Untitled

Funder

Organizational Unit

Publications

Metagenomic analysis of the bacterial microbiome, resistome and virulome distinguishes Portuguese Serra da Estrela PDO cheeses from similar non-PDO cheeses: An exploratory approach

Publication . Salamandane, Acácio; Leech, John; Almeida, Rita; Silva, Carolina; Crispie, Fiona; Cotter, Paul D.; Malfeito-Ferreira, Manuel; Brito, Luísa

This study aimed to evaluate the microbiome, resistome and virulome of two types of Portuguese cheese using high throughput sequencing (HTS). Culture-dependent chromogenic methods were also used for certain groups/ microorganisms. Eight samples of raw ewe’s milk cheese were obtained from four producers: two producers with cheeses with a PDO (Protected Designation of Origin) label and the other two producers with cheeses without a PDO label. Agar-based culture methods were used to quantify total mesophiles, Enterobacteriaceae, Escherichia coli, Staphylococcus, Enterococcus and lactic acid bacteria. The presence of Listeria monocytogenes and Salmonella was also investigated. The selected isolates were identified by 16S rRNA gene sequencing and evaluated to determine antibiotic resistance and the presence of virulence genes. The eight cheese samples analyzed broadly complied with EC regulations in terms of the microbiological safety criteria. The HTS results demonstrated that Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus plantarum, Lacticaseibacillus rhamnosus, Enterococcus durans and Lactobacillus coryniformis were the most prevalent bacterial species in cheeses. The composition of the bacterial community varied, not only between PDO and non-PDO cheeses, but also between producers, partic- ularly between the two non-PDO cheeses. Alpha-diversity analyses showed that PDO cheeses had greater bac- terial diversity than non-PDO cheeses, demonstrating that the diversity of spontaneously fermented foods is significantly higher in cheeses produced without the addition of food preservatives and dairy ferments. Despite complying with microbiological regulations, both PDO and non-PDO cheeses harbored potential virulence genes as well as antibiotic resistance genes. However, PDO cheeses exhibited fewer of these virulence and antibiotic resistance genes compared to non-PDO cheeses. Therefore, the combination of conventional microbiological methods and the metagenomic approach could contribute to improving the attribution of the PDO label to this type of cheese.

2024-08Journal article

Open access

Investigating the Influence of Vessel Shape on Spontaneous Fermentation in Winemaking

Publication . Malfeito-Ferreira, Manuel; Granja-Soares, Joana; Chandra, Mahesh; Asryan, Arman Asryan; Oliveira, Joana; Freitas, Victor; Loira, Iris; Morata, Antonio; Cunha, Jorge; Harutyunyan, Mkrtich

The earliest archaeological evidence of wine came from ceramic vessels of the Tran- scaucasian ‘Shulaveri-Shomutepe’ or ‘Aratashen-Shulaveri-Shomutepe culture’ (SSC/AShSh: c. 6000–5200 BC). Western European ‘Bell Beaker culture’ (BB: c. 2500–2000 BC) is characterized by bell- shaped pottery vessels but has so far not been found with residues consistent with wine. Knowing that wild grapes populated both habitats, the absence of wine during the Bell Beaker period remains to be explained. The main goal of this work was to investigate whether the shape of the vessels could influence the performance of spontaneous fermentation, specifically regarding the production of volatile acidity. Crushed grapes or juices from various grape cultivars were fermented in two types of vessels: (i) borosilicate glass beakers (4–5 L) to imitate bell beakers and (ii) Erlenmeyer flasks (5 L) to imitate SSC/AShSh vessels. Fermentations occurred spontaneously, and the wines were analyzed for their conventional physical–chemical parameters (e.g., ethanol content, total acidity, volatile acidity, pH), chromatic characteristics (e.g., wine color intensity, wine hue), and volatile composition by gas-chromatography-flame ionization detection (GC-FID). At the end of fermentation, the yeast species were identified by molecular methods. In addition, wine yields and phenolic composition (e.g., total phenols, anthocyanins, total pigments) were determined for wild grapes in comparison with six red varieties Vitis vinifera L. subsp. sativa (Vinhão, Marufo, Branjo, Melhorio, Castelão and Tempranillo Tinto), chosen as a function of their genetic relatedness with the wild counterpart. Wines produced from V. sylvestris grapes showed higher total acidity and color intensity when compared to the cultivated varieties. Saccharomyces cerevisiae dominated at the end of all spontaneous fermen- tations in all types of vessels and conditions. Wines fermented in Erlenmeyers showed ethanol concentrations as high as 14.30% (v/v), while the highest ethanol level was 12.30% (v/v) in beakers. Volatile acidity increased to a maximum of 4.33 g/L (acetic acid) in Erlenmeyers and 8.89 g/L in beakers. Therefore, the shape of the vessels influenced the performance of fermentation, probably due to the different exposures to air, leading to vinegary ferments more frequently in open mouths than in conical-shaped flasks. These results provide a hypothesis based on fermentation performance for the absence of wine produced in the Iberian Peninsula until the arrival of Phoenician settlers.

