Combined approach for the investigation of dominant fermenting microbiota in two traditional sourdoughs produced in sicily

In order to explore the community of lactic acid bacteria (LAB) and yeasts present in two major typical Sicilian sourdoughs, seven mature sourdoughs for "Pane Nero di Castelvetrano" (CV1 - CV3 samples) and "Pane di Monreale" (MR1 - MR4 samples) were analysed through a culturedependent and culture-independent approach. The highest values of microbial counts were revealed in MR1 sourdough. In particular, LAB counts were at about 109 CFU/g in media specific for typical sourdough LAB, such as SDB and SFM, while levels of 106 CFU/g were registered for yeasts. The total DNA form each sourdough sample was extracted and subjected to a multiplex-PCR in order to recognize the major groups of LAB. Seventy-six LAB with a rod shape, presumptively Lactobacillus, were phenotypically grouped and subjected to a genotypic identification by sequencing of the 16S rRNA gene and further confirmed by species-specific PCRs. Yeasts were isolated and identified by a combined genotypic approach consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and sequencing of D1/D2 domain of the 26S rRNA gene. The LAB species identified were Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus brevis and Lactobacillus coryniformis. Among yeasts, Saccharomyces cerevisiae, Pichia guiliermondii, Pichia segobiensis, Rhodotorula acuta and Rhodotorula mucilaginosa were the species hosted in Sicilian sourdough. The multiplex PCR carried out on total DNA of sourdoughs allowed the rapid identification of the majority of sourdough lactobacilli but the culture-dependent methodology was confirmed to be necessary for the detection of the species, such as Lactobacillus coryniformis not included in the system

Metabolomics study of human embryonic stem cell culture media

Self-renewal and pluripotency, the hallmarks of human embryonic stem cells (hESC), confer these cells with the capacity to expand indefinitely while maintaining the ability to differentiate into any cell type of the human body; thus, making hESC a valuable source of functional differentiated cells suitable for applications in regenerative medicine, drug discovery, biotechnology, biopharmaceuticals and developmental biology. However, the large-scale production of clinical-grade hESC, required for such applications, has been hampered by the current culture conditions in which hESC still depend on the use of mouse embryonic fibroblast-conditioned medium (MEF-CM) for their efficient growth. Therefore, investigation of the factors provided by MEFs is of the utmost importance to discover which components of MEF-CM allow the long-term expansion of undifferentiated hESC. While considerable progress has been made on the identification of the protein components of MEF-CM, very little is known about the small molecules (metabolites) secreted by MEFs. In this context, an untargeted metabolomics method was developed for the investigation of potential bioactive metabolites present in MEF-CM implicated in the proliferation and/or maintenance of pluripotency of hESC in vitro. A metabolomics method was applied and successfully identified a number of metabolites which were later confirmed in their identities with the use of authentic standards, to be further investigated for their effect on hESC culture. Interestingly, the addition of PGE2, 6-keto-PGF1α, 9, 12, 13-TriHOME, 7-Ketocholesterol and stearidonic acid (the metabolites found in MEF-CM) to the unconditioned medium (UM), a medium incapable of the maintenance of hESC, showed a delay in apoptosis when compared to the negative control UM; thus, suggesting that these metabolites could help with the proliferation of hESC. Increasing evidence that hESC secrete factors into their microenvironment that can also help them to proliferate or to maintain an undifferentiated state prompted the application of the same metabolomics method to the analysis of hESC spent culture media. The results identified lysophospholipids (LPLs) as potential molecules mediating some biological activities; however, the precise role of these LPLs still remains to be determined. Overall, the results of this thesis are expected to impact and add knowledge to the field of stem cell biology providing useful information for the creation and development of more efficient and defined culture conditions for the propagation of hESC with the appropriate quality to realise their widespread application in clinic and other research areas

Filamentous Fungi transported by birds during migration across the mediterranean Sea.

The potential for the transport and diffusion of some pathogenic microorganisms by migratory birds is of concern. Migratory birds may be involved in the dispersal of microorganisms and may play a role of mechanical and biological vectors. The efficiency of dispersal of pathogenic microorganisms depends on a wide range of biotic and abiotic factors that influence the survival or disappearance of a given agent in a geographical area. In the present study, 349 migratory birds were captured in four sites (Mazara del Vallo, Lampedusa, Ustica and Linosa), representing the main stop-over points during spring and autumnal migration, and analyzed for the presence of filamentous fungi. A total of 2,337 filamentous fungi were isolated from 216 birds and identified by a combined phenotypic-genotypic approach to species level. Twelve species were identified in the study, with Cladosporium cladosporioides, Alternaria alternata, and Aspergillus niger as the most abundant. The transport of these fungal species isolated in this study is of considerable importance because some of these species can create dangers to human health

