could be hypothesized: open field trials are necessary to assess the robustness of the obtained results.

Nanotechnology is a growing field with a major role in product and process innovations. This is an opportunity to develop new products, but it also harbors risks for the environment and health. Although it is a common knowledge that some nanoparticles (NPs) have antibacterial activity, there is little information on the influence of NPs on soil microbial community (Hänsch and Emmerling, 2010). The harmful properties of silver ions to bacteria, for example, are well investigated. Silver ions may inhibit bacterial respiratory activity and may negatively interact with the bacterial cell wall enzymes. Furthermore, Ag ions may inhibit DNA replication. Perturbations of soil microbial consortia can have significant consequences for ecosystems and soil microbial consortia are influenced by and also affect plant growth (Davidson and Janssens, 2006). The aim of this work was to assess the impact of Ag, CeO2, Fe3O4, SnO2 NPs on the soil microbial community, to evaluate if NPs can affect the soil quality. 500 mg of NPs / kg of soil were mixed to an A1 horizon of Epileptic Cambisols (sandy clay loam texture; subacid pH) deriving from a forest environment and incubated at constant temperature and controlled moisture (e.g. 25°C and 60% WHC). Aliquots of soil samples were analysed after 30, 60 and 90 days of incubation with NPs to evaluate the following parameters: viable bacterial and moulds/yeasts counts , total soil microbial biomass, estimation of soil respiration, DNA extraction and PCR-DGGE. Preliminary results of the biological assays shows an increment of the basal respiration and a decrease in the amount of carbon soil microbial biomass. The relation between these parameters determine a higher metabolic quotient (qCO2) compared to the control test, that identifies a stressful situation, most evident in the thesis with Ag-NPs. Microbial plate counts evidenced no significant differences among treated and control soil except for a 2 log decrease of viable bacterial count in the Ag-treated soil after 60 days (Ag-60). Moreover, the quantification of soil DNA extractions showed a gradual decrease of DNA concentration in Ag-treated soil during incubation. PCR-DGGE was performed to evaluate NPs impact on microbial soil community. DGGE profile of control soil was mainly conserved in CeO2, Fe3O4 and SnO2 treated samples. On the contrary, Ag-treated soil evidenced a clear different profile already after the first 30 days (Ag-30). Moreover, the profile changed again in Ag-60 and remain stable in Ag-90. It is interesting to note that some bands showed an increased intensity as the incubation with Ag NPs proceeded. This preliminary results show an important influence of the Ag NPs on soil microbial community, decreasing richness and diversity. Bands sequencing are in progress to identify the microbial populations that are positively/negatively affected by NPs treatment.

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems of newborns such as colics. The pathogenesis of colics remains largely unknown although evidences suggested multiple independent causes, including modification of the gut microbiota with an increased number of gas-forming coliforms. Positive effects on newborn colics have been evidenced after administration of Lactobacillus reuteri, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. The aim of this work is to select bifidobacteria to be used as probiotics in newborns for the treatment of enteric disorders with a special attention on colics. 46 Bifidobacterium strains were originally screened and, among them, 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 cytotoxic effects and adhesion to nontumorigenic 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 results obtained allowed to identify 3 B. breve strains and a B. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics.

The quality and safety of foods is of outmost importance for governments, industry and consumers. The EU project Pathogen Combat (www.pathogencombat.com) has provided new essential information and methods on how to reduce the prevalence of pathogens across the entire food chain. During the five years, researchers have obtained new results which have been disseminated and tested into SMEs belonging to dairy, poultry, pork and beef and lamb production chain. The unique achievements of Pathogen Combat will be presented. In addition, we will focus on the use of probiotics and prebiotics as an intervention measure to reduce the prevalence of pathogens at farm level. Campylobacter jejuni has now emerged as a leading bacterial cause of food-borne gastroenteritis in humans around the world. Epidemiological studies indicate poultry and poultry products as a significant source of human infection. We evaluated the effects of two probiotic strains and two prebiotic compounds to modulate the gut microbiota of broiler chickens to obtain a competitive reduction of C. jejuni colonization. The experiments workflow counts: faecal samples collection at three different times, samples processing and qPCR analysis. The best strain, resulting from the first treatment, was then microencapsulated, coupled with the best prebiotic compound and fed to broiler chickens as synbiotic mixture. Detection and quantification of the targeted bacterial groups (Bifidobacterium spp., Lactobacillus spp., Campylobacter spp., and C. jejuni) were performed on fecal samples by Real-Time PCR using SybrGreen chemistry. C. jejuni concentration in poultry faeces was significantly (p

