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- Quantifying the Impact of High‐Pressure Processing on the Phenolic Profile, Antioxidant Activity, and Pollen Morphology in HoneyPublication . Scepankova, Hana; Majtan, Juraj; Pospiech, Matej; Moreira, Manuela M.; Pinto, Carlos A.; Dias, Luís G.; Estevinho, Letícia M.; Delerue‐Matos, Cristina; Saraiva, Jorge A.Honey can benefit from non‐thermal processing techniques such as high‐pressure processing (HPP) to improve its quality and bioactivity. This study investigated the impact of HPP (600 MPa for 5, 10, and 15 min) on honey's quality, including the levels of hydroxymethylfurfural (HMF), antioxidant activity, total phenolic content (TPC), and phenolic profile. HPP treatment did not significantly affect HMF or TPC levels but led to selective changes in the phenolic profile. Despite a reduction in certain phenolic compound content, HPP for 5 and 15 min caused a significant increase in the antioxidant activity (2,2‐diphenyl‐1‐picrylhydrazyl [DPPH]) of honey from the mean value of 41.8% to values of 45.4% and 49.6%, respectively. On the other hand, HPP for 10 min did not change the antioxidant activity of tested honey. A 27.5% reduction in the equatorial diameter of pollen grains was observed after HPP combined with temperature at 75°C, suggesting an improved release of bioactive compounds. The content of specific phenolic compounds, including caffeic acid, p‐coumaric acid, sinapic acid, naringin, kaempferol, and the TPC, significantly affected the DPPH activity. The increment in the antioxidant activity of HPP honey may be attributed to selective changes in the content of certain phenolic compounds and improved their extraction from pollen grains.
- Antioxidant Properties and Microbiological Stability of Yogurt Enriched with Elderberry ExtractPublication . Pascariu, Oana-Elena; Estevinho, Letícia M.; Seixas, Natália; Dopcea, Ioan; Boiu-Sicuia, Oana A.; Geicu-Cristea, Mihaela; Israel-Roming, FlorentinaThis study examines the impact of added elderberry (EDB) extract on the physico-chemical, microbiological, and sensory properties of yogurt over a 21-day storage period. Two separate experiments were conducted: the first focused on testing concentrations of 0.05%, 0.1%, 0.2%, and 0.5% (w/w) EDB extract for impoving antioxidant properties and replacing potassium sorbate, a chemical preservative commonly used in dairy products, while the second aimed to enhance the bioactive compounds’ concentration by adding 0.5% EDB extract, and to assess the effect of a sweetener (agave syrup) on the sensory profile. Both experimental approaches demonstrated a significant increase (p < 0.01) in total phenolic content. In the first experiment, there was approximately 2.6 times more total phenolic content compared to the control (with a maximum of 11.71 mg GAE/100 g for the sample with 0.2% EDB extract), and about 6 times more with the addition of 0.5% EDB extract and agave syrup (with a maximum of 25.29 mg GAE/100 g). Additionally, the IC50 value decreased for all samples with EDB extract, suggesting enhanced antioxidant activity. Specifically, the antioxidant activity was approximately 3.3 times higher for the control for samples obtained from homemade yogurt with EDB extract, and about 8 times higher for samples obtained by adding EDB extract to commercial yogurt, compared to their respective controls. The high stability of bioactive compounds during the storage period highlights the potential of EDB extract as a functional antioxidant ingredient. Microbiological analysis confirmed the safety of the yogurt, with lactic acid and mesophilic bacteria showing stable counts and minimal declines over time. In the first experiment, a reduction of about 2.3 CFU/g was observed from day 0 to day 21 in samples with 0.1% and 0.2% EDB extract, while the control sample showed a reduction of 1.84 CFU/g. However, the control sample recorded the growth of psychrophilic bacteria, yeasts, and molds. In the second experiment, the samples with 0.5% EDB extract showed an average reduction of approximately 0.35 CFU/g, while the control showed a reduction of 0.51 CFU/g, maintaining stable counts and no detectable growth of psychrophilic bacteria, yeasts, molds, or coliforms. Additionally, the inclusion of different concentrations of EDB extract, along with the combination of a higher concentration of EDB extract and agave syrup, improved the yogurt’s sensory attributes, thereby enhancing consumer acceptance. For the first experiment, 68% of the panelists expressed their preference for the samples with EDB extract, and 10% preferred the control sample. In the second experiment, 49% preferred the sample with 0.5% EDB extract and agave syrup, while 23% preferred the control sample. These findings support the integration of EDB extract into yogurt formulations to boost antioxidant properties while preserving microbiological stability. Future research should explore the potential health benefits and long-term effects of these functional dairy products.
