DNA-based approaches for dairy products authentication: A review and perspectives

Background Food fraud has become an increasingly worldwide problem mainly driven by rapid innovation in the food sector and constantly changing consumer's choices. This led to an increased necessity to improve/establish reliable authentication processes, resulting in the replacement of protein-based techniques for dairy products authentication by higher sensitive and reproducible DNA-based methods. Most used molecular methods for dairy products authentication include PCR, Real-time PCR, multiplex PCR, and PCR-RFLP. Despite the several molecular methods available for species/breeds differentiation in dairy products, there is a need for the development of more efficient and reliable molecular tools. Scope and approach In this review, traditional and more recent DNA-based methods for dairy products authentication are discussed and analysed. Moreover, the increasingly important role of bioinformatic tools for analysis of large amount of data and for the development of DNA markers is also discussed. Key findings and conclusions DNA quality is one of the major factors affecting molecular-based dairy products authentication, which can be influenced by the manufacturing process, extraction method employed, chemical composition of the food matrix, among others. PCR-based methods continue to be the most important and successfully used for dairy products authentication. The DNA markers chosen for species/breed detection are an important factor for PCR success. Although there are several molecular methods for the detection of adulterant species, there is still an unmet demand for methods to detect adulterant breeds. Available public databases and bioinformatics revolutionized the analysis of large amount of data and will be pivotal for the development of effective DNA markers.This work was supported by the ProDOP Serra da Estrela (PDR2020-101-032096) funded by PDR2020 - Programa de Desenvolvimento Rural 2014–2020, Portugal 2020, European Agricultural Fund For Rural Development (EAFRD), and the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 and the MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to JTC).info:eu-repo/semantics/publishedVersio

Evaluation of yeast surface display of the endolysin Ply511 against Listeria monocytogenes

Yeast surface display, also known as yeast display, is a protein engineering technique that uses the expression of recombinant proteins of interest, fused with native cell wall proteins of yeast. Ply511 endolysin is a cell wall hydrolase with N-acetylmuramoyl-L-alanine amidase activity. Ply511 is naturally produced by the Listeria phage A511 to lyse bacterial cells and has been proposed as an alternative to antibiotics. Listeria monocytogenes is a Gram-positive pathogen causative of human infections, resulting in febrile gastroenteritis, perinatal infection, and central nervous system infections. These infections are frequently acquired through the ingestion of contaminated food. In our study, we employed CRISPR-Cas9 to genetically modify Saccharomyces cerevisiae to display the listeria endolysin Ply511 on its surface. Flow cytometer analyses confirmed the expression of the genetic construction integrated in the recombinant yeast. Also, the enzymatic peptidoglycan-degrading activity of the engineered yeast was confirmed by using heat-killed L. monocytogenes cells. In killing assays, the recombinant yeast did not show CFU reduction of Listeria in comparison with the wild-type. These results suggest that yeast display of endolysins needs to be improved to be used as an antimicrobial strategy in the context of engineered probiotics, to assure that bacterial killing is achieved.info:eu-repo/semantics/publishedVersio

Biotechnological production of xylitol: engineering industrial Saccharomyces cerevisiae for valorization of lignocellulosic biomass

The use of renewable biomass, such as lignocellulosic materials, for the production of biofuels and chemicals within a biorefinery scheme contributes to achieve a sustainable development. Xylitol has been identified as one of the top 12 value added chemicals to be obtained from biomass, and can be produced from hemicellulose-derived xylose through biotechnological processes [1]. In this work, xylitol was produced from xylose (using glucose as co-substrate for co-factor regeneration) in batch fermentations by the industrial Saccharomyces cerevisiae PE-2 strain (over)expressing (1) a wild type xylose reductase from Pichia spititis (XR); (2) a NADH-preferable xylose reductase mutant (mut-XR) from Pichia spititis and (3) the endogenous GRE3 gene which encodes for an unspecific aldose reductase (AR). Maximum yield (0.98 g g-1) was obtained by the strain overexpressing the GRE3 gene. Moreover, the recombinant strain PE-2-GRE3 showed significantly higher xylitol productivity than the laboratory strain, CENPK.113-5D overexpressing the same gene. This strain (PE-2-GRE3) was selected for bioconversion of 160 g L-1 of xylose in a fed-batch fermentation, which resulted in 149 g L-1 of xylitol concentration with a productivity of 1.2 g L-1 h-1. These results open new perspectives and opportunities for the valorisation of hemicellulosic hydrolysates through the production of xylitol within a biorefinery concept.info:eu-repo/semantics/publishedVersio

Multi-feedstock biorefinery concept: Valorization of winery wastes by engineered yeast

The wine industry produces significant amounts of by-products and residues that are not properly managed, posing an environmental problem. Grape must surplus, vine shoots, and wine lees have the potential to be used as renewable resources for the production of energy and chemicals. Metabolic engineering efforts have established Saccharomyces cerevisiae as an efficient microbial cell factory for biorefineries. Current biorefineries designed for producing multiple products often rely on just one feedstock, but the bioeconomy would clearly benefit if these biorefineries could efficiently convert multiple feedstocks. Moreover, to reduce the environmental impact of fossil fuel consumption and maximize production economics, a biorefinery should be capable to supplement the manufacture of biofuel with the production of high-value products. This study proposes an integrated approach for the valorization of diverse wastes resulting from winemaking processes through the biosynthesis of xylitol and ethanol. Using genetically modified S. cerevisiae strains, the xylose-rich hemicellulosic fraction of hydrothermally pretreated vine shoots was converted into xylitol, and the cellulosic fraction was used to produce bioethanol. In addition, grape must, enriched in sugars, was efficiently used as a low-cost source for yeast propagation. The production of xylitol was optimized, in a Simultaneous Saccharification and Fermentation process configuration, by adjusting the inoculum size and enzyme loading. Furthermore, a yeast strain displaying cellulases in the cell surface was applied for the production of bioethanol from the glucan-rich cellulosic. With the addition of grape must and/or wine lees, high ethanol concentrations were reached, which are crucial for the economic feasibility of distillation. This integrated multi-feedstock valorization provides a synergistic alternative for converting a range of winery wastes and by-products into biofuel and an added-value chemical while decreasing waste released to the environment.info:eu-repo/semantics/publishedVersio

Integrated approach for selecting efficient Saccharomyces cerevisiae for industrial lignocellulosic fermentations: importance of yeast chassis linked to process conditions

In this work, four robust yeast chassis isolated from industrial environments were engineered with the same xylose metabolic pathway. The recombinant strains were physiologically characterized in synthetic xylose and xylose-glucose medium, on non-detoxified hemicellulosic hydrolysates of fast-growing hardwoods (Eucalyptus and Paulownia) and agricultural residues (corn cob and wheat straw) and on Eucalyptus hydrolysate at different temperatures. Results show that the co-consumption of xylose-glucose was dependent on the yeast background. Moreover, heterogeneous results were obtained among different hydrolysates and temperatures for each individual strain pointing to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process.The authors acknowledge the financial support from the Strategic Project of UID/BIO/04469/2013 CEB Unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte

Xylose fermentation efficiency of industrial Saccharomyces cerevisiae yeast with separate or combined xylose reductase/xylitol dehydrogenase and xylose isomerase pathways

Xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have been extensively used to confer xylose assimilation capacity to Saccharomyces cerevisiae and tackle one of the major bottlenecks in the attainment of economically viable lignocellulosic ethanol production. Nevertheless, there is a lack of studies comparing the efficiency of those pathways both separately and combined. In this work, the XI and/or XR/XDH pathways were introduced into two robust industrial S. cerevisiae strains, evaluated in synthetic media and corn cob hemicellulosic hydrolysate and the results were correlated with the differential enzyme activities found in the xylose-pathway engineered strains.This study was supported by the Portuguese Foundation for Science and Technology (FCT, Portugal) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to Joana T. Cunha), the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte, the MultiBiorefinery project (POCI-01–0145FEDER-016403) and the Biomass and Bioenergy Research Infrastructure (PINFRA/22059/2016).info:eu-repo/semantics/publishedVersio

Towards a cost-effective bioethanol process: yeast development to overcome challenges derived from lignocellulosic processing

The use of renewable biomass to supply the increasing energetic needs and to partially replace fossil fuels is nowadays recognized as a suitable and desirable alternative to attain a sustainable growth based on a bioeconomy. In spite of the intensive research on lignocellulose-to-ethanol production processes, second generation (2G) bioethanol is still not cost competitive and specific challenges remain. These processes are as a whole substantially more complex than initially thought as several types of biomass may be used as substrate, each with specific challenges. A range of biomass pre-treatments may be applied for biomass fractionation, each with its own specificities and leading to different inhibitor profiles. Finally, different hydrolysis/fermentation schemes may be used. The optimization of these processes has to consider the integration of all the stages of the process and should be done together. One of the key aspects for the development of cost-effective lignocellulose-to-bioethanol processes is the engineering of the yeast strain. There is still a lack of robust and sugars-fast fermentation yeast strains for 2G bioethanol. Recently, we have screened and selected naturally robust yeast strains from industrial environments[1] and engineering some of the more promising strains with the xylose metabolic pathway[2] and inhibitor tolerance genes[3]. We will present the results of the simultaneous engineering of xylose metabolization pathway together with inhibitor tolerance in diverse robust background strains and its performance in different hydrolysates obtained from distinct types of biomass. The heterogeneous outcome of the genetic engineering in different hydrolysates show that tolerance and xylose engineering must be customized to the strain background and hydrolysate used in the process. The results obtained highlight that yeast development must not only be integrated in the process but it must also be tailor-made for each specific process. [1]Pereira et al.(2014)Bioresource Technology 161:192-199. [2]Romani et al.(2015)Bioresource Technology 179:150-158. [3]Cunha et al.(2015)Bioresource Technology 191:7-16

Atypical form of acute myocardial infarction with tamponade

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2019.Background: Nowadays it is well recognized that the absence of obstructive coronary artery disease in a patient presenting with symptoms suggestive of ischemia and ST-segment alterations does not preclude an atherothrombotic etiology. CMR is an essential method for the investigation of Myocardial infarction (MI) with non obstructive coronary artery disease (MINOCA). Clinical Case: A 66 years-old female patient was referred after an episode of acute oppressive chest pain, nausea and hypersudorese, followed by syncope. She had a previous medical history of rheumatoid arthritis, under immunosuppression, occlusion of the cilioretinal artery, hypertension and dyslipidemia. On admission she was hypotensive (80/60mmHg). The ECG showed sinus rhythm and mild ST depression in V2-V3 leads, and the echocardiogram a small circumferential pericardial effusion (10mm) with signs of hemodynamic compromise. The blood tests documented a slight leukocytosis and an elevated troponin (hs-TnT 619ng/L).info:eu-repo/semantics/publishedVersio

Understanding cost escalation in nuclear reactor construction projects

This work seeks to evaluate overnight construction costs (OCC) and lead time escalation of nuclear reactors from 1955 to 2016. To this end, a comprehensive database of commercial Light Water Reactors (LWR) was developed and a statistical analysis was conducted. Findings reveal that there is significant delay in lead time, especially for the last generation reactors constructed from 2010’s. This results in the escalation of capital costs rather than a decline. Average OCC of newer reactors are 60% higher than the ones implemented in the earlier stages of the nuclear era. This suggests a negative learning curve effect for both OCC and lead time, which threats the market and financial sustainability of current and future nuclear energy projects. Although this is a general trend, this negative effect is country specific and, thus, induced by national policies and regulatory frameworks. Therefore, the role of nuclear technology to cope with the decarbonisation of the power sector must be better evaluated, taking into account the real cost impacts of nuclear technology implementation.This work was also funded by the Brazilian research funding agency CNPq.This work was funded by the Brazilian research funding agency CNPq and the Marie Curie International Research Staff Exchange Scheme Fellowship within the 7th European Union Framework Programme, under the project NETEP- European Brazilian Network on Energy Planning (PIRSES-GA-2013-612263)

Banana starch nanocomposite with cellulose nanofibers isolated from banana peel by enzymatic treatment: In vitro cytotoxicity assessment

The potential use of cellulose nanofibers (CNFs) as a reinforcing agent in banana starch-based nanocomposite films was investigated. CNFs were isolated from banana peel (Musa paradisiaca) by enzymatic hydrolysis. Banana starch-based nanocomposite films were prepared with CNFs using the casting method. CNFs effect on cell viability and on nanocomposite films properties was investigated. The cytotoxicity of CNFs was assessed on Caco-2 cell line. CNFs were not cytotoxic at 502000??g/mL. However, CNFs above 2000??g/mL significantly decreased cell viability. Topography analysis showed that the incorporation of CNFs modified the film structure. The nanocomposites exhibited a complex structure due to strong interactions between CNFs and starch matrix, promoting a remarkable improvement on mechanical and water barrier properties, opacity and UV light barrier compared to the control film. CNFs can offer a great potential as reinforcing material for starch-based nanocomposite films, producing a value-added food packaging from a waste material.The authors would like to acknowledge the financial support provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (140274/2014-6), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (2952/2011) and CAPES/FCT número349/13 for Ph.D internship program. Joana T. Martins acknowledges the Foundation for Science and Technology (FCT) for her fellowship (SFRH/BPD/89992/2012). This study was supported by FCT under the scope of the strategic funding of UID/BIO/ 04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors would also like to acknowledge the Brazilian Nanotechnology National Laboratory (LNNano) for allocation of the TEM, AFM and AFM-Nano IR apparatus.info:eu-repo/semantics/publishedVersio