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- Lipoptena fortisetosa (Diptera: Hippoboscidae) in a Red Deer in the Center of PortugalPublication . Rodrigues, Isabel; Martins, Filipa; Mateus, Teresa LetraDeer keds (Insecta: Diptera: Hippoboscidae: Lipoptena spp.) are haematophagous ectoparasites of wild as well as domestic animals, being most often associated with cervids, but also can infest Bovidae (cattle, goats, chamois, mouflon, antelopes, etc.), and occasionally humans. Upon finding a suitable mammal host, deer keds shed their wings, remaining in a wingless form for the rest of their life. These flies are viviparous species and they generate a larvae that falls to the ground and pupates. In severe infestations, these ectoparasites can be responsible for dermatitis, allergic rhinoconjunctivitis, and in extreme cases, anaphylactic shock when they bite humans. They also potential vectors of several pathogens such as Babesia spp.,Theileria spp., Bartonella spp., Coxiella spp. and Anaplasma spp. Of over 30 species of Lipoptena insects worldwide, two of the six known species in Europe, Lipoptena cervi along with L. fortisetosa, are known to have a relatively wide distribution. We report for the first time, the presence of L. fortisetosa in a red deer (Cervus elaphus) in Castelo Branco district, in the Center of Portugal. Lipoptena fortisetosa was probably introduced in Europe with sika deer from the Eastern Palaearctic and is continuously expanding its range, especially in the Southern part of Europe. There are pertinent morphological differences between the two species, that must be taken in account to facilitate their accurate identification, among others, the external features of the head and antennae, features on the thorax, number and distribution of bristles, and in the external genitalia. This new finding may represent a new challenge to the epidemiology of this species in Portugal, and to the health of its wide range hosts, particularly because it can transmit several disease-causing pathogens to animals, as well as to humans. Further investigations are worthy of attention on Lipoptena species, in Portugal, with a One Health perspective.
- Evaluation of the preservative capacity of Brassica oleracea var. Acephala extract in a bakery product.Publication . Pepinelli, Ana Luísa; Oliveira, Tatiane C. G.; Caleja, Cristina; Heleno, Sandrina A.; Rodrigues, Paula; Gonçalves, Odinei Hess; Oliveira, María B. P. P.; Barros, Lillian; Pereira, ElianaOver the years, the consumer’s preference for more natural and healthier products have been noticed. These changes have led the industry and scientific community to focus on finding possible ingredients that can act as substitutes for synthetic preservatives in food products. In this context, the objective of this study was to evaluate the effect of a natural ingredient obtained from Brassica oleracea var. Acephala biowastes as a substitute for synthetic preservatives in milk bread. For this, a rich phenolic compound extract was obtained by maceration and then lyophilised and incorporated into the food matrix. Physical, nutritional, and microbiological parameters were evaluated at a 0 and 3 storage days, and the results were compared with a control milk bread (without additives) and milk bread with the synthetic preservative sorbic acid, which is commonly used in the food industry. The results showed that the addition of the natural ingredient increased the mineral content and had a positive effect in protecting from the growth mesophilic microorganisms in milk bread. However, an adjustment in the concentration of this ingredient is necessary, for better effectiveness in the preserving action, since in previous tests, the extract presented antioxidant and antimicrobial activity of great interest. Even so, it is possible to verify that the Brassica oleracea var. Acephala extract may be considered as a potential alternative to synthetic preservatives in bakery products.