- 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.
- Quality of bee pollen submitted to drying, pasteurization, and high-pressure processing-A comparative approachPublication . Anjos, Ofélia; Seixas, Natália; Antunes, Carlos A.L.; Campos, Maria G.; Paula, Vanessa B.; Estevinho, Leticia M.Bee Pollen is a valuable and useful natural food product that can be used for different purposes, among which medical ones. This matrix is deemed a superfood because of its chemical composition, which is rich in nutrients and possesses significant bioactivities, including antioxidant and microbiological properties. Nevertheless, the storage conditions and processing methods must be optimized to maintain their properties and maximise their application. This work investigates the best bee pollen conservation process and its impact on individual con-stituents. Monofloral bee pollen was analysed for 30 and 60 days after three different storage processes (drying, pasteurization, and high-pressure pasteurization). The results showed a decrease mainly in fatty acid and amino acid content for the dried samples. The best results were obtained with high-pressure pasteurization, maintaining the proteins, amino acids and lipids characteristics of pollen and the lowest microbial contamination.
- Use of Black Pepper Essential Oil to Produce a Healthier Chicken PâtéPublication . Rodrigues, Sandra; Leite, Ana; Vasconcelos, Lia; Pereira, Etelvina; Seixas, Natália; Estevinho, Letícia M.; Teixeira, AlfredoThis study aims to explore the effect of using black pepper essential oil (BPEO) to produce a healthier chicken pâté. Four different formulations were produced: a control formulation without back pepper and three with increasing BPEO contents. To test the effect of using BPEO, physiochemical analyses were performed at two different moments, 8 and 21 days after production. Microbiological analyses were performed 2, 9, 16, and 23 days after production. Sensory analysis to evaluate the pleasantness of the pâtés to consumers was performed 21 days after production. Finally, total phenol and flavonoid content and antioxidant activity were evaluated. Results show no significant physicochemical differences. Pâtés with no BPEO or black garlic were the most pleasant, but 0.3 or 0.5% of BPEO were not significantly less appreciated, while 1% of the EO caused a decrease in taste and global appreciation. The higher the BPEO content, the higher the phenol content, antioxidant (with an increase from 1.58 to 2.27 mg eq. Fe II/g of sample, in the Control at T23 and V3 at T23, respectively), and microbial activity (with total mesophiles count reduction from 5.91 to 5.21 log CFU/g sample in V3 from T9 to T16). The use of 1% of BPEO showed a significant effect on the reduction in mesophile counts for at least two weeks. These results highlight the potential for optimizing BPEO or black garlic content to ensure both consumer acceptance and enhanced functional properties. While further analysis will help pinpoint the best formulation, the current findings are a promising step towards achieving an optimal balance.
- 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.
- Post-transcriptional gene silencing of glucanase inhibitor protein in Phytophthora cinnamomiPublication . Ferreira, Patrick; Chahed, Abdessalem; Estevinho, Leticia M.; Seixas, Natália; Costa, Rodrigo Arthur Fonseca; Choupina, AltinoInk disease is considered one of the most significant causes contributing to the decline of chestnut orchards. The reduced yield of Castanea sativa Mill can be attributed to two main species: Phytophthora cinnamomi and Phytophthora cambivora, with the first being the main pathogen responsible for ink disease in Portugal. P. cinnamomi is a highly aggressive and widely distributed plant pathogen, capable of infecting nearly 1000 host species. This oomycete causes substantial economic losses and is accountable for the decline of numerous plant species in Europe and worldwide. To date, no effective treatments are available to combat these pathogens. Given chestnut’s economic and ecological significance, particularly in Portugal, it is crucial to investigate the molecular mechanisms underlying the interaction between Phytophthora species and host plants. This can be achieved through the study of the glucanase inhibitor protein (GIP) produced by P. cinnamomi during infection. The technique of RNA interference (RNAi) was employed to suppress the GIP gene of P. cinnamomi. The resulting transformants, carrying the silenced gene, were used to infect C. sativa, allowing for the assessment of the effects of gene silencing on the plant’s phenotype. Additionally, bioinformatics tools predicted the secretion of GIP protein. The obtained results validate RNAi as a potential alternative tool for studying molecular factors and for controlling and managing P. cinnamomi.
