ANALISI FUNZIONALE DI GENI REGOLATORI DELLO SVILUPPO EMBRIONALE DI PARACENTROTUS LIVIDUS

Parole chiave: TRIM, E3-ubiquitina ligasi, ectoderma orale, scheletogenesi, riccio di mare. La recente acquisizione di consistenti dati genomici ha confermato che organismi bilateri, semplici come i nematodi o complessi come l’uomo, fanno uso degli stessi strumenti di base, quali fattori di trascrizione e molecole segnale, per decodificare le informazioni necessarie allo sviluppo embrionale. Nel contesto di tali regolatori, la famiglia di proteine TRIM/RBCC (Tripartite motif/RING-Bbox-Coiled coil) rappresenta una delle principali classi di E3 ubiquitina ligasi putative, che svolgono ruoli essenziali nella regolazione di processi quali ciclo cellulare e sviluppo embrionale. Nel genoma di ric…

Development of an ad hoc natural whey starter culture for the production of Vastedda della valle del Belìce cheese

This work was aimed to characterize the autochthonous lactic acid bacteria (LAB) of PDO Vastedda della valle del Belìce cheeses, produced in several dairy factories, for the development of an ad hoc starter culture preparation for the production of this cheese. To this purpose, winter and spring productions were analysed, in order to isolate LAB adapted to perform the fermentation at low temperatures. Plate counts showed the total microbial counts (TMC) till levels of 9 -1almost 10 CFU g and all cheese samples were dominated by coccus LAB. Not all samples were positive for the presence of enterobacteria, but when they were found their concentrations were at similar levels in both seasons. A…

Microbial interactions in food model systems: In situ antilisterial activity of mundticin KS producing strains

It is known that microbial interactions are important for the success of food fermentations, as well as for the establishment of the safety of the final products. In this study, we characterized extensively three enterococci (WFE3, WFE20 and WFE31) of flour origin, which were found to be active against Listeria monocytogenes, in order to investigate on their ability to produce bacteriocins in complex food model systems for their future industrial/food applications. The identification of the three bacterial isolates was performed by means of a combined 16S rRNA gene sequencing and multiplex PCR approach. Two isolates belonged unequivocally to the species E. mundtii, while one isolate could n…

Identification, typing, and investigation of the dairy characteristics of lactic acid bacteria isolated from 'Vastedda della valle del Belìce' cheese

Traditional cheeses made without starter cultures can be characterised by the attribute of instability. The addition of autochthonous starter cultures can ensure stability without compromising the characteristics of the final product. This study aimed to characterise the autochthonous lactic acid bacteria (LAB) population in “Vastedda della valle del Belice” cheeses, which have a protected designation of origin (PDO) status, in order to develop an ad hoc starter culture to be used in its future production. Winter and spring productions were analysed to ensure isolation of specific LAB that had adapted to perform fermentation at low temperatures. Plate counts revealed total microbial numbers…

Influence of Citrus Essential Oils on the Microbiological, Physicochemical and Antioxidant Properties of Primosale Cheese

The aim of this study was to produce a fresh ovine pressed cheese within Pecorino “Primosale” typology with the addition of citrus essential oils (EOs). For this purpose, ewe’s pasteurized milk was added with EOs from the peel of lemons, oranges and tangerines. Seven cheese productions were performed at the pilot plant scale level, including one control production (CP) without the addition of EOs and six experimental productions obtained by the addition of two EO concentrations (100 and 200 µL/L) to milk. The acidification process was obtained by means of the starter cultures Lactococcus lactis CAG4 and PON36. All cheeses showed levels of lactic acid bacteria (LAB) a…

Le proprietà sensoriali delle birre artigianali legate ai microrganismi della fermentazione

Selected lactic acid bacteria as a hurdle to the microbial spoilage of cheese: Application on a traditional raw ewes' milk cheese

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes’ milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the nonstarter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains…