Metabolomics study of human embryonic stem cell culture media

Self-renewal and pluripotency, the hallmarks of human embryonic stem cells (hESC), confer these cells with the capacity to expand indefinitely while maintaining the ability to differentiate into any cell type of the human body; thus, making hESC a valuable source of functional differentiated cells suitable for applications in regenerative medicine, drug discovery, biotechnology, biopharmaceuticals and developmental biology. However, the large-scale production of clinical-grade hESC, required for such applications, has been hampered by the current culture conditions in which hESC still depend on the use of mouse embryonic fibroblast-conditioned medium (MEF-CM) for their efficient growth. Therefore, investigation of the factors provided by MEFs is of the utmost importance to discover which components of MEF-CM allow the long-term expansion of undifferentiated hESC. While considerable progress has been made on the identification of the protein components of MEF-CM, very little is known about the small molecules (metabolites) secreted by MEFs. In this context, an untargeted metabolomics method was developed for the investigation of potential bioactive metabolites present in MEF-CM implicated in the proliferation and/or maintenance of pluripotency of hESC in vitro. A metabolomics method was applied and successfully identified a number of metabolites which were later confirmed in their identities with the use of authentic standards, to be further investigated for their effect on hESC culture. Interestingly, the addition of PGE2, 6-keto-PGF1α, 9, 12, 13-TriHOME, 7-Ketocholesterol and stearidonic acid (the metabolites found in MEF-CM) to the unconditioned medium (UM), a medium incapable of the maintenance of hESC, showed a delay in apoptosis when compared to the negative control UM; thus, suggesting that these metabolites could help with the proliferation of hESC. Increasing evidence that hESC secrete factors into their microenvironment that can also help them to proliferate or to maintain an undifferentiated state prompted the application of the same metabolomics method to the analysis of hESC spent culture media. The results identified lysophospholipids (LPLs) as potential molecules mediating some biological activities; however, the precise role of these LPLs still remains to be determined. Overall, the results of this thesis are expected to impact and add knowledge to the field of stem cell biology providing useful information for the creation and development of more efficient and defined culture conditions for the propagation of hESC with the appropriate quality to realise their widespread application in clinic and other research areas

A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis.

The genomic grapevine (Vitis vinifera L.) DNA extraction is difficult because of secondary metabolites that interfere with DNA isolation procedures and subsequent applications. We developed a simple, rapid and efficient method for the extraction of genomic DNA from asymptomatic and pathogeninfected grape leaves. The protocol reported, based on a modified cetyl trimethylammonium bromide (CTAB) extraction procedure, allowed the rapid DNA extraction from little amounts of leaf material without employment of liquid nitrogen for initial tissue grinding. The protocol included polyvinylpyrrolidone (PVP) to bind phenolic compounds, β-mercaptoethanol to inhibit the oxidation of polyphenols, and a high concentration of NaCl (2.5 M) to increase the solubility of polysaccharides, thus reducing their co-precipitation with DNA. Final DNA solution did not contain polysaccharides, polyphenols and other major contaminants. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings. In addition, the quality of the DNA extracted from asymptomatic, Oidium tuckeri- and Plasmopara viticola-infected leaves of V. vinifera L. was evaluated in polymerase chain reaction (PCR) analyses by using different set of primers to be able to amplify vegetal, fungal and bacterial DNA

Cultivable microorganisms associated with honeys of different geographical and botanical origin

In this study, the composition of the cultivable microbial populations of 38 nectar honey and honeydew honey samples of different botanical and geographical origin were assessed. After growth in specific media, various colonies with different appearance were isolated and purified before phenotypic (morphological, physiological and biochemical traits) and genotypic [randomly amplified polymorphic DNA (RAPD), repetitive DNA elements-PCR (rep-PCR) and restriction fragment length polymorphism (RFLP)] differentiation. The identification was carried out by 16S rRNA gene sequencing for bacteria and, in addition to RFLP, by sequencing the D1/D2 region of the 26S rRNA gene for yeasts and the 5.8S-ITS rRNA region for filamentous fungi. The results showed the presence of 13 species of bacteria, 5 of yeasts and 17 of filamentous fungi; the species most frequently isolated were Bacillus amyloliquefaciens, Zygosaccharomyces mellis and Aspergillus niger for the three microbial groups, respectively. The highest microbial diversity was found in multifloral honeys. No correlation among the microbial species and the botanical/geographical origin was found, but some strains were highly adapted to these matrices since they were found in several samples of different origin

Shelf life evaluation of fresh-cut red chicory subjected to different minimal processes

Microbiological, chemical and physical parameters of minimally processed red chicory (Cichorium intybus L.) subjected to two different transformation processes were investigated. A classic ready-to-eat (RTE) process (P1) and a production without cutting (P2) were monitored during refrigerated (4 °C) storage (15 d). Total mesophilic microorganisms, total psychrotrophic microorganisms and pseudomonads were detected at the highest cell densities in all samples. Presumptive Pseudomonas population dominated the cultivable microbial community of RTE red chicory and were characterized genetically. Twenty-two randomly amplified polymorphic DNA (RAPD) types were investigated by 16S rRNA gene sequencing, resulting in members of Rahnella and Pseudomonas. The identification of Pseudomonas species was further determined by sequencing of gyrB, rpoB and rpoD genes resulting in 16 species. A highest visual quality and a lower weight loss and colour variation were registered for P2, while soluble solid, nitrate and ascorbic acid contents were not affected by processing and storage. The integrated microbiological, chemical and physical approach applied in this study demonstrated the longer shelf-life of P2 red chicory