- Phenolic Class Analysis in Honey: Comparison of Classical and Single UV Spectrum MethodologiesPublication . Paula, Vanessa B.; Sousa-Dias, Miguel L.; Seixas, Natália; Combarros-Fuertes, Patricia; Estevinho, Leticia M.; Dias, L.G.The analytical results from a study of 16 honey samples (extra white to dark honey color range) of phenolic compounds obtained using the single UV spectrum methodology and classical spectrophotometric methods (Folin–Ciocalteu and AlCl3 methods) are presented. The first method quantified all classes of phenolic compounds in honey’s SPE-C18 extract: the total hydroxybenzoic acid content (concentrations between 0.37 ± 0.05 and 4.46 ± 0.37 mg of gallic acid/g of honey), total hydroxycinnamic acid content (0.13 ± 0.03 and 2.76 ± 0.13 mg of ferulic acid/g of honey), and total flavonoid content (0.15 ± 0.03 and 1.63 ± 0.17 mg of quercetin/g of honey). The total phenolic contents were, on average, 1.86 ± 0.72 and 1.78 ± 0.79 times higher than the results obtained for raw honey and the SPE-C18 extract, respectively, using the classical Folin–Ciocalteu method. The total flavonoid contents, on average, were 6.02 ± 3.14 times larger and 0.66 ± 0.33 times smaller than the results obtained using the classical AlCl3 method for raw honey and SPE-C18 extract, respectively.
- Comparative study of different Portuguese samples of propolis: pollinic, sensorial, physicochemical, microbiological characterization and antibacterial activityPublication . Dias, L.G.; Pereira, Ana Paula; Estevinho, Leticia M.The aim of this work was to study four propolis samples from Trás-os-Montes region of Portugal. The propolis samples’ color was different, which pollen analysis showed to be due to different botanical sources: Populus sp., Pinus sp., Quercus sp. and Castanea sativa. The data from physicochemical analysis (moisture, soluble and insoluble solids content, pH, conductivity, ash content, wax, total phenolics and flavonoids content) was treated using multivariate statistical tools as cluster heat map, principal components analysis and linear discriminant analysis with the purpose of classifying the sample accordingly to the botanical/geographical origin. The discriminant analysis was applied with stepwise to select the variables that most contribute to sample identification accordingly to pollinic profile. The cross-validation technique was applied, using the leave-one-out procedure, which showed good prediction capabilities of the samples. Microbiologically, the commercial quality was satisfactory, since the samples didn’t contain deterioration or pathogenic microorganisms. All the samples studied presented antimicrobial activity against Methicillin-resistant Staphylococcus aureus, in a dose dependent way. The antimicrobial activity was strictly related to the physicochemical composition. This work will allow connecting a particular chemical propolis type to a specific type of biological activity, what is essential for the use in therapeutic applications.
- Evaluation of an electronic tongue for honey classification according to its pollen analysisPublication . Sousa, Mara E.B.C.; Dias, L.G.; Peres, António M.; Estevinho, Leticia M.; Machado, Adélio A.S.C.Electronic tongues (ET) have attracted great interest due to its potential to obtain global Information from complex samples that could hardly be obtained by traditional instrumental methods of analysis. These multi-sensor arrays provide a huge amount of sample information which, by applying chemometric methods, allows sample identification/ classification, taste evaluation as well as, multicomponent analysis. The method of operation consists In obtaining a signal pattern which corresponds to the overall Information on the sample using chemical sensors with high stability and cross sensitivity to different species in solution. In this work, a potenclometrlc electronic tongue or taste sensor array was used. The device had 20 sensors, based on all-solid-state electrodes with lipid polymeric membranes formed on solid conducting silver supports.