2024-08Journal article

Open access

Partial replacement of mineral fertilisers with animal manures in an apple orchard: Effects on GHG emission

Publication . Esteves, Catarina; Costa, Eva; Mata, Miguel; Mota, Mariana; Ribeiro, Henrique; Fangueiro, David

Partial replacement of mineral fertilisers (MF) with animal manures is a good alternative to reduce MF use and increase both nutrient cycling in agriculture and soil organic matter. However, the adoption of this practice must not lead to increased environmental impacts. In this two-year study conducted in an apple orchard, MF were partially replaced with various animal manures, including cattle slurry (CS), acidified cattle slurry (ACS), solid cattle manure (CsM), or poultry manure (PM), and their impacts on greenhouse gas emission (GHG: CO2, N2O and CH4) were examined. A control (CTRL) receiving only MF served as the baseline, representing the con- ventional scenario in orchard fertilisation. Overall, replacing MF with manures increased GHG emissions, with the magnitude of the impacts depending on the specific characteristics of the manures and the amount of nu- trients and organic matter applied. Comparing to the CTRL, application of ACS and CS led to higher CH4 and N2O emissions, while PM application increased both N2O and CO2 emissions. In contrast, replacement with PM and CsM decreased CH4 emissions. Nevertheless, results varied between the two years, influenced by several factors, including soil conditions. While acidification showed potential to mitigate CH4 emissions, it also led to increased N2O emissions compared to CS, particularly in 2022, suggesting the need for further investigation to avoid emission trade-offs. Replacement with CS (20.49 t CO2-eq ha 1) and CsM (20.30 t CO2-eq ha 1) showed com- parable global warming potential (GWP) to the conventional scenario (CTRL, 19.49 t CO2-eq ha 1), highlighting their potential as viable MF substitutes

2024-04Journal article

Open access

Impact of Chlorella vulgaris Intake Levels on Performance Parameters and Blood Health Markers in Broiler Chickens

Publication . Mendes, Ana R.; Spínola, Maria P.; Lordelo, Madalena; Prates, José A. M.

This systematic review examines the effects of cumulative Chlorella vulgaris intake levels on broiler chickens, focusing on growth performance and systemic health markers. The review establishes a clear relationship between cumulative C. vulgaris intake and significant outcomes in poultry nutrition and health through a detailed analysis of various studies. The correlation analysis revealed that cumulative C. vulgaris intake levels ranging from 0.8 to 718 g/bird influenced growth rates and feed efficiency, following sigmoid models. Specifically, intakes of approximately 20 g/bird maximized final body weight (R2 = 0.616, p < 0.001), cumulative body weight gain (R2 = 0.627, p < 0.001) and daily weight gain (R2 = 0.639, p < 0.001). The feed conversion ratio also improved with increasing C. vulgaris intakes up to this level, although this was non-significant (R2 = 0.289, p = 0.117). In addition, similar cumulative C. vulgaris intake levels impacted plasma health markers in broilers, leading to reductions in triacylglycerols and cholesterol and improvements in immunoglobulin levels. These findings underscore the importance of carefully calibrated C. vulgaris supplementation strategies to optimise poultry growth and health without adverse effects. Future research should focus on refining C. vulgaris dosing guidelines and further exploring its long-term effects and mechanisms of action to enhance poultry health and production sustainability.

2024-06Journal article

Open access

Impact of high Spirulina diet, extruded or supplemented with enzymes, on blood cells, systemic metabolites, and hepatic lipid and mineral profiles of broiler chickens

Publication . Spínola, Maria P.; Alfaia, Cristina M.; Costa, Mónica M.; Pinto, Rui M. A.; Lopes, Paula A.; Pestana, José M.; Tavares, João C.; Mendes, Ana R.; Mourato, Miguel; Tavares, Beatriz; Carvalho, Daniela F. P.; Martins, Cátia F.; Ferreira, Joana I.; Lordelo, Madalena; Prates, José A. M.

The impact of 15% dietary inclusion of Spirulina (Arthrospira platensis) in broiler chickens was explored, focusing on blood cellular components, systemic metabolites and hepatic lipid and mineral composition. From days 14 to 35 of age, 120 broiler chickens were divided and allocated into four dietary treatments: a standard corn and soybean meal-based diet (control), a 15% Spirulina diet, a 15% extruded Spirulina diet, and a 15% Spirulina diet super-dosed with an enzyme blend (0.20% porcine pancreatin plus 0.01% lysozyme). The haematological analysis revealed no significant deviations (p > 0.05) in blood cell counts across treatments, suggesting that high Spirulina inclusion maintains haematological balance. The systemic metabolic assessment indicated an enhanced antioxidant capacity in birds on Spirulina diets (p < 0.001), pointing toward a potential reduction in oxidative stress. However, the study noted a detrimental impact on growth performance metrics, such as final body weight and feed conversion ratio (both p < 0.001), in the Spirulina-fed treatments, with the super-dosed enzyme blend supplementation failing to alleviate these effects but with extrusion mitigating them. Regarding hepatic composition, birds on extruded Spirulina and enzyme-supplemented diets showed a notable increase in n-3 fatty acids (EPA, DPA, DHA) (p < 0.001), leading to an improved n-6/n-3 PUFA ratio (p < 0.001). Despite this positive shift, a reduction in total hepatic lipids (p = 0.003) was observed without a significant change in cholesterol levels. Our findings underscore the need for further exploration into the optimal inclusion levels, processing methods and potential enzymatic enhancements of Spirulina in broiler diets. Ultimately, this research aims to strike a balance between promoting health benefits and maintaining optimal growth performance in poultry nutrition.

2024-03Journal article

Open access