- Plant extracts: A study on the chemical characterization and bioactive potential to inhibit grape pathogens.Publication . Ardohain, Elizandra; Marx, Ítala; Rodrigues, Paula; Afonso, Andreia; Oliveira, Izamara; Pires, Tânia; Barros, Lillian; Heleno, Sandrina A.Orange (Citrus sinensis L.) peels are usually discarded as wastes; however, they are rich sources of bioactive compounds, including organic acids with biological properties[1]. In addition, fennel (Foeniculum vulgareMill.)and salvia (Salvia officinalis L.)are widespreadherbs, traditionallyused for the medicinal purposes and human consumption. Their leaves have been described as potential sources of polyphenols, such as flavonoids with antioxidant and antifungal activities[2,3]. In this sense, the present study aimed to perform the phenolic characterization of fennel and salvia leaves extracts through HPLC-DAD/ESI-MS after a maceration process. Additionally, the extraction and further characterization of orange peels’in terms of organic acids was also evaluated through HPLC-DAD. The antimicrobial potential of these plant extracts against food contaminants, including grapevine pathogens, such asBotrytis cinerea, was evaluated using the microdilution method. The results obtained showed thatthepolyphenols with the highest concentration in leave’s extractswerethe flavonoids luteolin-7-O-glucuronide and quercetin-3-O-glucuronide, for salvia and fennel, respectively. The total amount of phenolic compounds in salvia was266+1 g/mg of extract, while for fennel was30+0.7 mg/g of extract, comprising the sum of total flavonoids and phenolic acids. The concentration of determined organic acids in orange peels was 8+0.1g/100g dw, in which the main compound found was citric acid(6+0.1g/100g dw).In this study, allplant extracts have demonstratedthe ability to inhibit the growth of B. cinerea at a concentration of 10 mg/mL. Therefore, these plant extracts, due to their potential composition in bioactive agents, could be used as naturalantifungals, acting against B. cinerea. Natural antimicrobials, based on plant extracts, represent a promising alternative for disease control and could provide significant economic benefits for the wine industry.
- Raman measurements on gamma irradiated chestnut fruitsPublication . Antonio, Amilcar L.; Cabo Verde, Sandra; Cerqueira, M. FatimaIrradiation for food preservation is on use in several countries for different types of foods: fish, meat, vegetables, etc., with its application accepted and regulated by national and international food safety agencies [1]. In this work, Raman scattering measurements were performed on non-irradiated and gamma irradiated chestnut fruits at different irradiation doses (1, 2 and 5 kGy), identifying the peaks associated with main molecular structures present on chestnut fruits [2]. The intensity ratios of relevant peaks were used to correlate the obtained results with the dose and/or variety. From the spectra analysis it was possible to conclude that some molecular structures were affected by radiation, observing that bending modes and stretching modes of O– H and C– H bonds have a correlation with irradiation dose. In particular, for disaccharide molecules, such as sucrose, radiation could break glycoside bond, leading to the increase of fructose and glucose, which is in accord with previous chemical analysis for the effects of irradiation processing on chestnut fruits [3]. With the obtained results, following the appropriate peaks of Raman spectra for irradiated fruits, it was possible to identify chestnut fruits variety and, furthermore, discriminate irradiated from non-irradiated samples.
- Ethnobotanical Heritage and Flora Conservation in the Barroso Ecoregion: Sustaining Traditions in Transboundary areasPublication . Andrade, C.; Alves, P.; Serrano, M.; Pacheco, G.; Aguiar, Carlos; Vila-Viçosa, C.The Barroso region, located in northern Portugal near the Portugal-Spain border and adjacent to Galicia, is recognized for its rich ethnobotanical heritage, with around 100 plant species utilized for medicinal, culinary, and agricultural purposes. Part of the Eurosiberian Region, specifically the Cantabro-Atlantic Province, the region’s flora is dominated by the families Asteraceae, Fabaceae, and Poaceae, which together account for a significant portion of the cataloged species—Asteraceae making up 25%, Fabaceae 18%, and Poaceae 15%. These families are integral to the region’s traditional knowledge and practic es, and are used for their medicinal properties, Fabaceae species like Medicago and Trifolium for agricultural practices, and Poaceae species like Agrostis, Arrhenatherum, Poa and Festuca contributing to pasture and meadow ecosystems. Despite this botanical richness, several species face significant conservation threats, including