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- Exploring geographical influences on physicochemical characteristics of honey: the Montesinho Natural Park scenarioPublication . Soares, Sónia; Magalhães, Leandro; Moreira, Manuela M.; Rede, Diana; Fernandes, Virgínia Cruz; Viegas, Olga; Pinto, Edgar; Almeida, Agostinho; Azevedo, Rui; Delerue-Matos, CristinaIn recent years, there has been a substantial increase in the global consumption of honey, driven by the high demand for natural products that offer health benefits. Consequently, consumers show a preference for honey, recognised for its superior quality, namely honey with a Protected Designation of Origin (PDO) or honey produced in protected areas, as it is associated with superior attributes and characteristics. Portugal is one of the leading countries in the production of PDO honey, with Montesinho Natural Park (MNP) being an excellent example of high-quality Portuguese honey, known for its distinctive attributes. However, environmental threats pose a double challenge, leading to a decline in honey production and compromising its overall quality. This study aimed to assess the specific physicochemical parameters and nutritional characteristics of MNP honey and investigate their correlation with the different locations of apiaries. MNP honey samples (n=13) were obtained from local producers or purchased at supermarkets. Standard physicochemical parameters, such as 5-hydroxymethylfurfural, diastase activity, moisture and ash content, free acidity, electrical conductivity and pH, were determined according to honey legislation. Additionally, colour parameters, protein, low-molecular-weight carbohydrates (mono- and disaccharides), and mineral content were determined. The results obtained were consistent with the requirements outlined in the legislation and those described in the literature. The results suggest that geographical factors within the park and boundaries do not contribute to variations in the analysed parameters. A significant level of homogeneity was evident in all parameters evaluated among the MNP honey samples. This is the first comprehensive study of the physicochemical properties of honey from various apiaries within the MNP.
- High-value compounds in papaya by-products (Carica papaya L. var. Formosa and Aliança): Potential sustainable use and exploitationPublication . Vinha, Ana F.; Costa, Anabela S. G.; Santo, Liliana Espírito; Ferreira, Diana M.; Sousa, Carla; Pinto, Edgar; Almeida, Agostinho; Oliveira, Maria Beatriz P. P.Food waste is a global and growing problem that is gaining traction due to its environmental, ethical, social, and economic repercussions. Between 2022 and 2027, the worldwide papaya market is expected to have a huge increase, meaning a growth in organic waste, including peels and seeds. Thus, this study evaluated the potential use of peels and seeds of two mature papaya fruits as a source of bioactive compounds, converting these by-products into value-added products. Proximate analysis (AOAC methods), mineral content (ICP-MS), free sugars (HPLC-ELSD), fatty acid composition (GC-FID), vitamin E profile (HPLC-DAD-FLD), and antioxidant activity (DPPH and FRAP assays) were evaluated. Both by-products showed high total protein (20–27%), and dietary fiber (32–38%) contents. Papaya peels presented a high ash content (14–16%), indicating a potential application as a mineral source. 14 fatty acids were detected, with α-linolenic acid (30%) as the most abundant in the peels and oleic acid (74%) in the seeds. Both by-products showed high antioxidant activity. Conclusion: Papaya by-products display great potential for industrial recovery and application, such as formulation of new functional food ingredients.
- An eco-friendly approach for analysing sugars, minerals, and colour in brown sugar using digital image processing and machine learningPublication . Alves, Vandressa; Santos, Jeferson M. dos; Viegas, Olga; Pinto, Edgar; Ferreira, Isabel M.P.L.V.O.; Lima, Vanderlei Aparecido; Felsner, Maria L.Brown sugar is a natural sweetener obtained by thermal processing, with interesting nutritional characteristics. However, it has significant sensory variability, which directly affects product quality and consumer choice. Therefore, developing rapid methods for its quality control is desirable. This work proposes a fast, environmentally friendly, and accurate method for the simultaneous analysis of sucrose, reducing sugars, minerals and ICUMSA colour in brown sugar, using an innovative strategy that combines digital image processing acquired by smartphone cell with machine learning. Data extracted from the digital images, as well as experimentally determined contents of the physicochemical characteristics and elemental profile were the variables adopted for building predictive regression models by applying the kNN algorithm. The models achieved the highest predictive capacity for the Ca, ICUMSA colour, Fe and Zn, with coefficients of determination (R2) ≥ 92.33 %. Lower R2 values were observed for sucrose (81.16 %), reducing sugars (85.67 %), Mn (83.36 %) and Mg (86.97 %). Low data dispersion was found for all the predictive models generated (RMSE < 0.235). The AGREE Metric assessed the green profile and determined that the proposed approach is superior in relation to conventional methods because it avoids the use of solvents and toxic reagents, consumes minimal energy, produces no toxic waste, and is safer for analysts. The combination of digital image processing (DIP) and the kNN algorithm provides a fast, non-invasive and sustainable analytical approach. It streamlines and improves quality control of brown sugar, enabling the production of sweeteners that meet consumer demands and industry standards.
