Extraction of Biocompatible Collagen From Blue Shark Skins Through the Conventional Extraction Process Intensification Using Natural Deep Eutectic Solvents

ERC-2016-CoG 725034The disposal of large amounts of skin waste resulting from the blue shark fishing industry presents several industrial and environmental waste management concerns. In addition, these marine subproducts are interesting sources of collagen, a fibrous protein that shows high social and economic interest in a broad range of biomedical, pharmaceutical, and cosmetic applications. However, blue shark wasted skins are a poorly explored matrix for this purpose, and conventional collagen recovery methodologies involve several pre-treatment steps, long extraction times and low temperatures. This work presents a new green and sustainable collagen extraction approach using a natural deep eutectic solvent composed of citric acid:xylitol:water at a 1:1:10 molar ratio, and the chemical characterization of the extracted collagen by discontinuous electrophoresis, thermogravimetric analysis, Fourier transformed infrared spectroscopy and circular dichroism. The extracted material was a pure type I collagen, and the novel approach presented an extraction yield 2.5 times higher than the conventional one, without pre-treatment of raw material and reducing the procedure time from 96 to 1 h. Furthermore, the in vitro cytotoxicity evaluation, performed with a mouse fibroblasts cell line, has proven the biocompatibility of the extracted material. Overall, the obtained results demonstrate a simple, quick, cheap and environmentally sustainable process to obtain marine collagen with promising properties for biomedical and cosmetic applications.publishersversionpublishe

Oceanic circulation models help to predict global biogeography of pelagic yellowbellied sea snake

International audienceIt is well recognized that most marine vertebrates, and especially tetrapods,precisely orient and actively move in apparently homogeneous oceanicenvironments. Here, we investigate the presumptive role of oceanic currents in biogeographic patterns observed in a secondarily marine tetrapod, theyellow-bellied sea snake (Hydrophis [Pelamis] platurus). State-of-the-art world ocean circulation models show how H. platurus, the only pelagic species of sea snake, can potentially exploit oceanic currents to disperse and maintain population mixing between localities that spread over two-thirds of theEarth’s circumference. The very close association of these snakes with surface currents seems to provide a highly efficient dispersal mechanism that allowed this species to range extensively and relatively quickly well beyond the central Indo-Pacific area, the centre of origin, abundance and diversity of sea snakes. Our results further suggest that the pan-oceanic population of this species must be extraordinarily large

Cosmeceutical Potential of Extracts Derived from Fishery Industry Residues: Sardine Wastes and Codfish Frames

The fishery industry generates large amounts of waste (20–75% (w/w) of the total caught fish weight). The recovery of bioactive compounds from residues and their incorporation in cosmetics represents a promising market opportunity and may contribute to a sustainable valorisation of the sector. In this work, protein-rich extracts obtained by high-pressure technologies (supercritical CO2 and subcritical water) from sardine (Sardina pilchardus) waste and codfish (Gadus morhua) frames were characterized regarding their cosmeceutical potential. Antioxidant, anti-inflammatory and antibacterial activities were evaluated through chemical (ORAC assay), enzymatic (inhibition of elastase and tyrosinase), antimicrobial susceptibility (Klebsiella pneumoniae, Staphylococcus aureus and Cutibacterium acnes) and cell-based (in keratinocytes-HaCaT) assays. Sardine extracts presented the highest antibacterial activity, and the extract obtained using higher extraction temperatures (250 °C) and without the defatting step demonstrated the lowest minimum inhibitory concentration (MIC) values (1.17; 4.6; 0.59 mg/mL for K. pneumoniae, S. aureus and C. acnes, respectively). Codfish samples extracted at lower temperatures (90 °C) were the most effective anti-inflammatory agents (a concentration of 0.75 mg/mL reduced IL-8 and IL-6 levels by 58% and 47%, respectively, relative to the positive control). Threonine, valine, leucine, arginine and total protein content in the extracts were highlighted to present a high correlation with the reported bioactivities (R2 ≥ 0.7). These results support the potential application of extracts obtained from fishery industry wastes in cosmeceutical products with bioactive activities

On the role of components of therapeutic hydrophobic deep eutectic solvent-based nanoemulsions sustainably produced by membrane-assisted nanoemulsification for enhanced antimicrobial activity

Stringent regulations and growing industrial interests have ensued the development of sustainable processes. Membrane assisted-nanoemulsification based on microengineered membranes has been explored for the sustainable production of therapeutic hydrophobic deep eutectic solvent (DES)-based nanoemulsions for enhanced antimicrobial applications. Hydrophobic DESs were synthesised by employing therapeutic terpenes: DL-menthol and thymol. To understand the mechanism of oil droplet detachment from the membrane when producing such nanoemulsions, the effect of hydrophilicity/hydrophobicity partition of the active membrane surface on the process performance was evaluated. Using a continuous phase cross-flow velocity of 0.32 m.s−1, a dispersed phase flowrate of 0.02 ml.min−1 and 2% (w/w) Tween 80 resulted in a DES-in-water nanoemulsion of 81.2 ± 0.9 nm droplet size. Novel observations like intrinsically reduced interfacial tension of 7.5 mN.m−1 between water and hydrophobic DES, and a hydrophobic DES wetting behaviour to both hydrophilic and hydrophobic active membrane surfaces were witnessed. Compared to DL-menthol, thymol or synthesised DES, the optimised DES-in-water nanoemulsions exhibited enhanced synergetic antimicrobial effect against Escherichia coli, Staphylococcus aureus and superior potency against Cutibacterium acnes, an acne inducing bacterial strain. These nanoemulsions were also cytocompatible with human keratinocytes and dermal fibroblasts. Lastly, membrane-assisted nanoemulsification manifested controlled size and monomodally distributed nanoemulsions compared to traditional ultrasound-driven emulsification.The authors would like to acknowledge Executive Agency for Education, Audiovisual & Culture (EACEA) of the European Commission for the scholarship grant of Erasmus Mundus Doctorate in Membrane Engineering (EUDIME) program to Syed Usman Taqui. Prof. Reyes Mallada and Dr. Ruth Lahoz from Nanoscience Institute of Aragon (INA), University of Zaragoza are acknowledged for their unconditional support in fabrication and characterization of metallic membranes. This work is also supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020). Authors M.T.B. Crespo, F.B. Gaspar and I.C. Leonardo acknowledge the financial support from the project “MobFood – Mobilizing scientific and technological knowledge in response to the challenges of the agri-food market” (POCI-01-0247-FEDER-024524) financed by European Regional Development Fund (ERDF), through the Incentive System to Research and Technological development, within the Portugal2020 Competitiveness and Internationalization Operational Program; iNOVA4Health - UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement; and INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC). This research was partially funded by the Spanish Ministry of Economy and Competitiveness (grant number CTQ2014-52384-R). V. Sebastian acknowledges the financial support from Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00. G. Mendoza gratefully acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008 -2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III (Spain) with assistance from the European Regional Development Fund.Peer reviewe