Pinus sylvestris (EN), Valeriana o ici nalis subsp. o icinalis (EN), Sorbus aria (CR), Polygonum bistorta (CR), Selinum broteri (VU), Arnica montana subsp. atlantica (NT), and Veronica micrantha (NT). The border with Galicia hosts populations of some of the most endangered species of the Iberian northwest, such as Klasea legionensis (CR) or Thymelaea broteriana (EN), which have one of their main threats in the massive human depopulation and the loss of traditional management knowledge that a ects this mountain border area. Additionally, the region harbors some of Portugal’s best-preserved yew woodlands (habitat 9580), and large areas of mixed Quercus pyrenaica and Q. orocantabrica forests (habitat 9230), riparian Betula celtiberica woodlands (habitat 91E0) and hay-meadows (habitat 6510). Half of the territory’s surface consists of communal property, covered by a diverse mosaic of mountain mesic heathland (habitat 4030), humid heathlands (habitat 4010 and 4020), perennial herbaceous grassland (including Nardus stricta grassland, habitat 6230) and minerotrophic mires (habitat 7140). This complex landscape is main tained through a traditional grazing system featuring the Barrosã cow, an indigenous bovine breed. Empirical knowledge surrounding these species is deeply embedded in the cultural identity of local communities. However, habitat degradation and the decline of traditional agricultural and pastoral practices threaten both biodiversity and the preservation of ethnobotanical knowledge. Barroso is currently the Portuguese region with the highest number of fire ignitions and the highest ratio of annual wildfire area to total area. Conservation e orts are essential to sustain Barroso’s distinctive landscapes and habitats, including oak forests, high-altitude meadows, heathlands and mires, which support a range of endemic and endangered species. Geobotanical studies highlight the importance of specific plant communities in maintaining ecological balance and cultural heritage. The traditional land-use practices of northern Portuguese mountainsplay a crucial role in conserving these habitats. Traditional (indigenous) fire practices or, its substitute, o icial prescribed fires, and the maintenance of hay meadows (Habitat 6510) are critical for the preservation of peripheral habitats, including oak and birch woodlands and peatlands. These culturally rooted systems integrate traditional knowledge with modern conservation approaches, enhancing biodiversity, reducing wildfire risks, and preserving the cultural landscapes that define Barroso.The Barroso-Galicia transboundary region exemplifies a complex interplay between ethnobotanical richness and conservation challenges. A comprehensive understanding of the area’s biogeographical and cultural characteristics, combined with sustainable management practices, is essential to address these issues e ectively and secure the long-term preservation of biodiversity and traditional knowledge.
- Ciclagem de nutrientes num sistema misto de agricultura biológicaPublication . Fernandes, Marco; Aguiar, Carlos; Mourão, IsabelA gestão eficiente da ciclagem de nutrientes é crucial para o setor agrícola garantir uma produção estável, reduzir os impactos ambientais e otimizar a utilização da superfície agrícola. Além das tecnologias modernas, como a agricultura de precisão e o uso de bioestimulantes, as práticas tradicionais desempenham um papel fundamental na gestão de nutrientes, tais como a diversificação de culturas, rotações, o pastoreio e o uso de fontes orgânicas de nutrientes. Um estudo realizado na exploração mista de agricultura biológica (Fonte Quintela, Paçó, Vinhais), teve como objetivo principal explorar os ciclos do azoto e do fosforo à escala da exploração agrícola. Foram identificados e parcialmente quantificados os compartimentos, stocks e fluxos destes nutrientes no interior do sistema. A exploração abrange 20 parcelas maioritariamente submetidas a um regime extensivo de pastoreio, com foco na produção de ovinos de raça Churra Galega Bragançana e de castanha DOP Terra Fria Transmontana. A produção de biomassa forrageira é essencial para o funcionamento da exploração e determinante na ciclagem de nutrientes. Para cada parcela foram determinados parâmetros como área total, características químicas do solo, produção de biomassa forrageira, produtividade e concentração de nutrientes (N e P) na forragem. A biomassa e os estrumes são movimentados dentro do sistema por reboque e pelos animais em pastoreio. O estudo permitiu identificar as componentes do sistema em perda ou a acumular P, e as que mais importam e exportam N. A partir da informação obtida foram desenhadas alterações na gestão da ciclagem de nutrientes no sistema de modo melhorar o desempenho ambiental e económico da exploração agrícola.
