Extension of the shelf-life of fresh pasta using modified atmosphere packaging and bioprotective cultures.

Microbial stability of fresh pasta depends on heat treatment, storage temperature, proper preservatives, and atmosphere packaging. This study aimed at improving the microbial quality, safety, and shelf life of fresh pasta using modified atmosphere composition and packaging with or without the addition of bioprotective cultures (Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium spp., and Bacillus coagulans) into semolina. Three fresh pasta variants were made using (i) the traditional protocol (control), MAP (20:80 CO2:N2), and barrier packaging, (ii) the experimental MAP (40:60 CO2:N2) and barrier packaging, and (iii) the experimental MAP, barrier packaging, and bioprotective cultures. Their effects on physicochemical properties (i.e., content on macro elements, water activity, headspace O2, CO2 concentrations, and mycotoxins), microbiological patterns, protein, and volatile organic compounds (VOC) were investigated at the beginning and the end of the actual or extended shelf-life through traditional and multi-omics approaches. We showed that the gas composition and properties of the packaging material tested in the experimental MAP system, with or without bioprotective cultures, positively affect features of fresh pasta avoiding changes in their main chemical properties, allowing for a storage longer than 120 days under refrigerated conditions. These results support that, although bioprotective cultures were not all able to grow in tested conditions, they can control the spoilage and the associated food-borne microbiota in fresh pasta during storage by their antimicrobials and/or fermentation products synergically. The VOC profiling, based on gas-chromatography mass-spectrometry (GC-MS), highlighted significant differences affected by the different manufacturing and packaging of samples. Therefore, the use of the proposed MAP system and the addition of bioprotective cultures can be considered an industrial helpful strategy to reduce the quality loss during refrigerated storage and to increase the shelf life of fresh pasta for additional 30 days by allowing the economic and environmental benefits spurring innovation in existing production models.

Building essential biodiversity variables (EBVs) of species distribution and abundance at a global scale.

Much biodiversity data is collected worldwide, but it remains challenging to assemble the scattered knowledge for assessing biodiversity status and trends. The concept of Essential Biodiversity Variables (EBVs) was introduced to structure biodiversity monitoring globally, and to harmonize and standardize biodiversity data from disparate sources to capture a minimum set of critical variables required to study, report and manage biodiversity change. Here, we assess the challenges of a 'Big Data' approach to building global EBV data products across taxa and spatiotemporal scales, focusing on species distribution and abundance. The majority of currently available data on species distributions derives from incidentally reported observations or from surveys where presence-only or presence-absence data are sampled repeatedly with standardized protocols. Most abundance data come from opportunistic population counts or from population time series using standardized protocols (e.g. repeated surveys of the same population from single or multiple sites). Enormous complexity exists in integrating these heterogeneous, multi-source data sets across space, time, taxa and different sampling methods. Integration of such data into global EBV data products requires correcting biases introduced by imperfect detection and varying sampling effort, dealing with different spatial resolution and extents, harmonizing measurement units from different data sources or sampling methods, applying statistical tools and models for spatial inter- or extrapolation, and quantifying sources of uncertainty and errors in data and models. To support the development of EBVs by the Group on Earth Observations Biodiversity Observation Network (GEO BON), we identify 11 key workflow steps that will operationalize the process of building EBV data products within and across research infrastructures worldwide. These workflow steps take multiple sequential activities into account, including identification and aggregation of various raw data sources, data quality control, taxonomic name matching and statistical modelling of integrated data. We illustrate these steps with concrete examples from existing citizen science and professional monitoring projects, including eBird, the Tropical Ecology Assessment and Monitoring network, the Living Planet Index and the Baltic Sea zooplankton monitoring. The identified workflow steps are applicable to both terrestrial and aquatic systems and a broad range of spatial, temporal and taxonomic scales. They depend on clear, findable and accessible metadata, and we provide an overview of current data and metadata standards. Several challenges remain to be solved for building global EBV data products: (i) developing tools and models for combining heterogeneous, multi-source data sets and filling data gaps in geographic, temporal and taxonomic coverage, (ii) integrating emerging methods and technologies for data collection such as citizen science, sensor networks, DNA-based techniques and satellite remote sensing, (iii) solving major technical issues related to data product structure, data storage, execution of workflows and the production process/cycle as well as approaching technical interoperability among research infrastructures, (iv) allowing semantic interoperability by developing and adopting standards and tools for capturing consistent data and metadata, and (v) ensuring legal interoperability by endorsing open data or data that are free from restrictions on use, modification and sharing. Addressing these challenges is critical for biodiversity research and for assessing progress towards conservation policy targets and sustainable development goals.

