Planctomycetes as novel source of bioactive molecules

Marine environments are a fruitful source of bioactive compounds some of which are the newest leading drugs in medicinal therapeutics. Of particular importance are organisms like sponges and macroalgae and their associated microbiome. Planctomycetes, abundant in macroalgae biofilms, are promising producers of bioactive compounds since they share characteristics, like large genomes and complex life cycles, with the most bioactive bacteria, the Actinobacteria. Furthermore, genome mining revealed the presence of secondary metabolite pathway genes or clusters in 13 analyzed Planctomycetes genomes. In order to assess the antimicrobial production of a large and diverse collection of Planctomycetes isolated from macroalgae from the Portuguese coast, molecular, and bioactivity assays were performed in 40 bacteria from several taxa. Two genes commonly associated with the production of bioactive compounds, nonribosomal peptide synthetases (NRPS), and polyketide synthases (PKS) genes were screened. Molecular analysis revealed that 95% of the planctomycetes potentially have one or both secondary bioactive genes; 85% amplified with PKS-I primers and 55% with NRPS primers. Some of the amplified genes were confirmed to be involved in secondary metabolite pathways. Using bioinformatic tools their biosynthetic pathways were predicted. The secondary metabolite genomic potential of strains LF1, UC8, and FC18 was assessed using in silico analysis of their genomes. Aqueous and organic extracts of the Planctomycetes were evaluated for their antimicrobial activity against an environmental Escherichia coli, E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and a clinical isolate of Candida albicans. The screening assays showed a high number of planctomycetes with bioactive extracts revealing antifungal (43%) and antibacterial (54%) activity against C. albicans and B. subtilis, respectively. Bioactivity was observed in strains from Rhodopirellula lusitana, R. rubra, R. baltica, Roseimaritima ulvae, and Planctomyces brasiliensis. This study confirms the bioactive capacity of Planctomycetes to produce antimicrobial compounds and encourages further studies envisaging molecule isolation and characterization for the possible discovery of new drugs. © 2016 Graça, Calisto and Lage.This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT—Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020 and by the Structured Program of R&D&I INNOVMAR – Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), funded by the Northern Regional Operational Programme (NORTE2020) through the European Regional Development Fund (ERDF)

Tailloring deep eutectic solvents for the extraction of valuable compounds from natural sources using choline chloride and carboxylic acids mixtures: optimization of the extraction of phenolic compounds from Juglans regia L. leaves

Phenolic compounds are a wide group of plant secondary metabolites with increasing interest due to their antioxidant ability and marked effects on oxidative processes related to several chronic diseases. Nowadays, they find applications in pharmaceutical products, functional foods and natural-based cosmetics. Therefore, the extraction and identification of these valuable compounds from different plants have become a major research area. Nevertheless, conventional solid-liquid extraction of phenolics usually involves the use of flammable, toxic and volatile organic solvents. Deep eutectic solvents (DES) are gaining much interest as alternative solvents to extract valuable compounds from natural matrices. DES can be considered “designer solvents” due to the possibility of combining different HBA (hydrogen bond acceptor) and HBD (hydrogen bond donor) to obtain solvents with specific properties to the target application. In this study, DES composed of choline chloride (CC) and different groups of organic acids were prepared, in order to extract phenolic compounds from leaves of Juglans regia L. (walnut leaves). The initial screening involved monocarboxylic (acetic, propionic, butyric, valeric, lactic and glycolic acids), dicarboxylic (malonic, glutaric and malic acids), tricarboxylic (citric) and aromatic acids (phenylacetic acid, 3-phenylpropanoic acid). The initial extraction conditions were: 50 ºC, 60 min extraction time and 20% water content. The main phenolic compounds (neochlorogenic acid, quercetin 3-O-glucoside and quercetin O-pentoside) were quantified by high performance liquid chromatography coupled to a diode de array detector (HPLC-DAD at 280 and 370 nm). Higher extraction yields were obtained using CC:butyric acid and CC:3-phenylpropanoic acid. For these systems, the stoichiometric ratio of HBA and HBD as well as the water content in the DES solutions were further evaluated. Better results were obtained using DES compared to the conventional water + ethanol mixed solvent. The present work contributes to the valorization of walnut leaves extracts using alternative solvents that could be tailored for potential applications in the food and pharmaceutical areas.The authors thank the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros (SFRH/BPD/107855/2015) and V. Vieira (SFRH/BD/108487/2015) grants. To POCI-01-0145-FEDER-006984 (LA LSRE-LCM), funded by ERDF, through POCI-COMPETE2020 and FCT. To Xunta de Galicia for financial support for the post-doctoral researcher of M.A. Prieto.info:eu-repo/semantics/publishedVersio