Insights Into the Cultivable Microbial Ecology of “Manna” Ash Products Extracted From Fraxinus angustifolia (Oleaceae) Trees in Sicily, Italy

Microbial communities characterizing a specific food matrix, generally, strongly contribute to both its composition, and properties for food applications. To our knowledge, this is the first study to investigate the cultivable microbial ecology of Sicilian "Manna" ash products in order to acquire new information on the hygienic quality, shelf-life and potential application of this traditional food. To this purpose, several manna samples belonging to different commercial categories were collected and subjected to the analysis of bacteria, yeasts, and filamentous fungi. Furthermore, an investigation of the sugar content and physicochemical parameters was performed. The results of our study fo…

Computational prediction of the Helitron-N2 (HeN2) transposable element in the strim1 locus of the Paracentrotus lividus genome

Transposons constitute a significant component of the eukaryotic genome. Among others, Helitrons represent a novel major class of eukaryotic transposons, and are fundamentally different from classical ones in terms of their structure and mechanism of transposition [1]. In particular, Helitrons constitute ~1% of the sea urchin genome [2]. By a in silico approach focused on the genome of the Mediterranean sea urchin Paracentrotus lividus, we have predicted regions of high sequence identity to a Helitron-N2 (HeN2) element in the strim1 locus. Of interest, HeN2 sequences lies within the 5’ non coding region of the strim1a and strim1b genes, spanning from -105 to -2255 (the start codon ATG of st…

Spatially restricted expression of strim, a novel TRIM-containing gene, in the oral ectoderm of the developing sea urchin embryo Paracentrotus lividus.

The recent wealth of genomic data has confirmed that bilaterians as simple as nematodes and as complex as humans use the same basic toolkit of transcription factors and signaling molecules to process spatial and temporal information during development (1,2). Among these regulators, the TRIM/RBCC (Tripartite motif/RING-Bbox-Coiled Coil) family represents one of the largest classes of putative E3 ubiquitin ligases that play essential roles in cell cycle regulation and development (3). It consists of rapidly evolving genes, identified in eukaryotic organisms (4,5). In the sea urchin Paracentrotus lividus we have identified a locus containing two copies, probably originated from a duplication e…

Localized expression of Strim1, a novel member of the TRIM-containing family, guides the skeletal morphogenetic program of the sea urchin embryo

The building of the skeleton in the indirect developing sea urchin embryo is a complex morphogenetic process that is executed by the Primary Mesenchyme Cells or PMCs (Ettensohn et al, 1997; Wilt 2002). It is well known that the PMCs acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth (Armstrong 1993; Cavalieri et al, 2003; Röttinger et al, 2008). In this study, we analyze the function of a novel gene, encoding for a tripartite motif-containing (TRIM) protein denoted strim1, that adds up to the list of genes constituting the epithelial-mesenchymal signaling network. We show that strim1 is expressed in ectoderm regions adjacen…

A large factory-scale application of selected autochthonous lactic acid bacteria for PDO Pecorino Siciliano cheese production

The main hypothesis of this study was that the autochthonous lactic acid bacteria (LAB) selected for their dairy traits are able to stabilize the production of PDO (Protected Denomination of Origin) Pecorino Siciliano cheese, preserving its typicality. The experimental plan included the application of a multi-strain lactic acid bacteria (LAB) culture, composed of starter (Lactococcus lactis subsp. lactis CAG4 and CAG37) and non starter (Enterococcus faecalis PSL71, Lactococcus garviae PSL67 and Streptococcus macedonicus PSL72) strains, during the traditional production of cheese at large scale level in six factories located in different areas of Sicily. The cheese making processes were foll…

Characterization of kefir-like beverages produced from vegetable juices

The aim of this work was to develop new non-dairy fermented beverages using vegetable juices as fermentable substrates. Carrot, fennel, melon, onion, tomato and strawberry juices underwent back-slopping fermentations, carried out by water kefir microorganisms. Results indicated that lactic acid bacteria and yeasts were capable of growing in the juices tested. Melon juice registered the highest numbers of microorganisms. Almost all juices underwent a lactic fermentation. After fermentation, there was observance of a decrease of the soluble solid content and an increase of the number of volatile organic compounds. In particular, esters were present in high amounts after the fermentation, espe…

Genome-wide analysis of the repertoire of TRIM genes in sea urchins

The eukaryotic TRIM (TRIpartite Motif) super-family represents one of the largest classes of putative E3 ubiquitin ligases involved in several processes, including epigenetic control of development and disease. In the post-genomic era, new approaches allow genome-wide studies of gene family. In particular, we performed a comprehensive analysis of the TRIM repertoire in selected sea urchin species. By combining iterations of ab initio predictions and pairwise comparative methods, we first retrieved the full complement of TRIM genes in Strongylocentrotus purpuratus, whose full genome sequence was available. Interestingly, such a DNA sequence set includes not previously classified, echinoderm-…

Spoilage potential of Brettanomyces bruxellensis strains isolated from Italian wines

Abstract Brettanomyces bruxellensis is an important wine spoilage agent. In this study a population of Brettanomyces strains isolated from Italian wines was thoroughly investigated to evaluate adaptability to wine conditions and spoilage potential. The presumptive isolates of Brettanomyces were identified at species level with 26S rRNA gene sequencing and species-specific PCR, and subsequently subjected to analysis of intra-species variability through the study of intron splice sites (ISS-PCR). Although, some strains were tracked in wines from different regions, extensive genetic biodiversity was observed within the B. bruxellensis population investigated. All strains were evaluated for the…

Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread

The addition of active compounds to enhance the functional properties of foods is a quite common practice. Recently, bread became one of the target foods to incorporate functional ingredients such as those deriving from Opuntia spp. So far, only Opuntia ficus-indica cladodes in powder has been tested. The addition of fresh O. ficus-indica mucilage (in substitution to water) did not influence the biological leavening of the doughs. The resulting breads showed a biological role of the cactus mucilage, because their antioxidant activity was higher than that of control wheat bread. The sensory analysis indicated a general appreciation of the breads enriched with O. ficus-indica mucilage by the …

A survey of the main technology, biochemical and microbiological features influencing the concentration of biogenic amines of twenty Apulian and Sicilian (Southern Italy) cheeses

Abstract Twenty Apulian and Sicilian cheeses were analysed for their concentrations of eight biogenic amines (BAs), free amino acids, pH, water activity, and subjected to microbiological characterisation. In addition, lactic acid bacteria isolated from cheeses were assayed for their capacity to generate BAs. Principal component analysis was performed to find the effect of different parameters on the distribution of the cheeses. Although short-ripened (≤30 d) cheeses did not show significant BA concentrations, the only BA showing high positive correlation with time of ripening was histamine. Concentration of histidine and, especially, percentage of histidine-decarboxylase bacteria presumably…

In silico identification of the TRIM gene complement of the sea urchin and computational prediction of Helitron-N2 transposable element in the 5’ non-coding region of two strim1 genes

Development of new non-dairy beverages from Mediterranean fruit juices fermented with water kefir microorganisms

The aim of this work was to explore the use of several Mediterranean fruit juices as fermentable substrates to develop new non-dairy fermented beverages. Microbiological, chemical and sensory features of kefir-like beverages obtained after the fermentation of juices extracted from fruits cultivated in Sicily (southern Italy) with water kefir microorganisms were investigated. Results indicated that both lactic acid bacteria and yeasts were able to develop in the fruit juices tested, but the highest levels were registered with prickly pear fruit juice. All fruit juices underwent a lactic fermentation, since a lactic acid content was detected in the resulting kefir-like beverages. Except kiwif…

Ectoderm restricted expression of the TRIM-containing factor Strim1 directs the migration, aggregation and terminal differentiation of mesenchymal cells during skeletal morphogenesis of the sea urchin embryo

Biodiversity and oenological attitude of Saccharomyces cerevisiae strains isolated in the Montalcino district: biodiversity of S. cerevisiae strains of Montalcino wines.