- Effect of drying process and incorporation of natural preservatives in the viability of lactic acid bacteria in yogurtsPublication . Ueda, Jonata Massao; Rodrigues, Paula; Barros, Lillian; Morales, Patricia; Carocho, Márcio; Heleno, Sandrina A.Dairy products are considered one of the most consumed foods in the world, and yogurts are highly demanded for their flavour and nutritional properties, also including macronutrients, vitamins, minerals, or probiotics offered. To avoid food waste, alternatives to preserve food are highlighted, such as the addition of food preservatives or the production of powdered foods. Therefore, the objective of this work was to evaluate the survival of total lactic acid bacteria when natural extracts (rosemary, basil, and sage) were incorporated into yogurts, as well as to investigate their viability after the freezedrying process. Yogurts were prepared in the laboratory, and lyophilised plant extracts (30 mg/kg) were added. Total lactic acid bacteria were analysed for 14 days, in addition to evaluating the lactic acid and lactose contents by HPLC-DAD and HPLC-RI, respectively. Subsequently, the viability of Streptococcus thermophilus and Lactobacillus bulgaricus were evaluated in freeze-dried yogurts without the addition of vegetable extracts. Regarding the results obtained, there was a slight increase in the lactic acid bacteria counts, in addition to an increase in lactic acid and a reduction in the lactose content over 14 days, confirming the yogurt fermentation process. For the viability of S. thermophilus and L. bulgaricus during the lyophilisation process, a slight reduction of both bacteria was observed, but within the limits established by the Codex Standards (minimum of 107CFU/g). Therefore, the incorporation of plant extracts and the freeze-drying process did not significantly change the yogurts regarding the viability of the lactic acid bacteria, remaining a probiotic food.
- In vitro evaluation of plant extracts as antifungal agents against Botrytis cinerea.Publication . Marx, Ítala; Ardohain, Elizandra; Rodrigues, Paula; Barros, Lillian; Heleno, Sandrina A.Grapevine bunch rot, caused by Botrytis cinerea, causes important economic losses every year in grape production [1]. However, indiscriminate use of pesticides to control this disease leads to serious problems such as consumer intoxication and the emergence of populations of pathogens resistant to fungicides. In this sense, the development of new natural ingredients against B. cinerea is of paramount relevance. Plant extracts are a valuable source of a wide variety of biologically active chemical defense constituents. Several plant bioactive compounds have been identified as possible antifungal agents, also associated with their antioxidant properties [2]. The objective of this work was to evaluate the effect of plant extracts rich in phenolic compounds (concentration of 10 mg/mL, each) on mycelial growth of B. cinerea in vitro. In this study, three formulations of plant extracts were evaluated (PreVine 1, PreVine 2 and PreVine 3). Potato Dextrose Broth (PDB) medium was prepared with agar and enriched with the three formulations of plant extracts. Negative control (CTR -) was treated only with water, and the positive control (CTR +) was treated with the conventional fungi cide Teldor® (active substance fenhexamid), concentration of 0.075 mg/mL. The obtained results demonstrated that all plant extracts formulations revealed the capacity to inhibit the growth of B. cinerea. The inhibitory potential was greater for PreVine 1, being responsible for inhibiting the growth of B. cinerea by 64% at 9 days of incubation, when compared to the control samples. As regards to the growth rate, plant extracts were able to reduce this parameter from 1.8391 (CTR -) to 1.2051 cm/day (PreVine 1). The positive antifungal activity of the plant extracts assessed, especially for the PreVine 1, could be attributed to their varied bioactive composition, such as several classes of phenolic compounds (i.e., flavonoids, phenolic acids, and tannins). In fact, these plant extracts have been described as source of compounds with biological activity against grape pathogens, by promoting a disruption of fungal cell membranes, inhibiting fungal enzymes and/ or inducing oxidative stress in fungal cells [3,4]. Considering the potential antifungal properties of these plant extracts formulations, these natural formulations could be candidates for application grape and wine industries as biofungicides, acting against grape pathogens, such asB. cinerea, decreasing, or avoiding the use of chemical fungicides.
