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- Melipona scutellaris geopropolis: chemical composition and bioactivityPublication . Coutinho, Sónia; Matos, Vanessa; Seixas, Natália; Rodrigues, Hellen; Paula, Vanessa B.; Freitas, Lais; Dias, Teresa; Santos, Francisco de A.R.; Dias, L.G.; Estevinho, Leticia M.Geopropolis has been used in traditional medicine for centuries. In this study, the botanical origin, physicochemical profile, and biological activities of geopropolis from Melipona scutellaris harvested during rainy and dry seasons were investigated. Palynological analysis identified over 50 pollen types, with Schinus terebinthifolius and Cecropia being the predominant types. The analytical results were in line with those reported in the literature. Rainy-season geopropolis exhibited higher total phenol and flavonoid content (determined using High Performance Liquid Chromatography-25.13% and 3.92%, respectively) compared to the dry season (19.30% and 2.09%); the major peaks (naringin, gallic acid, and catechin) were similar among samples. Antioxidant capacity was assessed via DPPH, reducing power, and beta-carotene/linoleic acid discoloration assays. Rainy-season samples displayed superior antioxidant activity across methods. Antimicrobial effects were determined using microdilution, while the impact on the cholinesterase enzyme was quantified using 5-thio-2-nitrobenzoic acid accumulation. Anti-inflammatory and antimutagenic activities were assessed through hyaluronidase enzyme inhibition and by utilizing Saccharomyces cerevisiae ATCC-20113 cells. Both samples exhibited anti-inflammatory and antimutagenic properties. Moreover, a significant inhibition of acetylcholinesterase was observed, with IC50 values of 0.35 mu g/mL during the rainy season and 0.28 mu g/mL during the dry season. Additionally, the geopropolis displayed antimicrobial activity, particularly against Staphylococcus aureus. These findings suggest the therapeutic potential of M. scutellaris geopropolis in the context of inflammatory, oxidative, and infectious diseases.
- The Effect of Incorporating Fermented Elderberries (Sambucus nigra) into Bread: Quality, Shelf Life, and Biological EnhancementPublication . Seixas, Natália; Paula, Vanessa B.; Dias, Teresa; Dias, Luís G.; Estevinho, Letícia M.Elderberries, known for their antioxidant, anti-inflammatory, and antiviral properties, have traditionally been used to prevent and treat infections and boost the immune system. By increasing the quantity and quality of certain compounds, fermentation can potentially make them more effective as food additives. The aim of this study was to evaluate the effect of incorporating fermented elderberries on the bioactivity and shelf life of a traditional bread. The elderberry fermentation process was optimised using Saccharomyces cerevisiae, guided by a Plackett–Burman experimental design. The aim was to assess the impact of incorporating fermented elderberries into bread on its bioactive properties and shelf life. The fermentation of the elderberries was found to enhance their bioactive compounds and antioxidant activity. The total phenolic content ranged from 8.63 to 20.56 mg GAE/g (in samples without and with 2% extract, respectively). The antioxidant capacity, measured using the FRAP method, also showed a significant increase with the addition of the extract (from 9.16 to 26.66 mg Fe (II) E/g of the sample). Furthermore, bread enriched with fermented elderberry extracts demonstrated an extended shelf life during the study period.
- Eficácia da própolis no controlo da loque americana. Avaliação em zonas controladasPublication . Vilas-Boas, Miguel; Veloso, Ana C.A.; Peres, António M.; Dias, L.G.; Dias, Teresa; Cardoso, Susana M.; Kluskens, Leon; Rocha, Isabel
- Influence of the storage conditions (Frozen vs. Dried) in health-related lipid indexes and antioxidants of bee pollenPublication . Estevinho, Leticia M.; Dias, Teresa; Anjos, OféliaFollowing harvest, bee pollen must be submitted to processing in order to maintain properties for consumers’ health insurance. In this study, the changes on the lipid profile, contents of vitamin C, β-carotene and lycopene of bee pollen samples submitted to two conservation methods (freezing and drying) are evaluated. Eleven fatty acids, eight saturated, one monounsaturated, and two polyunsaturated are quantified. The PUFA/SFA ratio ranges from 1.18 to 3.95 g 100−1 g−1 and is significantly higher in the frozen extracts. On the other hand, the ratio n6:n3 (ranging between 0.36 and 0.86 g 100−1 g−1) did not differ among processing methodologies, for most of the cases. The atherogenicity (AI) and thrombogenecity (TI) indexes are similar among preservation processes and coherent with the found on other health-promoting foods. The contents of vitamin C, β-carotene and lycopene are, for all samples, significantly superior in the frozen bee pollen. Practical Applications: In the recent years the interest in natural products has mushroomed. Indeed, in addition to the good nutritional composition bee pollen possesses important bioactive compounds and promising health promoting activities that remain unstudied. In this study, it is evaluated the influence of the geographical origin and two storage methods (drying and freezing) in the fatty-acid profile, lycopene, vitamin C and β-carotene contents and on two lipid indexes of bee pollen. Results suggest that bee pollen may act as a complement for the prevention of atherosclerosis and thrombogenesis. Regarding storage it wi inferred that freezing allows preserving the bioactive compounds in a greater extent and, therefore, must be the preferred method. Further studies may be performed in order to take advantage of this natural products’ potential. From the nutritional point of view, it is better to consume bee pollen frozen in comparison to that of dried.