ABSTRACT The biodiversity of Saccharomyces cerevisiae was studied in the Montalcino area (Italy). Two wineries were involved in the study, which compared the genotypic and oenological characteristics of the S. cerevisiae strains isolated in spontaneous fermentations. After isolation yeasts were identified by 26S rRNA gene sequence analysis, and S. cerevisiae strains were characterized through interdelta sequence analysis (ISA). Oenological tests were performed in synthetic grape must by varying the magnitude of the main wine-imiting factors. The evolution of alcoholic fermentation was monitored by measuring sugar consumption and flow cytometry. The results revealed the prevalence of S. cere…

Selection of amine-oxidizing dairy lactic acid bacteria and identification of the enzyme and gene involved in the decrease of biogenic amines

ABSTRACT Accumulation of biogenic amines (BAs) in cheese and other foods is a matter of public health concern. The aim of this study was to identify the enzyme activities responsible for BA degradation in lactic acid bacteria which were previously isolated from traditional Sicilian and Apulian cheeses. The selected strains would control the concentration of BAs during cheese manufacture. First, 431 isolates not showing genes encoding the decarboxylases responsible for BA formation were selected using PCR-based methods. Ninety-four out of the 431 isolates degraded BAs (2-phenylethylamine, cadaverine, histamine, putrescine, spermine, spermidine, tyramine, or tryptamine) during cultivation on …

The sea urchin histone H2A enhancer-binding protein MBF-1 is needed for maximal expression also for the H3 gene, while is buffered by the sns5 insulator on its interaction with the downstream H1 promoter.

Enhancers are DNA elements which increase the transcription of associated gene in a position and distance independent manner relative to the transcription initiation site. Molecular mechanisms must operate to direct enhancers to specific promoters in complex genetic loci. The sea urchin a-histone genes are organized in several hundred tandem repeated units, each containing one copy of the five histone genes in the order 5’-H4-H2B-H3-H2A-H1-3’. Transcription is limited to the early cleavage and reaches its maximum at morula stage. After hatching these genes are repressed and maintained in the silenced state for whole life cycle of the animal. In Paracentrotus lividus, the MBF-1 activator bin…

Microbial Ecology of Retail Ready-to-Eat Escarole and Red Chicory Sold in Palermo City, Italy

Background: Ready-To-Eat (RTE) foods include any edible food that is commonly consumed raw. This study aimed at evaluation of microbial ecology of retail RTE escarole and red chicory sold in Palermo city, Italy. Methods: A total of 32 mono-varietal RTE samples, including escarole (n=16) and red chicory (n=16) samples were obtained from Palermo, Italy. Both RTE vegetables at expiry date were analyzed to quantify spoilage bacteria, pathogenic bacteria, and yeast. All different colonies were isolated and identified on the basis of phenotypic characteristics and genetic polymorphisms by random amplification of polymorphic DNA-Polymerase Chain Reaction (PCR) and further genotype by sequencing th…

Enhancer, chromatin insulator, non-coding RNA and α-histone gene expression during embryogenesis of the sea urchin Paracentrotus lividus.

Core promoters and chromatin insulators (ins) may direct a transcriptional enhancer (enh) to prefer a specific promoter in complex genetic loci. Enh and ins flank the sea urchin Paracentrotus lividus α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. In vivo competition assays of enh and ins functions reveal that the H2A enh-bound MBF-1 activator participates also in the expression of the H3 gene and that the sns5 ins buffers the downstream H1 promoter from the H2A enh. These results suggest that both the H2A enh and the sns5 ins may account for the diverse accumulation of the linker vs core nucleosomal histones during early development of the s…

Production, stability, gene sequencing and in situ anti-Listeria activity of mundticin KS expressed by three Enterococcus mundtii strains