Enhanced extraction of phenolic compounds using choline chloride based deep eutectic solvents from Juglans regia L.

The extraction of phenolic compounds from walnut leaves (Juglans regia L.) was optimized using heat-assisted extraction and deep eutectic solvents based on choline chloride and carboxylic acids. A preliminary solvent screening was performed using a selected group of carboxylic acids as hydrogen bond donors, showing that the highest extraction yield of phenolic compounds was obtained using choline chloride mixtures with butyric or phenylpropionic acid at a mole ratio 1:2, with 20% of water (w/w). The extraction conditions (time, temperature and water proportion) were then optimized by an experimental design, assisted by response surface methodology. To evaluate the response, the three most abundant compounds identified by HPLC (neochlorogenic acid, quercetin 3-O-glucoside and quercetin O-pentoside) were quantified. Additionally, the solid/liquid ratio effect at the optimal conditions, in dose-response format, was studied in view of its upscale, not showing any significant decrease until 140 g/L. The results here presented provide valuable information towards the design of a process in a pre-industrial form for the extraction of phenolic compounds from J. regia leaves using deep eutectic solvents.The authors thank the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), V. Vieira (SFRH/BD/108487/2015) grant and L. Barros contract. To project POCI-01-0145-FEDER-006984 – Associate Laboratory LSRE-LCM funded by FEDER through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT – Fundação para a Ciência e a Tecnologia. To project CICECO‐Aveiro Institute of Materials POCI‐01‐0145‐FEDER‐007679 (FCT Ref. UID/ CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate co‐financed by FEDER under the PT2020 Partnership Agreement. This work is funded by the European Structural and Investment Funds (FEEI) through POCI, within the scope of Project POCI nº 030463. To Xunta de Galicia for financial support for the postdoctoral researcher of M.A. Prieto.info:eu-repo/semantics/publishedVersio

Aquaculture as a circular bio-economy model with Galicia as a study case: How to transform waste into revalorized by-products

Background: World-wide aquaculture represents a very important sector capable of supplying huge amounts of animal protein. However its relevance has proportionally augmented its waste generation. In Europe, the geographical constitution of Galicia has prompted the instauration of many aquaculture-based systems along its coasts. Indeed aquaculture means a very relevant industry in Galicia, together with animal farming, agriculture and biotechnology. Scope and approach: Over the last decade Europe legislation encourages the proper management of wastes (mostly reutilization and reducing strategies) and the sustainable use of natural resources. The application of circular bio-economy (reuse of wastes) represents a feasible model to protect human and animal health and the environment. To achieve a more efficient production system that complies with European regulations, aquaculture wastes and sub-products need to be re-utilised to increase their throughput. This approach will positively impact on their economical yield while reducing their generation and thus protecting health and environment. Key findings and conclusions: Different applications have been considered for re-using aquaculture wastes and sub-products. One of the most efficient approaches is the establishment of models that allow the metabolic waste reduction, as the integrated multi-trophic aquaculture. For derived aquaculture sub-products, the most efficient process is recovering important biomolecules such as proteins (collagen, gelatine), polysaccharides (chitosan), lipids (omega 3) or pigments (astaxanthin or beta-carotene). Biomolecules can further be applied for human and animal consumption, food industry, cosmetics or pharmaceuticals. Due to the importance of this productive system in Galicia it is critical its update to include aquaculture into circular bio-economy.The research leading to these results received institutional and financial support from: Programa de Cooperaci´on Interreg V-A España—Portugal (POCTEP) 2014–2020 (projects Ref.: 0181_NANOEATERS_01_E and Ref: 0377_IBERPHENOL_6_E); Spanish Ministry of Economy, Industry and Competitiveness through the project AGL2015–67039–C3–1–R; MICINN supporting the Ram´on&Cajal grant for M.A. Prieto (RYC-2017-22891); Xunta de Galicia and University of Vigo for supporting the post-doctoral grant of María Fraga Corral (ED481B-2019/096) and the pre-doctoral grants of Antía Gonz´alez Pereira (ED481A-2019/0228) and P. García-Oliveira (ED481A-2019/ 295); Xunta de Galicia through the program EXCELENCIA-ED431F 2020/12 and the project ED431B 2019/24; Ibero-American Program on Science and Technology (CYTED - AQUA-CIBUS, P317RT0003); Axudas Conecta Peme (Xunta de Galicia) supporting the IN852A 2018/ 58 NeuroFood Project; AlgaMar (www.algamar.com); EcoChestnut Project (Erasmus+ KA202); Bio Based Industries Joint Undertaking (JU) under grant agreement No 888003 UP4HEALTH Project (H2020-BBIJTI- 2019), the JU receives support from the European Union’s Horizon 2020 research and innovation program and the Bio Based Industries Consortium. Funding for open access charge: Universidade de Vigo/ CISUG.info:eu-repo/semantics/publishedVersio

Multifunctions of Pleurotus sajor-caju (Fr.) Singer: a highly nutritious food and a source for bioactive compounds

A study with Pleurotus sajor-caju was conducted to: evaluate the nutritional and chemical composition of the fruiting bodies; optimize the preparation of bioactive phenolic extracts; and characterize the optimized extract in terms of bioactive compounds and properties. P. sajor-caju revealed an equilibrated nutritional composition with the presence of hydrophilic (sugars and organic acids) and lipophilic (tocopherols and PUFA) compounds. p-Hydroxybenzoic, p-coumaric and cinnamic acids were identified in the extract obtained with ethanol (30 g/l ratio) at 55 °C for 85 min. This extract showed antioxidant properties (mainly reducing power and lipid peroxidation inhibition), antibacterial activity against MRSA and MSSA and cytotoxicity against NCI-H460, MCF-7 and HeLa. Furthermore, as the extract showed capacity to inhibit NO production in Raw 264.7 macrophages, molecular docking studies were performed to provide insights into the anti-inflammatory mechanism of action, through COX-2 inhibition by the phenolic acids identified.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Program PT2020 for financial support to CIMO (UID/AGR/00690/2013) and for L. Barros and R. Calhelha contracts. T.C. Finimundy thanks CAPES Foundation, Ministry of Education of Brazil (CAPES fellow, process number 88881.134581/2016-01). To Xunta de Galicia for financial support for the post-doctoral researcher of M.A. Prieto.info:eu-repo/semantics/publishedVersio

Pink‐ and orange‐pigmented Planctomycetes produce saproxanthin‐type carotenoids including a rare C45 carotenoid

Planctomycetes, are ubiquitous and environmentally important Gram-negative aquatic bacteria with key roles in global carbon and nitrogen cycles. Many planctomycetal species have a pink or orange colour and have been suggested to produce carotenoids. Potential applications as food colorants or anti-oxidants have been proposed. Hitherto, the planctomycetal metabolism is largely unexplored and the strain pigmentation has not been identified. For a holistic view on the complex planctomycetal physiology we analyzed carotenoid profiles of the pink-pigmented strain Rhodopirellula rubra LF2T and of the orange strain Rubinisphaera brasiliensis Gr7. During LC-MS/MS analysis of culture extracts we were able to identify three saproxanthin-type carotenoids including a rare C45 carotenoid. These compounds, saproxanthin, dehydroflexixanthin and 2’-isopentenyldehydrosaproxanthin, derive from the common carotenoid precursor lycopene and are characterized by related end groups, namely a 3-hydroxylated β-carotene-like cyclohexene ring as one end group and simple hydration on the other end of the molecule. Based on the observed molecule structure we present putative pathways for their biosynthesis. Results support Planctomycetes as a promising, yet mostly untapped source of carotenoids

An optimized nanoparticle delivery system based on chitosan and chondroitin sulfate molecules reduces the toxicity of amphotericin B and is effective in treating tegumentary leishmaniasis

Amphotericin B (AmpB) is active against leishmaniasis, but its use is hampered due to its high toxicity observed in patients. In this study, a nanoparticles-delivery system for AmpB (NQC-AmpB), containing chitosan and chondroitin sulfate molecules, was evaluated in BALB/c mice against Leishmania amazonensis. An in vivo biodistribution study, including biochemical and toxicological evaluations, was performed to evaluate the toxicity of AmpB. Nanoparticles were radiolabeled with technetium-99m and injected in mice. The products presented a similar biodistribution in the liver, spleen, and kidneys of the animals. Free AmpB induced alterations in the body weight of the mice, which, in the biochemical analysis, indicated hepatic and renal injury, as well as morphological damage to the kidneys of the animals. In general, no significant organic alteration was observed in the animals treated with NQC-AmpB. Mice were infected with L. amazonensis and treated with the nanoparticles or free AmpB; then, parasitological and immunological analyses were performed. The NQC-AmpB group, as compared to the control groups, presented significant reductions in the lesion size and in the parasite burden in all evaluated organs. These animals presented significantly higher levels of IFN-γ and IL-12, and low levels of IL-4 and IL-10, when compared to the control groups. The NQC-AmpB system was effective in reducing the infection in the animals, and proved to be effective in diminishing the toxicity evoked by AmpB, which was observed when it was administered alone. In conclusion, NQC-AmpB could be considered a viable possibility for future studies in the treatment of leishmaniasisThis work was supported by grants from Pró-Reitoria de Pesquisa from UFMG (Edital 01/2014), Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00496-11 and CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9 and APQ-482976/2012-8). MACF is a grant recipient of FAPEMIG/CAPES. EAFC, VNC, and AAGF are grant recipients of CNPq. Eduardo AF Coelho and André AG Faraco are co-senior authors of this stud

Prevalence and risk factors for bovine leptospirosis in Mato Grosso do Sul, Brazil

Foi investigada a prevalência de anticorpos antileptospira em fêmeas bovinas com idade igual ou superior a 24 meses, provenientes de 178 rebanhos de 22 municípios do estado de Mato Grosso do Sul, bem como identificados fatores de risco associados à infecção. Foram analisadas 2.573 amostras de soro sangüíneo por meio do teste de soroaglutinação microscópica perante 10 sorovares de leptospira. Títulos iguais ou superiores a 100 para um ou mais sorovares foram detectados em 1.801 fêmeas (98,8%) de 161 (96,5%) rebanhos. O sorovar Hardjo (65,6%) foi apontado como o mais provável, seguido do sorovar Wolffi (12,3%). Os resultados demonstram que a leptospirose bovina se encontra presente em todos os municípios estudados, com alta prevalência, tanto em animais como em rebanhos. Os fatores de risco identificados neste estudo e associados à infecção por bactérias do gênero lepstopira foram o tipo de exploração pecuária de corte e a raça Zebu. _____________________________________________________________________________________________________________ ABSTRACTThe prevalence of anti-Leptospira spp. antibodies was estimated for female cattle aged 24 months or older. The sample comprised 178 herds from 22 counties in the state of Mato Grosso do Sul, Brazil. The risk factors associated with the presence of infeccion were investigated. A total of 2,573 blood serum samples were tested against 10 leptospira serovars using the microagglutination test (MAT). Titers of 100 or higher for one or more serovars were detected in 1,801 females (98.8%) from 161 herds (96.5%). Serovar Hardjo (65.6%) was the most frequent, followed by serovar Wolffi (12.3%). These results suggest that bovine leptospirosis is widespread in all the counties under study, with a high prevalence both at the animal and the herd level. Beef farms and the Zebu breed were associated to the higher risk of herd infection by leptospiras