Browsing by Author "Lopes, Ana I."
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- Alginate edible films containing essential oils: characterization and bioactive potentialPublication . Lopes, Ana I.; Melo, Adma; Afonso, Tiago B.; Silva, Sara; Barros, Lillian; Tavaria, Freni K.; Pintado, ManuelaEssential oils (EOs) are natural substances rich in phenolic compounds with notable antimicrobial and antioxidant properties. However, they present some limitations, such as low stability and bioavailability. Incorporating EOs into polymeric films offers a novel approach to overcome these challenges while enhancing their efficacy. In this study, we produced and thoroughly characterized alginate-based edible films incorporated with five different EOs—rosemary, eucalyptus, oregano, sage, and thyme. This is the first comprehensive investigation to include this diverse range of EOs in alginate films. Their antimicrobial and antioxidant activities were also evaluated. The results demonstrated that alginate films containing EOs exhibited significant bioactive properties. Notably, the film incorporated with oregano EO completely inhibited the growth of all tested bacteria and fungi and showed the highest antioxidant activity. Based on these findings, alginate films containing EOs present promising bioactive potential and could serve as biodegradable alternatives to conventional packaging materials, reducing environmental impact. However, further studies are necessary to assess their safety profile and confirm their viability as replacements for traditional food packaging. Future research should focus on evaluating cytotoxicity, genotoxicity, and the practical application of these films in food matrices.
- Antimicrobial impact of essential oils on human skin colonizers and pathogens: targeting Staphylococcus speciesPublication . Lopes, Ana I.; Oliveira, Cláudia S.; Pintado, Manuela E.; Tavaria, Freni K.Background: Staphylococcus spp., one of the most abundant bacterial genera of the human skin microbiome, comprises commensal microorganisms – Staphylococcus epidermidis (S. epidermidis) – and opportunistic pathogens – Staphylococcus aureus (S. aureus). Staphylococcus-caused diseases are typically treated with antibiotics, but the antimicrobial resistance underscores the need for alternative treatments. Essential oils (EOs) are natural compounds made of terpenes and terpenoid molecules. Their antimicrobial activity results from the effect upon several cell targets, making the appearance of resistances unlikely. Objective: To study the antimicrobial activity of three EOs (eucalyptus, lavender, and thyme) on two Staphylococcus species (S. epidermidis and S. aureus). Methods: To assess the antimicrobial potential of EOs against the selected bacteria, the following assays were performed: i) determination of the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations; ii) inhibition curves for each oil defined for the MIC, to understand how the bacteria respond to the oils; iii) antibiofilm formation assays using MIC, ½ MIC and ¼ MIC dilutions for each oil; and iv) evaluation of the membrane damage through flow cytometry, by staining the bacterial cells exposed to the oils, with propidium iodide (PI) and thiazole orange (TO). Results: The MIC and MBC values for the selected EOs ranged from 0.078% and 2.5% (v/v). Thyme presented the lower MIC for both bacteria. The inhibition curves corroborated the MIC results for all EOs. However, thyme was the one with a bactericidal effect on both bacteria. Thyme also presented higher antibiofilm activity compared to eucalyptus and lavender. The flow cytometry assay showed that all oils were able to damage the cell membrane of both bacteria (positive PI staining), but S. epidermidis showed less cell damage than S. aureus. Conclusion: The selected EOs presented antibacterial activity against S. epidermidis and S. aureus. Thyme EO was the one with the best antibacterial activity. S. epidermidis is more resistant to the oils than S. aureus.
- Antimicrobial impact of essential oils on human skin colonizers and pathogens: targeting Staphylococcus speciesPublication . Lopes, Ana I.; Oliveira, Cláudia S.; Pintado, Manuela E.; Tavaria, Freni K.Introduction: Staphylococcus spp. is one of the most abundant bacterial genera of the human skin microbiome (Joglekar et al. 2023). It comprises commensal microorganisms – Staphylococcus epidermidis (S. epidermidis) – and opportunistic pathogens – Staphylococcus aureus (S. aureus) (Imam and Luqman 2024). Staphylococci-caused diseases are typically treated with antibiotics, but the antimicrobial resistance underscores the need for alternative treatments. Essential oils (EOs) are natural compounds made of terpenes and terpenoid molecules. Their antimicrobial activity targets multiple cellular components, which reduces the likelihood of resistance development (Zuzarte et al. 2011). Objective: To study the antimicrobial activity of three EOs (eucalyptus, lavender, and thyme) on two Staphylococcus species (S. aureus and S. epidermidis). Conclusions: Eucalyptus, lavender, and thyme EOs present antibacterial activity against S. aureus and S. epidermidis. Thyme possesses the lowest MIC; the inhibition curves corroborated this result. The flow cytometry assay showed that all oils were able to damage bacterial cells; thyme oil presented the highest percentages of dead and injured cells. All EOs also showed anti-biofilm activity; thyme presented the highest anti-biofilm activity when compared to eucalyptus and lavender. S. epidermidis is more resistant to the oils than S. aureus.
- Application of chitosan in the control of fungal infections by dermatophytesPublication . Lopes, Ana I.; Tavaria, Freni K.; Pintado, Manuela E.Dermatophytes are a group of fungi that can invade keratinized tissues of humans and other animals and produce an infection called Dermatophytosis. As chitosan possesses antimicrobial activity, it can potentially be used to treat dermatophytic infections. The main objective of this work was therefore, to evaluate the antifungal activity of chitosan upon some dermatophytes, namely Microsporum canis and Trychophyton rubrum. In view of this, Minimum Inhibitory (MICs) and Minimum Fungicidal Concentrations (MFCs) of chitosans upon the fungi were determined. Moreover, in order to understand the effect of chitosan on fungal activity, hair was infected with these fungi in the presence and absence of chitosan and Scanning Electron Microscopy (SEM) images were obtained and analyzed. Lastly, keratin-azure was used as substrate to evaluate the effect of chitosan on keratin degradation by M. canis and T. rubrum. The results showed that chitosan possesses antifungal activity against T. rubrum and M. canis, presenting MICs and MFCs ranging from 1.1 to 2.2 mg/mL. The antifungal activity of chitosan is concentration dependent. The analysis of SEM images of hair infected with these dermatophytes revealed that chitosan seems to have a protective effect on the hair, reducing the extent of damage when compared to the control. Chitosan also displayed important activity in preventing proteases’ action and in preventing hair damage. Based on the obtained results, it’s possible to conclude that chitosan showed relevant antifungal activity against dermatophytes, which opens good prospects to the use of chitosan as an alternative for the conventional fungal treatments
- Assessing the antimicrobial activity of essential oils against skin microorganisms using flow cytometryPublication . Lopes, Ana I.; Oliveira, Cláudia S.; Pintado, Manuela E.; Tavaria, Freni K.Introduction: The skin microbiome is a dynamic ecosystem vital for skin health, comprising bacteria like Staphylococcus spp. And Cutibacterium acnes, and fungi such as Candida and Malassezia spp. Dysbiosis, or microbial imbalance, can lead to conditions like acne and dermatitis. Current treatments, including anti-inflammatory drugs and antimicrobials, help manage dysbiosis but have limitations, such as microbiome disruption and antimicrobial resistance. Consequently, interest in natural alternatives,particularly essential oils (EOs), is increasing. Rich in bioactive terpenes and terpenoids, EOs target multiple cellular structures, reducing microbial adaptation and resistance. Flow cytometry, a powerful analytical tool, enables precise assessment of antimicrobial activity by distinguishing live and dead microorganisms using fluorescent staining. This study evaluates the antimicrobial effects of eucalyptus, lavender, and thyme EOs, offering insights into their potential as microbiome-friendly therapeutic agents. Methods: flow cytometry assay was used to assess the antimicrobial activity of eucalyptus, lavender, and thyme EOs against seven skinassociated microorganisms: methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), Cutibacterium acnes (C. acnes), Candida albicans (C. albicans), Candida tropicalis (C. tropicalis), and Malassezia furfur (M. furfur). Firstly, minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined. Then, to further explore EOs antimicrobial effects, microbial cells were exposed to MIC, ½MIC, and ¼MIC of each oil, followed by staining with propidium iodide (PI) and thiazole orange (TO). Flow cytometry was then used to assess viability, membrane integrity, and cell death. Additionally, fluorescence microscopy was performed on yeasts exposed to eucalyptus oil at ¼MIC, providing complementary validation of the flow cytometry findings. Results: The MIC and MBC values of the tested EOs ranged from 0.06% to 2.5% (v/v), with thyme EO showing the lowest values for all microorganisms, indicating its strongest antimicrobial activity. Among bacteria, S. epidermidis had the highest MIC/MBC values, suggesting greater resistance, while C. acnes had the lowest, indicating higher susceptibility. Among yeasts, C. albicans exhibited the highest values, whereas C. tropicalis and M. furfur were more susceptible. Flow cytometry confirmed these findings, validating thyme EO as the most effective, with the highest percentages of dead and injured cells. It also demonstrated that EOs antimicrobial activity is concentration-dependent. S. epidermidis had the highest percentage of live cells, reinforcing its resistance, while C. acnes had the lowest, confirming its susceptibility. C. albicans was more resistant than C. tropicalis. For M. furfur, fluorescence microscopy clarified an apparent lack of TO staining in flow cytometry, confirming viable but unstained cells. Conclusion: The selected EOs exhibited antimicrobial activity against all tested microorganisms. Thyme EO showed the strongest effect. Flow cytometry confirmed the MIC/MBC results, revealing that thyme EO induced the highest percentages of dead and injured cells at all tested concentrations. Among bacteria, S. epidermidis was the most resistant, while C. albicans was the most resistant yeast. For M. furfur, flow cytometry suggested an absence of live cells due to the lack of TO staining, even in untreated samples. However, fluorescence microscopy confirmed that M. furfur cells remained viable but did not stain green like Candida spp. This discrepancy likely results from the yeast’s lipid-rich membrane interfering with TO uptake, highlighting potential staining limitations. This work reinforces flow cytometry as a powerful tool to validate EO’s antimicrobial effects and support the potential of thyme, eucalyptus, and lavender EO’s as natural alternatives for managing skin dysbiosis while preserving microbiome balance.
- Assessing the antimicrobial activity of essential oils against skin microorganisms using flow cytometryPublication . Lopes, Ana I.; Oliveira, Cláudia S.; Pintado, Manuela E.; Tavaria, Freni K.The skin microbiome, composed of bacteria like Staphylococcus spp. and Cutibacterium acnes and fungi such as Candida spp. and Malasseziaspp., plays a key role in skin health (Byrd et al., 2018). Dysbiosis, or microbial imbalance, is linked to conditions like acne and dermatitis (Egert etal., 2017). While conventional treatments, such as anti-inflammatory drugs and antimicrobials, can help, they may disrupt the microbiome andpromote resistance (Sfriso et al., 2020).Natural alternatives like essential oils (EOs) are gaining attention due to their terpene-rich composition, which targets multiple microbial structuresand reduces resistance risk (Zuzarte et al., 2011). Flow cytometry offers a precise method to assess antimicrobial effects by distinguishing livefrom dead cells through fluorescent staining (Shapiro, 2003).
- Biodegradable films with polysaccharides, proteins, and bioactive compounds from lobosphaera sp.: antioxidant and antimicrobial activitiesPublication . Martins, Valter F. R.; Lopes, Ana I.; Machado, Manuela; Costa, Eduardo M.; Ribeiro, Tânia B.; Poças, Fátima; Pintado, Manuela; Morais, Rui M. S. C.; Morais, Alcina M. M. B.Microalgae are a sustainable source of bioactive compounds and nutrients that do not compete with crops for arable land. Lobosphaera sp. was used to produce biodegradable films. Bioactive compounds, polysaccharides, and proteins were extracted from this microalga. The total phenolic content (TPC) and antioxidant activity (ABTS, DPPH, and ORAC) of the bioactive-rich extract were determined, and its composition was analyzed for phenolics using LC-ESI-QqTOF-HRMS and for lipids using GC-FID. The cytotoxicity of this extract on Caco-2 cells was also assessed. Different types of films were produced based on alginate (2%) (film A) and alginate with polysaccharides-rich (PS-rich) extract (0.5%) (film B); PS-rich extract and bioactive-rich extract (0.25%) (film C); protein-rich (P-rich) extract (0.5%) (film D); and P-rich extract and bioactive-rich extract (film E). The antioxidant activity and physical parameters of the films, such as thickness, color, water vapor permeability, solubility, tensile strength (TS), and elongation at break (EAB), were determined. The TPC of the bioactive-rich extract was 1.07 ± 0.05 mg GAE/100 mg DW, and its antioxidant activity was 2.44 ± 0.27, 1.67 ± 0.15, and 11.90 ± 1.22 µmol TE/100 mg DW for ABTS, DPPH, and ORAC, respectively. The extract showed no cytotoxicity to gut cells at concentrations equal to or below 1.0 mg/mL. Film E obtained the best results for the antioxidant activity, 451.06 ± 14.68 and 212.81 ± 39.12 µM TE/mg film for ABTS and DPPH, respectively. In addition, the films enriched with the bioactive-rich extract (films C and E) presented antimicrobial activity against Listeria monocytogenes. These films controlled the mold and yeast growth in strawberries during a four-day storage at 25 °C. All films were completely soluble in water and hydroethanolic solutions but only partially solubilized in acetic acid (3%). TS and EAB were not significantly different among the films. It was possible to produce biodegradable films using microalga Lobosphaera sp. with good bioactivity and physical characteristics.