Sustainable production of nanoemulsions by membrane-assisted nanoemulsification using novel aroma-based hydrophobic deep eutectic solvents for enhanced antifungal activities

Hydrophobic deep eutectic solvents (DESs), a recent class of green solvents, offer 100% atom economy, low cost, potential biodegradability, negligible toxicity and promising bioactivities. In this work, novel aroma-based therapeutic hydrophobic DESs were prepared and dispersed in aqueous media as nanoemulsions to potentiate biomedical applications, where polar media is encountered. A reusable microengineered stainless-steel isoporous membrane was fabricated by laser drilling technique. Three hydrophobic DESs, namely DES A (menthol and vanillin), DES B (menthol and raspberry ketone), and DES C (thymol and raspberry ketone) were prepared and emulsified in aqueous media by sustainable membrane emulsification technique. The optimised nanoemulsion (DES C-in-water) exhibited a monomodal size distribution with Zavg (size average) of 147 nm and polydispersity index of 0.22. From the application perspective, the formulated DES-in-water nanoemulsions and their constituents were evaluated for their antibacterial properties against Escherichia coli and Staphylococcus aureus. Additionally, antifungal properties of the DES-based emulsions were reported for the first time by testing them against four fungal strains, Aspergillus fumigatus, Candida albicans, Candida krusei, and Trichophyton mentagrophytes. The nanoemulsions were found to be exhibit antimicrobial effect and lesser quantities of individual compounds were needed in nanoemulsified state to show similar effects. Different 1D and 2D NMR techniques were successfully used to investigate the structural orientation and the inter and intramolecular interactions in the DES and emulsion systems, which revealed a probable cause for higher antimicrobial activity of DES C-based emulsions compared to its peers. Lastly, a synergistic effect of the components in nanoemulsions led to enhanced antimicrobial activities.SM acknowledges financial support from Fundação para a Ciência e a Tecnologia (FCT), Portugal for PhD grant SFRH/BD/146967/2019. This research was supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020). EP acknowledges support from national funds through FCT (Foundation for Science and Technology) within the scope of Base Funding UIDB/04423/2020 and UIDP/04423/2020 (CIIMAR). IL, FG, TC acknowledge funding from INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC), as well as iNOVA4Health [UIDB/04462/2020 and UIDP/04462/2020] and LS4FUTURE [LA/P/0087/2020] programs financially supported by FCT and the Ministério da Ciência, Tecnologia e Ensino Superior (MCTES). PR acknowledges financial support Grant PID2021-126132NB-I00 by MCIN/AEI/10.13039/501100011033 and the NMR Service of CEQMA (CSIC-UZ). VS acknowledges financial support Grant PID2021-127847OB-I00 and PDC2022-133866-I00 by MCIN/AEI, as well the ELECMI (LMA node) and NANBIOSIS ICTSs.Peer reviewe

Development and international validation trial of an advanced, multi-locus DNA metabarcoding metho to identify endangered species in complex samples

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance

Development and international validation trial of an advanced, multi-locus DNA metabarcoding metho to identify endangered species in complex samples

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance

Potential Ophthalmological Application of Extracts Obtained from Tuna Vitreous Humor Using Lactic Acid-Based Deep Eutectic Systems

A green technique was developed to extract hyaluronic acid (HA) from tuna vitreous humor (TVH) for its potential application in managing dry eye disease. Deep eutectic solvents (DES) were used to extract HA and were synthesized using natural compounds (lactic acid, fructose, and urea). The DES, the soluble fraction of TVH in DES (SF), and the precipitated extracts (PE) were evaluated for their potential use in dry eye disease treatment. In vitro experiments on human corneal epithelial cell lines and the effect on dry eye-associated microorganisms were performed. The influence of the samples on the HCE viability, their intracellular reactive oxygen species (ROS) scavenging capacity, inflammatory response, and antimicrobial properties were studied. According to the results, all samples displayed an antioxidant effect, which was significantly higher for PE in comparison to SF. Most of the tested samples did not induce an inflammatory response in cells, which confirmed the safety in ophthalmic formulations. In addition, the DES and SF proved to be efficient against the studied bacterial strains, while PE did not show an antimicrobial effect. Hence, both DES and SF at defined concentrations could be used as potential compounds in dry eye disease management

Development and international validation trial of an advanced, multi-locus DNA metabarcoding metho to identify endangered species in complex samples

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance