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- A biorefinery approach to the biomass of the seaweed Undaria pinnatifida (Harvey Suringar, 1873): obtaining phlorotannins-enriched extracts for wound healingPublication . Ferreira, Carolina A. M.; Félix, Rafael; Félix, Carina; Januário, Adriana P.; Alves, Nuno; Novais, Sara C.; Dias, Juliana R.; Lemos, Marco F.L.Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities (e.g., antioxidant, anti-inflammatory, and antimicrobial) with potential to improve wound healing. To obtain phlorotannins enriched extracts from Undaria pinnatifida, a biorefinery was set using lowcost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 +- 10.04 mmol mg-1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 +-1.13mMeq. Fe II mg-1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 ug mL-1. NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.
- Faecal indicator bacteria and Pseudomonas aeruginosa in marine coastal waters: is there a relationship?Publication . Januário, Adriana P.; Afonso, Clélia; Mendes, Susana; Rodrigues, Maria J.To estimate the quality of coastal waters, European Union Directive 2006/7/EC provides guidelines to assess levels of faecal bacteria, including Escherichia coli and intestinal enterococci. These microbiological criteria are based on studies that determine the risk of bathers having diseases caused by enteric bacteria, not necessarily measuring the potential danger associated with the presence of nonenteric pathogens. The association between the presence of faecal contaminant indicators and nonenteric pathogenic microorganisms has not been well defined yet. The purpose of this study is to establish a relationship between Pseudomonas aeruginosa and microbiological indicators of faecal contamination. Presence of microbiological contamination in the coastal waters near the sewage treatment plant (STP) of Peniche (Portugal) was confirmed (P. aeruginosa 135.8 Colony Forming Unit/100 mL, Escherichia coli 1100.1 Most Probable Number/100 mL, intestinal enterococci 2685.9 MPN/100 mL) with much lower levels in the areas located south of the STP, along the main water coastal current (beach 1: 0.7 CFU/100 mL, 16.5 MPN/100 mL, 100.5 MPN/100 mL; beach 2: 0.3 CFU/100 mL, 74.0 MPN/100 mL, 145.9 MPN/100 mL, respectively). Analysis of Pearson’s correlation revealed a strong positive correlation between E. coli and P. aeruginosa, suggesting E. coli as an indicator of P. aeruginosa presence.
- Cosmeceutical potential of grateloupia turuturu: using low-cost extraction methodologies to obtain added-value extractsPublication . Félix, Carina; Félix, Rafael; Carmona, Ana M.; Januário, Adriana P.; Dias, Pedro D.M.; Vicente, Tânia F. L.; Silva, Joana; Alves, Celso; Pedrosa, Rui; Novais, Sara C.; Lemos, Marco F.L.The invasive macroalga Grateloupia turuturu is known to contain a diversity of bioactive compounds with different potentialities. Among them are compounds with relevant bioactivities for cosmetics. Considering this, this study aimed to screen bioactivities with cosmeceutical potential, namely, antioxidant, UV absorbance, anti-enzymatic, antimicrobial, and anti-inflammatory activities, as well as photoprotection potential. Extractions with higher concentrations of ethanol resulted in extracts with higher antioxidant activities, while for the anti-enzymatic activity, high inhibition percentages were obtained for elastase and hyaluronidase with almost all extracts. Regarding the antimicrobial activity, all extracts showed to be active against E. coli, S. aureus, and C. albicans. Extracts produced with higher percentages of ethanol were more effective against E. coli and with lower percentages against the other two microorganisms. Several concentrations of each extract were found to be safe for fibroblasts, but no photoprotection capacity was observed. However, one of the aqueous extracts was responsible for reducing around 40% of the nitric oxide production on macrophages, showing its anti-inflammatory potential. This work highlights G. turuturu’s potential in the cosmeceutical field, contributing to the further development of natural formulations for skin protection.