- Honeybee-collected pollen from five Portuguese Natural Parks: palynological origin, phenolic content, antioxidant properties and antimicrobial activityPublication . Dias, Teresa; Morais, Margarida; Moreira, Leandro; Estevinho, Leticia M.The aim of this study was to determine the palynological origin, phenolic content, antioxidant properties and antibacterial activity of honeybee- collected pollen from five Portuguese Natural-Parks [Parque Nacional Peneda Geres (PNPG); Parque Natural do Montesinho (PNM); Parque Natural do Alvao (PNA); Parque Natural da Serra da Estrela (PNSE) and Parque Natural do Douro Internacional (PNDI)]. Eight families were found in the mixture of bee pollen: Rosaceae, Cistaceae, Boraginaceae, Asteraceae, Fagaceae, Ericaeae, Myrtaceae and Fabaceae. The phenolic compounds content, determined by the Folin-Ciocalteu method, varied between 10.5 and 16.8 mg of gallic acid equivalents/ g of extract (mg GAE/ g) found in bee pollen from PNM and PNDI, respectively. The free radical scavenging measured showed the highest effective extract - PNM with ECSO 2.16, followed by PND with 2.24 mg/ mL. In the 0-carotene bleaching assay the same behaviour as in the DPPH method was verified. We also verified that the presence of pollen differentially affected the growth of bacteria Gram-positive, Gram-negative and fetmentative yeasts under study, depending on the microorganism and the type of bee pollen used. This is an important study since, as far we know, it is the first time that Portuguese bee pollen from Natural Parks was studied, and their characterization can increase their economic value.
- Assessment of physiological conditions in e. Coli fermentations by epifluorescent microscopy and image analysisPublication . Carneiro, Sónia; Amaral, António L.; Veloso, Ana C.A.; Dias, Teresa; Peres, António M.; Ferreira, Eugénio C.; Rocha, IsabelThe development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes.
- An assessment of the processing and physicochemical factors contributing to the microbial contamination of salpicão, a naturally-fermented Portuguese sausagePublication . Cadavez, Vasco; Gonzales-Barron, Ursula; Pires, P.; Fernandes, E.; Pereira, Ana Paula; Gomes, Aline; Araújo, José Pedro; Lopes-da-Silva, M.F.; Rodrigues, Paula; Fernandes, Conceição; Saavedra, Maria José; Butler, Francis; Dias, TeresaComplete microbiological, physicochemical and environmental longitudinal data were obtained from factory surveys in order to elucidate risk factors leading to the survival of Enterobacteriaceae, Staphylococcus aureus and Listeria monocytogenes in salpicão sausage. A total of six batches were surveyed from two factories; one added nitrite and polyphosphates in their formulation (Factory II). The addition of polyphosphates slowed down fermentation, which favoured the increase in Enterobacteriaceae (∼0.5 log CFU/g), S. aureus (∼0.5 log CFU/g) and L. monocytogenes (∼70 CFU/g) until maceration. Maceration should be regarded as a critical stage in the control of S. aureus in salpicão, as longer maceration (p = 0.033), higher temperatures of maceration (p = 0.018) and pH of macerated meat (p = 0.079) led to higher S. aureus counts in the final product. The main hurdle controlling S. aureus in salpicão is the pH. For L. monocytogenes and Enterobacteriaceae, at least three hurdles hinder their viability: low Aw (p = 0.010; 0.012), smoking (p < 0.0001; 0.020) and nitrite (p = 0.158; 0.105). Cross-contamination, especially during mixing, should be avoided as it is an important factor explaining the increase in Enterobacteriaceae (p = 0.041), S. aureus (p = 0.015) and L. monocytogenes (p = 0.082) on a batch basis. There is a need to optimise the acidification process and reinforce good hygiene practices