- Natural solutions for grape pathogen control: chemical analysis and bioactivity evaluation of plant extractsPublication . Ardohain, Elizandra; Marx, Ítala; Rodrigues, Paula; Afonso, Andreia; Oliveira, Izamara; Pires, Tânia C.S.P.; Barros, Lillian; Heleno, Sandrina A.Peels from orange (Citrus sinensis L.) are often considered processing waste. Citrus peels contain bioactive compounds such as organic acids that have important biological properties [1]. In addition, fennel (Foeniculum vulgare Mill.) and sage (Salvia officinalis L.) are widely used herbs that have been used for medicinal purposes and human consumption for centuries. The leaves of these herbs contain polyphenols, which are antioxidant and antifungal compounds such as flavonoids [2,3]. To explore the potential of these plants as natural antifungals, this study aimed to analyze the phenolic compounds in extracts of fennel and sage leaves using HPLC-DAD/ESI-MS, after a maceration process. The study also evaluated the organic acids present in orange peels using HPLC-DAD. Finally, the antimicrobial potential of the plant extracts against food contaminants, including grapevine pathogens such as Botrytis cinerea, was tested using the microdilution method. This study found that the highest concentration of polyphenols in the leaves of sage and fennel were the flavonoids luteolin-7-O-glucuronide and quercetin-3-O-glucuronide, respectively. Sage leaves extract contained 69% of flavonoids and 31% of phenolic acids, while fennel leaves extract was richer in phenolic acids, corresponding to 61%, and the flavonoids were found in lower concentrations, corresponding to 39% of total phenolic composition. The concentration of organic acids in orange peels was 8±0.1g/100g dw, with citric acid being the main compound found, corresponding to 75%, while ascorbic acid corresponding to 18% of total organic acids determined. In this study, oxalic and quinic acids were also found in orange peels extract. All plant extracts demonstrated the ability to inhibit the growth of B. cinerea at a concentration of 10 mg/mL, indicating their potential as natural antifungals against this grapevine pathogen. Thus, these plant extracts have the potential to act as natural antimicrobials, showing promising results for disease control in grapevines. In this sense, the valorization of these plant extracts could generate significant economic benefits for the wine industry.
- Chemical characterization of Cistus ladanifer L. phenolic extract and its antifungal activity against Botrytis cinerea.Publication . Marx, Ítala; Ardohain, Elizandra; Rodrigues, Paula; Afonso, Andreia; Pires, Tânia C.S.P.; Dias, Maria Inês; Barros, Lillian; Heleno, Sandrina A.Botrytis bunch rot, caused by Botrytis cinerea, is an important disease of grapevines in temperate climates worldwide. It can cause extensive economic losses through grape desiccation, rot, and biochemical changes that reduce wine quality. Therefore, it is crucial to identify new compounds, especially natural ones, that are active against B. cinerea. Biofungi cides from plant origin have been recommended to reduce viticulture’s dependence upon synthetic fungicides for the last decades [1]. Cistus (Cistus ladanifer L.) is a perennial shrub from the Cistaceae family that can be found in abundance in the Mediterranean's marginal fields [2]. Moreover, cistus phenolic extract has been described as a potential antifungal agent, due to its interesting phenolic composition, including ellagitannins and flavonoids [3]. In this sense, the present study evaluated the antifungal potential of a phenolic extract of C. ladanifer from Northeastern Portugal, against B. cinerea. Dry leaves from cistus were extracted by maceration using ethanol: water (80:20 v/v) as solvent. The phenolic extracts were characterized by HPLC-DAD/ESI-MS and evaluated for their capacity to inhibit B. cinerea using the microdilution method. Ellagic acid derivatives (24.3 mg/g extract; 83.8%), flavonoids (3.93 mg/g extract; 13.6%), such as flavonols and flavones; phenolic acids, and derivatives (0.76 mg/g extract; 2.6%), were found in the sample (Figure 1). The most abundant group was ellagic acid derivatives in which punicalagin and punicalagin gallate, were found in the highest amounts (13.3±0.9 and 11±2 mg/g extract, respectively), being these results in line with previous studies [3]. The extract revealed an interesting capacity to inhibit the growth of B. cinerea at a concentration of 10 mg/mL, which could be related with the high composition in ellagitannins. Considering the bioactivity of cistus phenolic extract, this natural product could be applied as an alternative to synthetic fungicides used in the prevention and treatment of fungal infections in grapevine, minimizing the environmental and health impacts caused by these chemical agents.