- Toxic and non-toxic cyanobacterial biomass as a resource for sustainable agriculture: A lettuce cultivation experimentPublication . Massa, Anabella; Santos, Érica; Martins, Diogo; Azevedo, Joana; Reimão, Mariana; Almeida, Agostinho; Azevedo, Rui; Pinto, Edgar; Vasconcelos, Vítor; Campos, Alexandre; Freitas, MarisaCyanobacteria represent a promising resource for sustainable agriculture, as they have demonstrated the ability to restore soil fertility even after death and decay. However, several cyanobacteria can also release secondary metabolites, such as cyanotoxins, which may compromise the quality of agricultural products and pose a potential risk to human health. Depending on the concentration of exposure, few studies reported deleterious effects on plant species when irrigated with cylindrospermopsin (CYN) contaminated water, impairing plant growth and leading to food product contamination, while other studies show promoting effects on plant yield. To evaluate the potential of cyanobacterial biomass (cyanotoxin-containing or not) as a sustainable resource for soil amendment, biostimulants or fertilizers for lettuce cultivation, a study was carried out that consisted of the culture of lettuce plants under controlled conditions, in soil: (1) with no extra nutrient addition (control) and supplemented with 0.6 g of freeze-dried Raphidiopsis raciborskii biomass of (2) a non-CYN-producing strain, (3) a CYN-producing strain, and (4) the same CYN-producing strain pasteurized. Results showed no significant differences in photosystem II efficiency with the amendment addition. On the contrary, shoot fresh weight significantly increased in lettuce plants grown with the cyanobacterial biomass addition, especially in condition (3). In addition, there were significant differences in mineral concentrations in lettuce leaves after the cyanobacterial biomass addition, such as K, Na, Ca, P, Mg, Mn, Zn, Cu, Mo, and Co. CYN accumulation was detected under conditions (3) and (4), with concentrations observed in descending order from roots > soil > shoot. Nevertheless, the CYN concentration in edible tissues did not exceed the WHO-proposed tolerable daily intake of 0.03 μg/kg/day. These findings suggest that incorporating cyanobacterial biomass as a soil amendment, biostimulant or fertilizer for lettuce cultivation, even with trace amounts of CYN (1–40 μg/g), may enhance plant yield without leading to cyanotoxin accumulation in edible tissues above the WHO-recommended tolerable daily intake.
- Application of real-time PCR in the assessment of the toxic cyanobacterium cylindrospermopsis raciborskii abundance and toxicological potentialPublication . Moreira, Cristiana; Martins, António; Azevedo, Joana; Freitas, Marisa; Regueiras, Ana; Vale, Micaela; Antunes, Agostinho; Vasconcelos, VítorCyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.