In Europe, campylobacteriosis is the most common infectious disease transmissible from animals to humans through food and the bacterial species most frequently implicated is Campylobacter jejuni (Humprey et al. 2007). Epidemiological data obtained by the European Food Safety Authority (EFSA) evidenced that poultry meat appears to be a major, if not the largest, source of human infection (Hugas et al., 2009). Risk assessment studies concluded that the most effective intervention measures is the reduction of Campylobacter concentration in poultry, which can effectively reduce the contamination throughout the whole food chain (Nauta et al. 2009). An alternative and effective approach to antibiotic administration to livestock to reduce Campylobacter contamination is the use of probiotics, which have to be carefully selected both to meet the safety requisites established by EFSA and to survive in the feed as well as in the gastro-intestinal tract of the host. The aim of the present work is to select probiotics to be used as additives in feed for livestock poultry in order to reduce C. jejuni contamination and therefore enhance the safety of poultry meat. The first part of the work regarded the screening of 55 lactic acid bacteria and bifidobacteria for desirable properties for their use as probiotics in poultry: anti-microbial activity against C. jejuni strains, survival in simulated gastro intestinal tract (pH 2.5 and 1% bile salts) and food/feed processing conditions (50 and 55 °C); moreover, susceptibility to the most diffused antibiotics was checked. Secondly, the two most interesting strains, namely Lactobacillus plantarum PCS 20 and Bifidobacterium longum PCB 133, were separately administered to poultry by oral gavages to evaluate their capability of colonizing the GI tract and to estimate their effect on C. jejuni population. The results obtained evidenced that B. longum PCB 133 significantly increased in the feces after 15 days administration and its amount was still high after a wash-out period of 6 days. In the same period, C. jejuni concentration in poultry faeces was significantly reduced. Then, B. longum PCB 133 was microencapsulated in a lipid matrix to obtain optimal survival in the feed, coupled with a prebiotic compound and fed to broiler chickens as a synbiotic mixture. C. jejuni concentration faeces was significantly (p

Biotechnological processes for the production of vanillin from substrates of natural origin are getting increasing interest since the obtained flavor can be labeled as “natural” according to the EU legislation. In this work, we investigated the possibility of producing vanillin from ferulic acid, i.e., an abundant cell wall constituent in higher plants available from bran processing by-products, by employing resting cells of Pseudomonas fluorescens strain BF13 carrying an insertional inactivation of vdh gene and ech and fcs BF13 genes under the control of native Pfer promoter on a low copy number plasmid. Process parameters were optimized both for the biomass production phase (pH of the medium, catabolic induction) and the bioconversion phase (pH of the buffer, cells concentration, substrate concentration). Vanillin up to 8.46 mM could be obtained under optimized conditions after 24h of bioconversion with ferulic acid 10 mM as substrate, whereas the highest productivity (0.53 mmoles vanillin L-1 h-1) was obtained using ferulic acid 5 mM.

Despite significant investment in food safety, the incidence of food-borne disease is still on the rise in the European Union. Probiotic cultures and prebiotics, as additives in animal feed, may be an effective strategy to prevent or reduce the incidence of pathogens in the gastrointestinal tract of food-producing animals, improving food safety and consumer health. Thanks to the EU project Pathogen Combat, new probiotics cultures has been selected for application in animal nutrition. The selected strains were added to the feed of goats and broilers as lyophilized powder or microencapsulated to better resist to harsh environmental conditions. Following 15 days of treatment, the intestinal microbiota was analysed to evaluate the influence of probiotics on the composition of the microbiota and on pathogen presence.

An aerobic bacterial consortium (Consortium A) was recently isolated from textile wastewater and tested to be able of degrading a variety of nonylphenol polyethoxylates (NPnEOs) congeners as well as an industrial mixture of branched side-chain isomers of 4-nonylphenol (4-NP) using them as the sole carbon and energy source. In the perspective of developing a biotechnological process for the treatment of effluents from activated sludge plants fed with NPnEO contaminated wastewater, the capability of Consortium A of biodegrading two industrial mixtures of NPnEOs in the condition of immobilized cells was investigated. A packed-bed bioreactor has been developed by immobilizing the consortium on an innovative ceramic material (Vukopor® S10) by re-circulating Consortium A cells growing on a 4-NP contaminated water under batch conditions. Two different NPnEO mixtures were subsequently tested as the only carbon sources. The experimental approach was then defined as follows: the reactor was fed with the first mixture (IgepalCO-520: average ethoxylation degree of 5), operated under batch conditions for a brief period and then it was switched to the continuous mode. The same was done feeding the reactor with the second mixture (IgepalCO-210: average ethoxylation degree of 1,5). The reactor was monitored through chemical, microbiological and molecular integrated methodology. Both experiments displayed comparable NPnEO mineralization under batch and continuous conditions. DGGE analyses were running on the DNA extracted from the liquid phases and from the biofilm developed on supports at the end of the two experiments, to determine the spatial distribution of Consortium A microorganisms mostly involved in NPnEO biodegradation. The data collected in this study provide a preliminary strong indication on the feasibility of Consortium A-based biofilm technology for the decontamination of NPnEO containing effluents.