- Isolation and sequence analysis of alfa-tubulin gene from Phytophora cinnamomiPublication . Dias, Teresa; Andrade, Maria; Jorge, Lurdes; Vaz, Madalena; Martins, Fátima; Dominguez, Ángel; Choupina, AltinoPhytophthora diseases cause widespread economic and environmental losses worldwide. Thousands of plant species are susceptible. In Portugal, Phytophthora cinnamomi is responsible for chestnut ink disease. Despite the differences there are a number of key steps common to most infection strategies, including adhesion to the plant surface, plant penetration through the secretion of a diverse range of cell wall-degrading enzymes and hyphal growth. The cell cytoskeleton plays a critical role in these processes. Microtubules are a major constituent of the cell cytoskeleton. They participate in a wide range of cellular functions, such as motility, division, maintenance of cell shape, and intracellular transport. However, microtubule role is variable depending on the organism, cell type and other factors. Tubulin is the major constituent of microtubules and is composed of a heterodimer of two closely related proteins, alpha and beta tubulin. In S. cerevisiae cells, the essential TUB1 gene is the major gene, while the nonessential gene TUB3 is a minor gene, encoding α-tubulin. The β-tubulin subunit is encoded by the TUB2 gene. In Magnaporthe grisea both α-and β-tubulins are found as single-copy genes. The Oomycetes are, however, phylogenetically quite distinct from the fungi. Analysis of structural, biochemical and molecular characteristics have led to the Oomycetes being grouped with the chromophyte algae. In order to elucidated the role of cytoskeleton in pathogenicity mechanisms of Phytophthora cinnamomi, was cloned a gene encoding alpha-tubulin from P. cinnamomi. To isolated this gene, the existing Tub1 nucleotide sequences were retrieved from the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank). These sequences were aligned in Clustal and degenerate primers Tub1 and Tub2 were designed. A 1200bp fragment was generated from genomic DNA by PCR and subsequently cloned into pGEM-T vector. To complete the open reading frame it was used the HE-TAIL PCR. The complete ORF was sequenced and submitted in EMBL databases (Accession number AM412177.1). Based on the computational analysis through BioEdit software, TUB1 has a 1362 bp ORF and encodes a 453 a.a protein with a molecular weight of 49,911kDa. Phylogenetic analysis of deduced amino acid sequence using FASTA programs from EMBL databases revealed that Tub1 revealed 99.6% identity with alpha-tubulin of P. infestans T30.4 and 98.9% identity with P. capsici, but only 68,1 % with alpha-tubulin of S. cerevisiae.
- Dietary sugars analysis: quantification of fructooligossacharides during fermentation by HPLC-RI methodPublication . Correia, Daniela M.; Dias, L.G.; Veloso, Ana C.A.; Dias, Teresa; Rocha, Isabel; Rodrigues, Lígia R.; Peres, António M.In this work, a simple chromatographic method is proposed and in-house validated for the quantification of total and individual fructooligossacharides (e.g., 1-kestose, nystose, and 1F-fructofuranosylnystose). It was shown that a high-performance liquid chromatography with refractive index detector could be used to monitor the dynamic of fructooligossacharides production via sucrose fermentation using Aspergillus aculeatus. This analytical technique may be easily implemented at laboratorial or industrial scale for fructooligossacharides mass-production monitoring allowing also controlling the main substrate (sucrose) and the secondary by-products (glucose and fructose). The proposed chromatographic method had a satisfactory intra- and inter-day variability (in general, with a relative standard deviation lower than 5%), high sensitivity for each sugar (usually, with a relative error lower than 5%), and low detection (lower than 0.06+/-0.04 g/L) and quantification (lower than 0.2+/-0.1 g/L) limits. The correct quantification of fructooligossacharides in fermentative media may allow a more precise nutritional formulation of new functional foods, since it is reported that different fructooligossacharides exhibit different biological activities and effects.
- Encapsulation efficiency of Lactobacillus plantarum microencapsulado in Acrycoat S100Publication . Vasconcelos, Lia; Silva-Buzanello, Rosana; Kalschne, Daneysa Lahis; Dias, Teresa; Canan, Cristiane; Corso, Marinês PaulaSeveral studies have attributed health benefits to probiotics, as the contribution to intestinal microflora activity (Khan et al. 2013). However, adverse conditions in gastrointestinal transit can reduce the viability of probiotics as Lactobacillus plantarum. Acrycoat S100 is a co-polymer from methacrylic acid and methyl methacrylate, water insoluble and soluble in pH ≥ 7. Therefore, microencapsulation of probiotic in Acrycoat S100 could allow microorganism protection until it reach the intestine. The objective of this study was to determine the encapsulation efficiency of L. plantarum microencapsulated in Acrycoat S100.