- Multifunctional Gelatin/Chitosan Electrospun Wound Dressing Dopped with Undaria pinnatifida Phlorotannin-Enriched Extract for Skin RegenerationPublication . Ferreira, Carolina A. M.; Januário, Adriana P.; Félix, Rafael; Alves, Nuno; Lemos, Marco F.L.; Dias, Juliana R.The similarities of electrospun fibers with the skin extracellular matrix (ECM) make them promising structures for advanced wound dressings. Moreover, infection and resistance in wounds are a major health concern that may be reduced with antibacterial wound dressings. In this work, a multifunctional wound dressing was developed based on gelatin/chitosan hybrid fibers dopped with phlorotannin-enrich extract from the seaweed Undaria pinnatifida. The intrinsic electrospun structure properties combined with the antimicrobial and anti-inflammatory properties of phlorotannin-enrich extract will enhance the wound healing process. Electrospun meshes were produced by incorporating 1 or 2 wt% of extract, and the structure without extract was used as a control. Physico-chemical, mechanical, and biological properties were evaluated for all conditions. Results demonstrated that all developed samples presented a homogenous fiber deposition with the average diameters closer to the native ECM fibrils, and high porosities (~90%) that will be crucial to control the wound moist environment. According to the tensile test assays, the incorporation of phlorotannin-enriched extract enhances the elastic performance of the samples. Additionally, the extract incorporation made the structure stable over time since its in vitro degradation rates decreased under enzymatic medium. Extract release profile demonstrated a longstanding delivery (up to 160 days), reaching a maximum value of ~98% over time. Moreover, the preliminary antimicrobial results confirm the mesh’s antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In terms of biological characterization, no condition presented cytotoxicity effects on hDNF cells, allowing their adhesion and proliferation over 14 days, except the condition of 2 wt% after 7 days. Overall, the electrospun structure comprising phlorotannins-enriched extract is a promising bioactive structure with potential to be used as a drug delivery system for skin regeneration by reducing the bacterial infection in the wound bed.
- Red seaweed-derived compounds as a potential new approach for acne vulgaris carePublication . Januário, Adriana P.; Félix, Rafael; Félix, Carina; Reboleira, João; Valentão, Patrícia; Lemos, Marco F.L.Acne vulgaris (AV) is a chronic skin disease of the pilosebaceous unit affecting both adolescents and adults. Its pathophysiology includes processes of inflammation, increased keratinization, sebum production, hormonal dysregulation, and bacterial Cutibacterium acnes proliferation. Common AV has been treated with antibiotics since the 1960s, but strain resistance has emerged and is of paramount concern. Macroalgae are known producers of substances with bioactive properties, including anti-viral, antibacterial, antioxidant, and anti-inflammatory properties, among several others. In particular, red algae are rich in bioactive compounds such as polysaccharides, phenolic compounds, lipids, sterols, alkaloids, and terpenoids, conferring them antioxidant, antimicrobial, and anti-inflammatory activities, among others. Thus, the exploration of compounds from marine resources can be an appealing approach to discover new treatment options against AV. The aim of this work is to provide an overview of the current knowledge of the potentialities of red macroalgae in the treatment of AV by reviewing the main therapeutic targets of this disease, and then the existence of compounds or extracts with bioactive properties against them.
- Detection and enumeration of Pseudomonas aeruginosa in coastal waters (Peniche, Portugal)Publication . Januário, Adriana Pereira; Rodrigues, Maria José Ribeiro Machado; Afonso, Clélia Paulete Correia NevesPseudomonas aeruginosa é um microrganismo que possui requisitos mínimos para sobreviver, tendo uma habilidade notável para se adaptar a uma variedade de condições ambientais, estando apto para sobreviver em água destilada ou desionizada durante meses. Desde as últimas três décadas, P. aeruginosa tem vindo a ser uma das bactérias mais estudadas em saúde pública devido à sua capacidade de se poder tornar uma ameaça significativa como patogénico humano, causando infeções em quase todos os órgãos ou tecidos. Está associada a infeções no ouvido, olhos e pele, e causa infeções hospitalares sérias em pacientes com queimaduras ou com fibrose cística. A Diretiva 2006/7/EC estabelece disposições para a monitorização e a classificação da qualidade das águas balneares. Tem como propósito preservar, proteger e melhorar a qualidade do ambiente e proteger a saúde humana. No que toca a águas costeias, os parâmetros microbiológicos utilizados para classificar a qualidade das águas balneares, são enterococos intestinais (CFU/100 ml) e Escherichia coli (CFU/100 ml). P. aeruginosa não é incluído como critério microbiológico, nem tão pouco mencionado. Apesar