Functional foods provide health benefits beyond basic nutrition. A relevant health aspect regards the equilibrium between nutrients and beneficial gut-flora, especially Bidifobacterium spp. and Lactobacillus spp. Flavonoids are biologically active polyphenols with high antioxidant properties and demonstrated role in prevention of not-heritable diseases (cardiovascular diseases and cancer). Flavonoids are present in legumes mainly as O-glycosides: generally intestinal enzymes cannot hydrolyze the bond between the sugar moiety and the bioactive compound thus avoiding its uptake and subsequent health benefits. However, for some human intestinal Bifidobacterium species beta-glycosidase activity on flavonoids has been described (Marotti et al., 2007). The aim of the present research is to extend the monitoring of beta-glycosidase activity to lactic acid bacteria (LAB) commonly used in the food sector. The first step was to optimize the beta-glucosidase assay in MRS substrates in order to tune the experimental conditions in a medium suitable for LAB growth. For the experimental conditions setting up, two Bifidobacterium species were chosen on the basis of the results obtained in a previous work (Marotti et al., 2007): one strain of B. pseudocatenutalum for its high beta-glucosidase activity and one strain of B. longum lacking detectable activity. The beta-glycosidase activity was assayed by adding p-NPG (p-nitrophenyl- beta-D-glucopyranoside) in the growth medium and evaluating the pNP (p-nitrophenol) concentration after 14-24 hrs of incubation. The beta-glycosidase activity in B. pseudocatenulatum was independent from the growing medium (TPY or MRS) and from cell density; but it was influenced by substrate concentration. The optimized protocol was applied to 15 strains of LAB, including the following species: L. fermentum, L. buchneri, L. fructivorans, L. helveticus , L. acidophilus, L. plantarum, L. kefiri, L. jugurti, L. lactis, L. bulgaricus. The maximal beta-glycosidase activity was observed for L. buchneri, L. acidophilus and L. plantarum. These strains were previously isolated from tomato sauce and milk derived foods: they are therefore good candidates for developing functional food. Further studies are in progress with the aim to confirm the beta-glycosidase activity of these strains on typical legume flavonoids.

Wheat bran contains about 5 g/kg of dry matter of ferulic acid, which can be released from cell wall polysaccharides through chemical or enzymatic hydrolysis. Ferulic acid is a precursor of vanillin, one of the most used flavouring compound in the food industry. It has previously been shown that ferulic acid occurring in wheat bran can be successfully bioconverted into vanillin by resting cells of Escherichia coli JM109/pBB1. In this work the role of ferulic acid concentration in wheat bran hydrolyzates is explored in order to evidence possible inhibitory effect on the bioconversion process by high substrate concentration. Moreover, different ferulic acid purification methods are compared in terms of vanillin molar yields obtained by the bioconversion of the recovered ferulic acid. In addition, the possibility of exploiting the carbohydrate rich water phase obtained after ferulic acid recovery as the growth substrate for the microorganism operating the bioconversion is explored. The results obtained have demonstrated that the high carbohydrate concentration present in crude bran hydrolyzates contribute to the low vanillin yields rather than the high concentration of ferulic acid. Purification of ferulic acid allowed in fact to increase the yields of the bioconversion process. The best ferulic acid recovery method was solid-liquid extraction with ISOLUTE ENV+® cartridges as it allowed the maximum ferulic acid recovery (95%) and good bioconversion yields (up to 75%); moreover the water phase rich in reducing carbohydrates resulting from this extraction is a good growth substrate for E.coli JM109/pBB1.

Nonylphenol ethoxylates (NPnEO) are synthetic non ionic surfactants widely used in textile industries. Metabolites produced by their degradation, such as short chain NPnEO and 4-nonyl-phenol (4-NP) tend to accumulate in the environment. In this work two immobilized cell bioreactors, one packed with granular activated carbon and the other with silica beads have been developed and tested for the degradation of nonylphenol ethoxylates, using an artificially NPnEO contaminated wastewater. Initially the bioreactors were operated in batch mode and fed with 4-NP and Igepal CO-520 (commercial mixture of NPnEO with an average degree of ethoxylation of five) as the sole carbon and energy source, showing excellent removal performances. Then they were forced in continuous mode and assayed at three different organic loads. Also in this condition, good results in terms of pollutant removing and mineralization were achieved. Therefore the results of this study showed that packed bed bioreactors could be usefully employed for the depuration of industrial wastewaters contaminated by NPnEO and their degradation metabolites.