Three enterococci (WFE3, WFE20 and WFE31) selected as presumptive bacteriocin producers were found to be active against Listeria monocytogenes. In this study, due to their potential industrial/food applications, the three bacterial isolates were extensively characterized. Identification was performed by means of a combined 16S rRNA gene sequencing and multiplex PCR approach, and was confirmed with the sequencing of a partial region of a protein-encoding gene, namely pheS. The three isolates belonged unequivocally to the species Enterococcus mundtii. The randomly amplified polymorphic DNA (RAPD) analysis recognized three distinct strains. The supernatants were mainly active against Listeria …

Addition of selected starter/non-starter lactic acid bacterial inoculums to stabilise PDO Pecorino Siciliano cheese production

Abstract The present study was carried out to produce Protected Denomination of Origin (PDO) Pecorino Siciliano cheese with a multi-species lactic acid bacteria (LAB) culture, composed of starter and non-starter strains in order to reduce the microbiological variability of the products derived without LAB inoculums. To this end, cheese samples produced in six factories located in five provinces (Agrigento, Catania, Enna, Palermo and Trapani) of Sicily, and previously characterised for physicochemical, microbiological and sensory aspects, have been investigated in this work for bacterial microbiome, fatty acid (FA) composition as well as volatile organic compound (VOC) profiles. Analysis of …

Anti-Listeria activity of citrus essential oils in a fresh ovine cheese

The aim of this study was to evaluate the anti-Listeria activity of essential oils (EOs) extracted from the peel of lemons, oranges and tangerines in a fresh cheese produced with pasteurized ewe’s milk. Four cheesemaking trials were performed at the pilot plant scale level, including one control production without the addition of EOs and three experimental productions obtained by the addition of 200 μL/L of each EOs to milk. Before the addition of EOs, the milk of all trials was inoculated with 107 CFU/mL of the starter cultures (Lactococcus lactis NT1 and NT5) and 104 CFU/mL of pathogenic bacteria (Listeria monocytogenes ATCC19114). Plate counts confirmed the dominance of the starter cultu…

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere tran…

Valorisation of Dairy Wastes Through Kefir Grain Production

The main aim of this work was to investigate on kefir grains (KGs) increase using different dairy by-products. To this purpose, whey and deproteinized whey from cow, goat and sheep milk were compared to the pasteurized milk of the corresponding animal species. Each substrate was inoculated with 3% (w/v) of KGs cultivated in ultra-high temperature cow milk and evalu- ated for pH decrease, total titratable acidity increase and development of lactic acid bacteria (LAB) and yeasts released in the matrices after 24 h incubation at three different temperatures (20, 25 and 30 °C). The genotypic characterization of the dominating microbial populations resulted in the identification of two LAB belon…

Microbiological, chemical and sensory aspects of bread supplemented with different percentages of the culinary mushroom Pleurotus eryngii in powder form

Pleurotus eryngii (DC.) Quél. powder was used in bread production. Three dough trials (0, 5 and 10% of mushroom) were obtained with commercial baker's yeast. P. eryngii powder was first tested against several yeast species; 10% P. eryngii trial was characterised by the highest pH and total titratable acidity. P. eryngii did not influence negatively the fermentation process, since all trials reached yeast levels of 10 8 CFU g −1 . Mushroom powder decreased bread height and softness, increased crust redness and crumb void fraction and cell density and, although the breads were scored diverse, the overall assessment was comparable. The final breads provided higher concentrations of thiamin, ri…

Yeast ecology of vineyards within Marsala wine area (western Sicily) in two consecutive vintages and selection of autochthonous Saccharomyces cerevisiae strains.

In this work, the yeast ecology associated with the spontaneous fermentation of Grillo cultivar grapes from 10 vineyards was analyzed from grape harvest till complete consumption of must sugars. The microbiological investigation started with the plate count onto two culture media to distinguish total yeasts (TY) and presumptive Saccharomyces (PS). Yeasts were randomly isolated and identified by a combined genotypic approach consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 14 species belonging to 10 genera. The distribution of the yeasts within the vineyards s…