disto, alguns autores recomendam limites para este microrganismo, sugerindo ausência em volumes de 100 ml de água. Mas, a associação entre a presença de indicadores de contaminação fecal (enterococos intestinais e E. coli) e a presença de microrganismos patogénicos não-entéricos parece não estar bem definida na literatura e, por essa razão, o propósito da primeira parte deste estudo foi estabelecer uma relação entre P. aeruginosa e esses indicadores. Os resultados revelaram uma relação positiva e moderada entre P. aeruginosa e E. coli. Assim sendo, aconselha-se a enumeração de P. aeruginosa, uma bactéria não-entérica, juntamente e completando a enumeração de E. coli e enterococos intestinais, sendo bactérias entéricas, já que são parâmetros definidos na Diretiva já mencionada. Desde os anos 60 que se têm vindo a utilizar técnicas convencionais microbiológicas para detetar e enumerar P. aeruginosa, sendo a filtração por membrana, o método mais utilizado. O procedimento na ISO 16266:2006 descreve o método de filtração por membrana para a deteção e enumeração de P. aeruginosa em amostras de água. Possuindo várias limitações, principalmente a dificuldade e morosidade da técnica, existe uma necessidade de desenvolver métodos alternativos que ultrapassem algumas das limitações encontradas nas metodologias clássicas. O novo método Pseudalert®/Quanti-Tray® MPN teste, desenvolvido pelos laboratórios da IDEXX, sinaliza a presença de P. aeruginosa em 24 horas. Esta metodologia foi formulada para água potável, de consumo e de piscina. Os resultados do presente estudo mostraram que não havia diferenças significativas quando comparado o método Pseudalert®/Quanti-Tray® MPN teste com a filtração por membrana, sugerindo que o novo teste pode ser considerado uma metodologia rápida para avaliar a contaminação por P. aeruginosa em águas costeiras. A principal limitação dos métodos de cultura microbiológicos, sejam eles tradicionais ou alternativos, é a sua incapacidade de detetar microrganismos viáveis, mas não cultiváveis. Ensaios que se baseiam em técnicas de PCR têm vindo a tornar-se mais atrativos, mas a maioria dos autores continua a utilizar procedimentos de enriquecimento ou crescimento em meio de cultura. Por isso, o objetivo da terceira parte do trabalho foi apontar uma metodologia de PCR para detetar P. aeruginosa sem qualquer crescimento bacteriano posterior. O tempo estimado do ensaio foi de 6 horas, permitindo a deteção da bactéria em estudo através da amplificação do gene ecfX utilizando a solução de lise TZ para extrair o ADN genómico presente nas membranas de filtração. Este ensaio pode ser uma alternativa viável às metodologias convencionais, quando aplicado à deteção de P. aeruginosa em águas costeiras marinhas.
- Tailoring shrimp aquafeed to tackle Acute Hepatopancreatic Necrosis Disease by inclusion of industry-friendly seaweed extractsPublication . Félix, Rafael; Félix, Carina; Januário, Adriana P.; Carmona, Ana M.; Baptista, Teresa; Gonçalves, Rui A.; Sendão, João; Novais, Sara C.; Lemos, Marco F.L.Shrimp farming has been severely affected by Vibrio infections in southeastern Asia, with severe economic impacts. Invasive seaweeds are an increasing threat that can be strategically valorized by biotechnology, promoting economic development while contributing to ecologic remediation. Asparagopsis armata and Sargassum muticum are two such species that have been frequently reported as excellent bioactive compounds producers, namely anti-bacterial and antioxidant. Thus, A. armata and S. muticum were extracted in an industry-focused approach, using low amounts of ethanol and short extraction times at room temperature. Yield, in vitro antioxidant capacity by the 2,2-diphenyl−1-picrylhydrazyl radical scavenging assay, and in vitro antibacterial activity against Vibrio parahaemolyticus (microdilution) were optimized using Response Surface Methodology (RSM). Optimal extracts (one from each seaweed species) were incorporated in feed, and an in vivo trial using the whiteleg shrimp Penaeus vannamei was performed in industrial facilities using the supplemented feed (1.5, 3.5 and 7.5 g extract per kg feed). Feed quality (along an 8-week period under regular storage conditions) and shrimp performance (40-day trial) were studied, as well as post-challenge (infection by virulent Vibrio parahaemolyticus) mortality rate and hepatopancreas condition. The results from the RSM showed that lower extraction times and ethanol volumes maximized extracts' bioactive properties (A. armata – 20 min, 10 mL solvent per gram seaweed; S. muticum – 100 min, 10 mL.g−1). The seaweed extracts did not impact shrimp performance (no statistically significant differences in weight gain, feed conversion rate or survival). Asparagopsis armata selected extract at 7.5 g per kg feed was able to decrease feed contamination by fungi along time and reduce shrimp mortality by up to 50% upon challenge with V. parahaemolyticus. Despite lacking statistical significance, a difference in hepatopancreas condition after challenge could be identified in shrimp fed the supplemented feed.