Development of a Synbiotic Beverage Enriched with Bifidobacteria Strains and Fortified with Whey Proteins.

The objective of this study was to develop a new synbiotic beverage evaluating the ability of some bifidobacteria strains to grow in this beverage which was fortified with whey proteins up to 20 g L-1, and enriched with 10 g L-1 of prebiotic inulin or resistant starch. The ability of Bifidobacterium strains to survive for 30 days at 4°C was evaluated in two synbiotic whey protein fortified beverages formulated with 2% of whey proteins and 1% of inulin or resistant starch. Microbial growth was significantly affected by the whey protein amount as well as by the kind of prebiotic fiber. Resistant starch promoted the growth of the Bifidobacterium pseudocatenulatum strain and its viability under cold storage, also conferring higher sensory scores. The development of this new functional beverage will allow to carry out in vivo trials in order to validate its pre- and probiotic effects.

BioVeL: a virtual laboratory for data analysis and modelling in biodiversity science and ecology.

BACKGROUND: Making forecasts about biodiversity and giving support to policy relies increasingly on large collections of data held electronically, and on substantial computational capability and capacity to analyse, model, simulate and predict using such data. However, the physically distributed nature of data resources and of expertise in advanced analytical tools creates many challenges for the modern scientist. Across the wider biological sciences, presenting such capabilities on the Internet (as "Web services") and using scientific workflow systems to compose them for particular tasks is a practical way to carry out robust "in silico" science. However, use of this approach in biodiversity science and ecology has thus far been quite limited. RESULTS: BioVeL is a virtual laboratory for data analysis and modelling in biodiversity science and ecology, freely accessible via the Internet. BioVeL includes functions for accessing and analysing data through curated Web services; for performing complex in silico analysis through exposure of R programs, workflows, and batch processing functions; for on-line collaboration through sharing of workflows and workflow runs; for experiment documentation through reproducibility and repeatability; and for computational support via seamless connections to supporting computing infrastructures. We developed and improved more than 60 Web services with significant potential in many different kinds of data analysis and modelling tasks. We composed reusable workflows using these Web services, also incorporating R programs. Deploying these tools into an easy-to-use and accessible 'virtual laboratory', free via the Internet, we applied the workflows in several diverse case studies. We opened the virtual laboratory for public use and through a programme of external engagement we actively encouraged scientists and third party application and tool developers to try out the services and contribute to the activity. CONCLUSIONS: Our work shows we can deliver an operational, scalable and flexible Internet-based virtual laboratory to meet new demands for data processing and analysis in biodiversity science and ecology. In particular, we have successfully integrated existing and popular tools and practices from different scientific disciplines to be used in biodiversity and ecological research.

Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum.

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 µg of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 µg of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system.

Changes in morphology, cell wall composition and soluble proteome in Rhodobacter sphaeroides cells exposed to chromate.

The response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants.

Genome walking in eukaryotes.

Genome walking is a molecular procedure for the direct identification of nucleotide sequences from purified genomes. The only requirement is the availability of a known nucleotide sequence from which to start. Several genome walking methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies, including the recent coupling with next generation sequencing technologies. This review focuses on the use of genome walking strategies in several aspects of the study of eukaryotic genomes. In a first part, the analysis of the numerous strategies available is reported. The technical aspects involved in genome walking are particularly intriguing, also because they represent the synthesis of the talent, the fantasy and the intelligence of several scientists. Applications in which genome walking can be employed are systematically examined in the second part of the review, showing the large potentiality of this technique, including not only the simple identification of nucleotide sequences but also the analysis of large collections of mutants obtained from the insertion of DNA of viral origin, transposons and transfer DNA (T-DNA) constructs. The enormous amount of data obtained indicates that genome walking, with its large range of applicability, multiplicity of strategies and recent developments, will continue to have much to offer for the rapid identification of unknown sequences in several fields of genomic research.

The photosynthetic membrane proteome of Rhodobacter sphaeroides R-26.1 exposed to cobalt.