- Environmentally friendly and cost-effective approaches to reduce toxin content in toxic cyanobacterial biomassesPublication . Loss, Letícia; Azevedo, Joana; Azevedo, Tomé; Freitas, Marisa; Vasconcelos, Vítor; Campos, AlexandreCyanobacterial outgrowths are naturally occurring processes in eutrophic aquatic ecosystems. Furthermore, as a result of climate change and anthropogenic pollution, cyanobacteria harmful algal blooms (CyanoHABs) are expanding worldwide. CyanoHABs are considered a threat to human health and environment due to the production of potent toxic substances, but at the same time, valuable products can be obtained from these microorganisms. The main objective of this study was to test straightforward and cost-effective methods to reduce the toxin content of cyanobacterial biomass for the exploitation of this important biological resource. To carry out this study, lyophilized or hydrated biomass from microcystin-LR (MC-LR) producing Microcystis aeruginosa and cylindrospermopsin (CYN) producing Chrysosporum ovalisporum strains were subjected to the following treatments: (1) thermal (50 °C); (2) ultraviolet (UV) radiation; (3) ozone; and (4) sunlight, for periods varying between 2 and 12 h. MC-LR and CYN concentrations were quantified by LC-MS and compared between experimental groups. The results show a significant reduction in the amount of MC-LR in M. aeruginosa biomass (lyophilized and hydrated) exposed to sunlight. Since no other treatment reduced MC-LR in M. aeruginosa biomass, this molecule was demonstrated to be very stable. Regarding CYN, the concentration of this toxin in C. ovalisporum biomass was significantly reduced with the exposure to UV radiation, to approximately 51% of the initial concentration after 2 h of exposure; 86% reduction after 5 h of exposure; and 77% reduction after 12 h of exposure. Overall, this study demonstrates that the toxicity of cyanobacterial biomass can be reduced by employing environmentally friendly and cost-effective treatments with sunlight and UV radiation.
- Unraveling the role of metals and organic acids in bacterial antimicrobial resistance in the food chainPublication . Rebelo, Andreia; Almeida, Agostinho; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaAntimicrobial resistance (AMR) has a significant impact on human, animal, and environmental health, being spread in diverse settings. Antibiotic misuse and overuse in the food chain are widely recognized as primary drivers of antibiotic-resistant bacteria. However, other antimicrobials, such as metals and organic acids, commonly present in agri-food environments (e.g., in feed, biocides, or as long-term pollutants), may also contribute to this global public health problem, although this remains a debatable topic owing to limited data. This review aims to provide insights into the current role of metals (i.e., copper, arsenic, and mercury) and organic acids in the emergence and spread of AMR in the food chain. Based on a thorough literature review, this study adopts a unique integrative approach, analyzing in detail the known antimicrobial mechanisms of metals and organic acids, as well as the molecular adaptive tolerance strategies developed by diverse bacteria to overcome their action. Additionally, the interplay between the tolerance to metals or organic acids and AMR is explored, with particular focus on co-selection events. Through a comprehensive analysis, this review highlights potential silent drivers of AMR within the food chain and the need for further research at molecular and epidemiological levels across different food contexts worldwide.
- Exploring peracetic acid and acidic pH tolerance of antibiotic-resistant non-typhoidal Salmonella and Enterococcus faecium from diverse epidemiological and genetic backgroundsPublication . Rebelo, Andreia; Duarte, Bárbara; Freitas, Ana R.; Peixe, Luísa; Antunes, Patrícia; Novais, CarlaAcid stress poses a common challenge for bacteria in diverse environments by the presence of inorganic (e.g., mammals’ stomach) or organic acids (e.g., feed additives; acid-based disinfectants). Limited knowledge exists regarding acid-tolerant strains of specific serotypes, clonal lineages, or sources in human/animal pathogens: namely, non-typhoidal Salmonella enterica (NTS) and Enterococcus faecium (Efm). This study evaluated the acidic pH (Mueller–Hinton acidified with HCl) and peracetic acid (PAA) susceptibility of Efm (n = 72) and NTS (n = 60) from diverse epidemiological/genetic backgrounds and with multiple antibiotic resistance profiles. Efm minimum growth/survival pH was 4.5–5.0/3.0–4.0, and for NTS it was 4.0–4.5/3.5–4.0. Efm distribution among acidic pH values showed that only isolates of clade-non-A1 (non-hospital associated) or the food chain were more tolerant to acidic pH compared to clade-A1 (hospital-associated clones) or clinical isolates (p < 0.05). In the case of NTS, multidrug-resistant (MDR) isolates survived better in acidic pH (p < 0.05). The PAA MIC/MBC for Efm was 70–120/80–150 mg/L, and for NTS, it was 50–70/60–100 mg/L. The distribution of Efm among PAA concentrations showed that clade-A1 or MDR strains exhibited higher tolerance than clade-non-A1 or non-MDR ones (p < 0.05). NTS distribution also showed higher tolerance to PAA among non-MDR and clinical isolates than food chain ones (p < 0.05) but there were no differences among different serogroups. This unique study identifies specific NTS or Efm populations more tolerant to acidic pH or PAA, emphasizing the need for further research to tailor controlled measures of public health and food safety within a One Health framework.