- Comparison among different green extraction methods of polyphenolic compounds from exhausted olive oil pomace and the bioactivity of the extractsPublication . Martins, Valter F. R.; Ribeiro, Tânia B.; Lopes, Ana I.; Pintado, Manuela E.; Morais, Rui M. S. C.; Morais, Alcina M. M. B.The use of by-products as a source of bioactive compounds with economic added value is one of the objectives of a circular economy. The olive oil industry is a source of olive pomace as a by-product. The olive pomace used in the present study was the exhausted olive pomace, which is the by-product generated from the air drying and subsequent hexane extraction of residual oil from the olive pomace. The objective was to extract bioactive compounds remaining in this by-product. Various types of green extraction were used in the present study: solvent extraction (water and hydroalcoholic); ultrasound-assisted extraction; Ultra-Turrax-assisted extraction; and enzyme-assisted extraction (cellulase; viscoenzyme). The phenolic profile of each extract was determined using HPLC-DAD and the total phenolic content (TPC) and antioxidant activity (ABTS, DPPH, and ORAC) were determined as well. The results showed significant differences in the yield of extraction among the different methods used, with the enzyme-assisted, with or without ultrasound, extraction presenting the highest values. The ultrasound-assisted hydroethanolic extraction (USAHE) was the method that resulted in the highest content of the identified phenolic compounds: 2.021 ± 0.29 mg hydroxytyrosol/100 mg extract, 0.987 ± 0.09 mg tyrosol/100 mg extract, and 0.121 ± 0.005 mg catechol/100 mg extract. The conventional extraction with water at 50 °C produced the best results for TPC and antioxidant activity of the extracts. The extracts from the USAHE were able to inhibit Gram-positive bacteria, especially Bacillus cereus, showing 67.2% inhibition at 3% extract concentration.
- Conventional and natural compounds for the treatment of dermatophytosisPublication . Lopes, Ana I.; Tavaria, Freni K.; Pintado, Manuela E.Dermatophytes are a group of pathogenic fungi that exclusively infect the stratum corneum of the skin, nails, and hair, causing dermatophytosis. Superficial skin infections caused by dermatophytes have increased in the last decades. There are conventional antifungals that treat these infections, such as terbinafine, fluconazole, and others. However, the limitations of these treatments (resistance, side effects and toxicity) along with the increasing over-prescription, the misuse of these antifungals and the high treatment costs led to the search for new, alternative, natural-based antifungal drugs. These have multiple mechanisms of action, which works to their advantage, making it difficult for a fungus to create resistance mechanisms against all of them at the same time. The main objective of this work is to provide a state-of-the-art review on dermatophytes, dermatophytosis, and the existing treatments, both conventional and natural, such as chitosan and essential oils.
- Development and bioactive potential of plant residue films enriched with eucalyptus extract and oregano essential oilPublication . Melo, Adma; Lopes, Ana I.; Barros, Lillian; Tavaria, Freni; Pintado, ManuelaIntroduction: Traditional plastic food packaging is typically non-renewable and non-biodegradable [1], creating a need for new eco-friendly and sustainable alternatives, such as plant-based films. Plant agro-industrial wastes, such as peels, stalks and bagasse, have been explored as sources of polymeric matrices in film development [2]. Additionally, natural compounds from plants, like plant extracts (PEs) and essential oils (EOs) can provide these films with antimicrobial and antioxidant activities, conferring them the ability to act as food preservatives against a broad spectrum of food poisoning/contaminants microorganisms and taking the opportunity of their antioxidant properties to avoid the deterioration of fats and other food constituents [3]. Objectives: To extract and characterize the bioactive molecules from licorice residues. To produce polymeric films with licorice-based polymers incorporated with eucalyptus extract and oregano EO. To evaluate the antimicrobial and antioxidant activity of the films. Conclusions: Licorice residues are rich in insoluble fibers. These fibers were used to produce edible films incorporated with oregano EO and eucalyptus extract. Licorice residue film incorporated with eucalyptus extract and oregano essential oil inhibited the growth of S. aureus and E. coli after 2h; the film with eucalyptus extract inhibited S. aureus after 4h. All films presented antioxidant activity; the film with oregano EO presented higher antioxidant activity by ABTS, whereas the film with oregano EO and eucalyptus extract presented higher antioxidant values by DPPH.