Cells of the carotenoidless strain R-26.1 of Rhodobacter sphaeroides were grown in the presence of a high concentration (5 mM) of cobalt ions. The photosynthetic intracytoplasmic membranes were isolated and investigated by proteomic analysis using non-denaturating blue native electrophoresis in combination with LC-ESI-MS/MS. Comparison with intracytoplasmic membranes of cells grown under control conditions showed a change in the relative amount of proteins belonging to the photosynthetic apparatus, with net downregulation of light-harvesting complexes and increased concentration of the nude reaction center (RC), as well as upregulation of enzymes related to chemoorganotrophy. These effects represent possible bacterial adaptation so as to retrieve energy for metabolic processes from sources alternative to less efficient photosynthesis. The influence of cobalt on the photochemistry of the RC in cell extracts was also investigated by charge recombination. The kinetics of the charge recombination reaction was found to be slower in extracts from cells exposed to Co(2+), indicating that the reorganization of the photosynthetic apparatus also involves its photochemical core.

Cystatins, serpins and other families of protease inhibitors in plants.

Plant protease inhibitors (PIs) are generally small proteins present in high concentrations in storage tissues (tubers and seeds), and to a lower level in leaves. Even if most of them are active against serine and cysteine proteases, PIs active against aspartic proteases and carboxypeptidases have also been identified. Inhibitors of serine proteases are further classifiable in several families on the basis of their structural features. They comprise the families known as Bowman-Birk, Kunitz, Potato I and Potato II, which are the subject of review articles included in this special issue. In the present article we aim to give an overview of other families of plant PIs, active either against serine proteases or other class of proteases, describing their distribution, activity and main structural characteristics.

PlantPIs--an interactive web resource on plant protease inhibitors.

PlantPIs is a web querying system for a database collection of plant protease inhibitors data. Protease inhibitors in plants are naturally occurring proteins that inhibit the function of endogenous and exogenous proteases. In this paper the design and development of a web framework providing a clear and very flexible way of querying plant protease inhibitors data is reported. The web resource is based on a relational database, containing data of plants protease inhibitors publicly accessible, and a graphical user interface providing all the necessary browsing tools, including a data exporting function. PlantPIs contains information extracted principally from MEROPS database, filtered, annotated and compared with data stored in other protein and gene public databases, using both automated techniques and domain expert evaluations. The data are organized to allow a flexible and easy way to access stored information. The database is accessible at http://www.plantpis.ba.itb.cnr.it/.

Assessment of an internal reference gene in Rhodobacter sphaeroides grown under cobalt exposure.

Aim of this study is the identification of an appropriate internal reference gene to quantify gene transcripts isolated from Rhodobacter (R.) sphaeroides cells grown in presence of high concentrations of cobalt ions. RNA was isolated using a commercial kit protocol ad-hoc modified. Several primer pairs were used to perform reverse transcription PCR and real-time PCR to assess the suitable internal reference gene whose expression is not affected by cobalt ions, identified with the gene rsp0154.This finding can be of definite help in the investigation of the response to heavy metals of the chosen strain, a potential candidate for environmental applications.

Identification and characterization of protease inhibitors in Diplotaxis species.

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.

Production of active angiotensin-I converting enzyme inhibitory peptides derived from bovine beta-casein by recombinant DNA technologies.

A wide range of anti-hypertensive peptides potentially able to lower blood pressure through the inhibition of vasoactive enzymes such as angiotensin-I converting enzyme (ACE) are known. Currently, ACE-inhibitory peptides can be produced from precursor proteins via enzymatic hydrolysis by proteolytic enzymes, or food fermentation with proteolytic starter cultures. These approaches are neither selective nor easy. In this study a novel procedure has been developed, based on recombinant DNA technologies, for the production of highly purified fractions of three polypeptides derived from bovine beta-casein active as ACE inhibitors in vitro. The procedure includes peptide expression in Escherichia coli cells as recombinant fusion proteins, purification by affinity chromatography, cleavage by proteinase from a selected strain of Lactobacillus helveticus and isolation of bioactive peptides (BPs). The reported concentration of inhibitor needed to reduce at 50% ACE activity (IC(50)) values for single BP calculated in inhibiting the ACE enzyme gave results in agreement with the same parameters available in literature for other milk-derived BPs. This procedure could be used to obtain quantities of pure peptides to determine their interactions with ACE, with the aim of designing peptides that have stronger inhibitory properties and exhibit new pharmacological profiles. Moreover, its scale up would be of commercial interest for the production of functional foods, e.g., milk beverages with blood pressure-lowering effects.

One of the three proteinase inhibitor genes newly identified in the Brassica napus genome codes for an inhibitor of glutamyl endopeptidase.

Three proteinase inhibitor genes have been identified in the rapeseed (Brassica napus) genome. They are highly homologous to other genes of the mustard inhibitor (MSI) family of proteinase inhibitors characteristic of Cruciferae. In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti-2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI-2 protein strongly inhibits (Ki=44 nM) a glutamyl proteinase from Streptomyces griseus.