- Decoding Klebsiella pneumoniae in poultry chain: Unveiling genetic landscape, antibiotic resistance, and biocide tolerance in non-clinical reservoirsPublication . Mourão, Joana; Magalhães, Mafalda; Ribeiro-Almeida, Marisa; Rebelo, Andreia; Novais, Carla; Peixe, Luísa; Novais, Ângela; Antunes, PatríciaThe rise of antibiotic resistance in the food chain is influenced by the use of antimicrobial agents, such as antibiotics, metals, and biocides, throughout the entire farm-to-fork continuum. Besides, non-clinical reservoirs potentially contribute to the transmission of critical pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae. However, limited knowledge exists about the population structure and genomic diversity of K. pneumoniae circulating in conventional poultry production. We conducted a comprehensive characterization of K. pneumoniae across the whole chicken production chain (flocks/environment/meat, 2019-2022), exploring factors beyond antibiotics, like copper and quaternary ammonium compounds (QACs). Clonal diversity and adaptive features of K. pneumoniae were characterized through cultural, molecular (FT-IR), and whole-genome-sequencing (WGS) approaches. All except one flock were positive for K. pneumoniae with a significant increase (p < 0.05) from early to pre-slaughter stages, most persisting in chicken meat batches. Colistin-resistant K. pneumoniae rates were low (4%), while most samples carried MDR strains (67%) and copper-tolerant isolates (63%; sil+pco clusters; MICCuSO4≥16mM), particularly at pre-slaughter. Benzalkonium chloride consistently exhibited activity in K. pneumoniae (MIC/MBC range=4-64mg/L) from diverse and representative strains independently of the presence/absence of genes linked to QACs tolerance. A polyclonal K. pneumoniae population, discriminated by FT-IR and WGS, included various lineages dispersed throughout the chicken’s lifecycle at the farm (ST29-KL124, ST11-KL106, ST15-KL19, ST1228-KL38), until the meat (ST1-KL19, ST11-KL111, ST6405-KL109, and ST6406-CG147-KL111), or over years (ST631-49 KL109, ST6651-KL107, ST6406-CG147-KL111). Notably, some lineages were identical to those from human clinical isolates. WGS also revealed F-type multireplicon plasmids carrying sil+pco (copper) co-located with qacEΔ1±qacF (QACs) and antibiotic resistance genes like those disseminated in humans. In conclusion, chicken farms and their derived meat are significant reservoirs for diverse K. pneumoniae clones enriched in antibiotic resistance and metal tolerance genes, some exhibiting genetic similarities with human clinical strains. Further research is imperative to unravel the factors influencing K. pneumoniae persistence and dissemination within poultry production, contributing to improved food safety risk management. This study underscores the significance of understanding the interplay between antimicrobial control strategies and non-clinical sources to effectively address the spread of antimicrobial resistance.
- Frontiers in occupational health and safety managementPublication . Ramos, Delfina; Cotrim, Teresa; Arezes, Pedro; Baptista, João; Rodrigues, Matilde; Leitão, JoãoThis Special Issue of the International Journal of Environmental Research and Public Health is devoted to the “Frontiers in Occupational Health and Safety Management”. This issue intends to contribute to the knowledge in the field regarding the new challenges for Occupational Safety and Health (OSH) management. This concern is stated in the EU Strategic Framework on Health and Safety at Work for 2021–2027, which sets out the key actions needed to improve the health and safety of workers in the years to come. This new strategy focuses on three cross-cutting objectives, namely, anticipating and managing change in the context of green, digital, and demographic transitions; improving the prevention of work-related accidents and diseases and striving towards a Vision Zero approach to workrelated deaths; and increasing the preparedness to respond to current and future health crises [1]. This strategy is expected to have a significant impact on the management of OSH at all levels, including companies from all sectors. It is also a basis for increasing the awareness and funding support for the improvement of the health and safety of workers. The European Agency for Safety and Health at Work (EU-OSHA) has an important role in implementing this strategy at the European level, but also in coordination with the numerous National Agencies.