In this work the possibility of producing vanillin through microbial bioconversion from ferulic acid obtained from the enzymatic hydrolysis of wheat bran has been explored. The biocatalyst employed was Escherichia coli JM109/pBB1, a recombinant strain containing the genes from Pseudomonas fluorescens BF13 for the transformation of ferulic acid into vanillin. The substrate of the bioconversion, performed with resting cells of E.coli JM109/pBB1 grown on LB rich medium, was the crude hydrolizate obtained from wheat bran or a buffer containing ferulic acid recovered from the crude matrix with different methods, as adsorption on ion exchange resins, liquid-liquid extraction and solid phase extraction; these operations allowed the selective recovery of ferulic acid from carbohydrates present in the hydrolizates, thus obtaining an aqueous phase rich in reducing sugars. The removal of reducing sugars allowed to obtain higher bioconversion yields. The results of this work showed that vanillin can be obtained at interesting yields from ferulic acid extracted from wheat bran.

In healthy people the acidity of the stomach kills most of microbial agents. However, if gastric acidity is diminished [e.g. in atrophic autoimmune gastritis (AAG) or reflux oesophagitis treated with omeprazole] the stomach can be colonized. This study investigates the gastric microbiota, in particular total anaerobes and Bifidobacteriaceae, in 29 individuals 13 of which treated with omeprazole, 11 affected by AAG and 5 controls. Samples deriving from biopsies and gastric juice were cultivated anaerobically in non selective and Bifidobacteriaceae selective media. Bifidobacteriaceae family identification was carried out by fructose-6-phosphate phosphoketolase (F6PPK) test. Species identification was obtained with fermentation tests, polyacrylamide gel-electrophoresis of soluble proteins and DNA-DNA homology. Differently from normal acid stomach, in achloridric stomach an important growth of anaerobes was detected. In selective medium colonies showed typical Bifidobacteriaceae morphology. However, F6PKK tests of all 127 isolates revealed that only 45 belonged to Bifidobacteriaceae while others were described as “actinomyces-like group” basing on their irregular morphology. All F6PPK positive isolates were found to belong to Bifidobacteriaceae typical of oral cavity (Scardovia inopinata, Parascardovia denticolens and Bifidobacterium dentium). The presence of an “actinomyces-like group”, probably originated from oral cavity too, suggests that colonization of achloridric stomach derived from oral cavity.

Lactic acid bacteria and bifidobacteria are natural inhabitants of the human intestinal tract with health–promoting effects. In this work, the antimicrobial activity of 92 LAB and bifidobacteria strains (Lactobacillus spp., Bifidobacterium spp., Pediococcus spp., Leuconostoc spp) was tested in vitro against 3 strains of Campylobacter jejuni. The results evidenced that 9 LAB and 2 bifidobacteria strains possessed antimicrobial activity against all C. jejuni strains, which was not caused by acid formation. The nature of the compounds responsible for this activity has been studied by treatment with different enzymes, thus evidencing a possible proteinaceus nature of them. The eleven strains have been characterized for their antibiotic resistance profile and assayed for their survival in the GI tract (viability at pH 2.5 and resistance to bile salts). Furthermore, their ability to survive under two different food processing conditions (heat and osmotic stress) was evaluated. The results showed that most of the strains rapidly loose their viability at low pH, whereas treatment with bile salts did not affect the survival of the majority of them. Almost all strains survived well at both processing conditions tested. Tests are being performed to evaluate the colonization of the selected strains in poultry and their in vivo activity against C. jejuni.

Textile wastewaters are usually subjected to secondary activated sludge treatment before being discharged in the environment or re-used. In order to improve the efficiency of this treatment, four reactors possessing different configurations and fluid dynamic characteristics (i.e. a Bioflotation® system; a fixed bed biofilm reactor (FBBR) filled with polypropylene balls; a flow-jet aeration system and a conventional activated sludge reactor) were developed and fed with a textile industry wastewater. COD, TC and non ionic surfactant concentrations were monitored in the reactor effluents and the cultivable heterotrophic biomass was isolated from the effluents. The results evidenced that the fluid dynamic characteristics and the air supply system can greatly influence the degradation efficiency of the secondary treatment. In particular, Bioflotation® and FBBR technologies appear to be the most efficient in term of pollutant removal. An highly specialized biomass, rich in strains belonging to the Pseudomonas and Stenotrophomonas genera, was evidenced in these reactors, thus allowing to conclude that there is a direct correlation among reactor configuration, biomass specialization and reactor performances.