- A Biorefinery Approach to the Biomass of the Seaweed Undaria pinnatifida (Harvey Suringar, 1873): Obtaining Phlorotannins-Enriched Extracts for Wound HealingPublication . Ferreira, Carolina A. M.; Félix, Rafael; Félix, Carina; Januário, Adriana P.; Alves, Nuno; Novais, Sara C.; Dias, Juliana R.; Lemos, Marco F.L.Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities (e.g., antioxidant, anti-inflammatory, and antimicrobial) with potential to improve wound healing. To obtain phlorotannins enriched extracts from Undaria pinnatifida, a biorefinery was set using low cost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 ± 10.04 mmol·mg−1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 ± 1.13 mM eq. Fe II·mg−1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 µg·mL−1 . NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.
- Uncovering the bioactivity of Aurantiochytrium sp.: a comparison of extraction methodologiesPublication . Reboleira, João; Félix, Rafael; Vicente, Tânia F. L.; Januário, Adriana P.; Félix, Carina; Melo, Marcelo M.R. de; Silva, Carlos M.; Ribeiro, Ana C.; Saraiva, Jorge A.; Bandarra, Narcisa M.; Sapatinha, Maria; Paulo, Maria C.; Coutinho, Joana; Lemos, Marco F.L.Aurantiochytrium sp. is an emerging alternative source of polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA), and squalene, playing an important role in the phasing out of traditional fish sources for these compounds. Novel lipid extraction techniques with a focus on sustainability and low environmental footprint are being developed for this organism, but the exploration of other added-value compounds within it is still very limited. In this work, a combination of novel green extraction techniques (high hydrostatic pressure extraction (HPE) and supercritical fluid extraction (SFE)) and traditional techniques (organic solvent Soxhlet extraction and hydrodistillation (HD)) was used to obtain lipophilic extracts of Aurantiochytrium sp., which were then screened for antioxidant (DPPH radical reduction capacity and ferric-reducing antioxidant potential (FRAP) assays), lipid oxidation protection, antimicrobial, anti-aging enzyme inhibition (collagenase, elastase and hyaluronidase), and anti-inflammatory (inhibition of NO production) activities. The screening revealed promising extracts in nearly all categories of biological activity tested, with only the enzymatic inhibition being low in all extracts. Powerful lipid oxidation protection and anti-inflammatory activity were observed in most SFE samples. Ethanolic HPEs inhibited both lipid oxidation reactions and microbial growth. The HD extract demonstrated high antioxidant, antimicrobial, and antiinflammatory activities making, it a major contender for further studies aiming at the valorization of Aurantiochytrium sp. Taken together, this study presents compelling evidence of the bioactive potential of Aurantiochytrium sp. and encourages further exploration of its composition and application.
- Multifunctional gelatin/chitosan electrospunwound dressing dopped with Undaria pinnatifida phlorotannin-enriched extract for skin regenerationPublication . Ferreira, Carolina A. M.; Januário, Adriana P.; Félix, Rafael; Alves, Nuno; Lemos, Marco F. L.; Dias, Juliana R.The similarities of electrospun fibers with the skin extracellular matrix (ECM) make them promising structures for advanced wound dressings. Moreover, infection and resistance in wounds are a major health concern that may be reduced with antibacterial wound dressings. In this work, a multifunctional wound dressing was developed based on gelatin/chitosan hybrid fibers dopped with phlorotannin-enrich extract from the seaweed Undaria pinnatifida. The intrinsic electrospun structure properties combined with the antimicrobial and anti-inflammatory properties of phlorotannin-enrich extract will enhance the wound healing process. Electrospun meshes were produced by incorporating 1 or 2 wt% of extract, and the structure without extract was used as a control. Physico-chemical, mechanical, and biological properties were evaluated for all conditions. Results demonstrated that all developed samples presented a homogenous fiber deposition with the average diameters closer to the native ECM fibrils, and high porosities (~90%) that will be crucial to control the wound moist environment. According to the tensile test assays, the incorporation of phlorotannin-enriched extract enhances the elastic performance of the samples. Additionally, the extract incorporation made the structure stable over time since its in vitro degradation rates decreased under enzymatic medium. Extract release profile demonstrated a longstanding delivery (up to 160 days), reaching a maximum value of ~98% over time. Moreover, the preliminary antimicrobial results confirm the mesh’s antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In terms of biological characterization, no condition presented cytotoxicity effects on hDNF cells, allowing their adhesion and proliferation over 14 days, except the condition of 2 wt% after 7 days. Overall, the electrospun structure comprising phlorotannins-enriched extract is a promising bioactive structure with potential to be used as a drug delivery system for skin regeneration by reducing the bacterial infection in the wound bed.