Biological activity of Bacillus spp. evaluated on eggs and larvae of red palm weevil Rhynchophorus ferrugineus

This study was conducted to characterize the Bacillus populations associated with dead Rhynchophorus ferrugineus, to develop a biological control for the red palm weevil. Dead adult beetles, collected throughout Sicily, were used for isolating internal and external spore forming bacteria (SFB) microbiota. The isolates, preliminarily allotted to the Bacillaceae family, were tested at 4 concentrations (103 to 106 CFU/mL) for their ability to inhibit hatching of eggs of R. ferrugineus and were used at 106 CFU/mL to monitor their insecticidal activity against 10 day-old larvae. Total amounts of SFB measured outside the skeleton and in the inners part of the beetles were 5.59-6.94 and 5.17-7.05 Log CFU/g, respectively. Hatching was inhibited markedly by 9 isolates, representing 9 distinct strains of 7 species (Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, and Lysinibacillus sphaericus), especially by the strains B. pumilus GC43 and GC51, which exhibited lethal concentrations 50 (LC50) values of 1.60 × 103 and 9.84 × 103 CFU/mL, respectively. Among all the strains tested, only B. licheniformis CG62 exhibited significant insecticidal activity against red palm weevil larvae. The Bacillus isolates characterized and tested in this study inhibited the hatching of red palm weevils in a contact-dependent manner. Thus, these isolates can be used as a preventive rather than as a curative treatment. Keywords Bacillus, Rhynchophorus ferrugineus, hatching assays, larvae, Pal

Development of a method for the direct fermentation of semolina by selected sourdough lactic acid bacteria

Three obligately heterofermentative lactic acid bacteria (LAB) strains (Lactobacillus sanfranciscensis PON100336, Leuconostoc citreum PON10079 and Weissella cibaria PON10030) were used in this study as a multi-species starter culture for sourdough production. The starter inoculum was prepared and propagated in sterile semolina extract (SSE) broth. Acidification kinetics, microbiological counts detected on specific media for sourdough LAB, polymorphic profile comparison and species-specific PCRs evidenced a stability of the liquid inoculum over time determining its suitability for direct addition to semolina. In order to validate this innovative method for the production of durum wheat (Triticum durum Desf) sourdoughs, 15 semolinas (from ten old and five modern genotypes cultivated in Sicily, southern Italy) were used to prepare the SSEs and to produce sourdoughs and finally breads. Chemical and microbiological analyses of the sourdoughs and the evaluation of the quality parameters (weight loss, height, crumb and crust colour, image analysis and volatile organic compound generation) of the resulting breads indicated that the direct addition of the liquid inocula propagated in SSE is a valuable method to stabilise the production of sourdoughs. The differences registered during the technological characterisation of the breads were underlined by the sensory tests and the multivariate analysis and are mainly imputable to the type of semolina

Codominance of Lactobacillus plantarum and obligate heterofermentative lactic acid bacteria during sourdough fermentation

Fifteen sourdoughs produced in western Sicily (southern Italy) were analysed by classical methods for their chemico-physical characteristics and the levels of lactic acid bacteria (LAB). pH and total titratable acidity (TTA) were mostly in the range commonly reported for similar products produced in Italy, but the fermentation quotient (FQ) of the majority of samples was above 4.0, due to the low concentration of acetic acid estimated by high performance liquid chromatography (HPLC). Specific counts of LAB showed levels higher than 108CFUg-1 for many samples. The colonies representing various morphologies were isolated and, after the differentiation based on phenotypic characteristics, divided into 10 groups. The most numerous group was composed of facultative heterofermentative isolates, indicating a relevance of this bacterial group during fermentation. The genetic analysis by randomly amplified polymorphic DNA (RAPD)-PCR, 16S rRNA gene sequencing and species-specific PCRs identified 33 strains as Lactobacillus plantarum, Lactobacillus curvatus and Lactobacillus graminis. Due to the consistent presence of L.plantarum, it was concluded that this species codominates with obligate heterofermentative LAB in sourdough production in this geographical area. In order to evaluate the performances at the basis of their fitness, the 29 L.plantarum strains were investigated for several technological traits. Twelve cultures showed good acidifying abilities invitro and L.plantarum PON100148 produced the highest concentrations of organic acids. Eleven strains were positive for extracellular protease activity. Bacteriocin-like inhibitory substances (BLIS) production and antifungal activity was scored positive for several strains, included L.plantarum PON100148 which was selected as starter for experimental sourdough production. The characteristics of the sourdoughs and the resulting breads indicated that the best productions were obtained in presence of L.plantarum PON100148