CBQF - Contribuições em Revistas Científicas / Contribution to Journals
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- Understanding the impact of different post-digestion enzymatic inhibition methods and storage conditions on standardized gastrointestinal model (INFOGEST): a case study of Pleurotus ostreatus mushroom biomassPublication . Araújo-Rodrigues, Helena; Salsinha, Ana Sofia; Coscueta, Ezequiel R.; Relvas, João Bettencourt; Tavaria, Freni K.; Pintado, Manuela E.INFOGEST became the standard digestion protocol to address inconsistencies in gastrointestinal tract (GIT) digestion protocols. However, sample collection, enzymatic inactivation methods, and storage conditions can significantly impact the accurate assessment of food compounds. This study investigates for the first time the impact of inactivation methods (thermal, pH-based, and specific inhibitors) and storage conditions (freezing and freeze-drying), following INFOGEST protocol on macronutrients of Pleurotus ostreatus biomass (used as a model food matrix). The results revealed that groups of macromolecules are differently affected by inactivation and storage methods. For ?-glucans and glucans with different linkages than (1 ? 3)(1 ? 6)-?-glucans, enzyme inactivation seemed to prevent overestimation of enzymatic activity, with freezing emerging as the most promising storage method. For this carbohydrate group, no significant differences were found between inactivation strategies. The results suggested that freezing and thermal inactivation preserve peptide solubility. Additionally, freeze-drying may promote phenolic degradation and reduce antioxidant potential. Depending on the GIT phase, pH-based and specific inhibitors negatively impact these variables. The storage seemed to have no significant effect on fatty acids, enabling the selection of storage conditions based on the stability requirements of other macronutrient groups. Overall, thermal inactivation combined with freezing emerged as an effective, low-cost, and straightforward approach, minimizing post-digestion enzymatic activity and protecting food compounds from degradation. These findings enhance the reproducibility of digestion studies, facilitating inter-laboratory comparisons. Future studies should explore additional macronutrient-rich matrices, particularly those with diverse protein and lipid profiles, to expand the applicability of these findings.
- Efficient screening and enhanced exopolysaccharide production by functional lactic acid bacteria (LAB) in lactose supplemented mediaPublication . Pintado, Ana I. E.; Barbosa, Catarina C. R.; Pintado, Manuela E.; Malcata, F. Xavier; Gomes, Ana M. P.Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) can be considered as natural biological thickeners that have attracted considerable attention in the food industry. This study aimed to evaluate and select potentially EPS-producing strains LAB and to assess the influence of carbon source and aeration on EPS production. Nine LAB strains were assessed as potential EPS producers, and Rahnella aquatilis ATCC 55046 was employed as the positive control strain for EPS. The compaction test and the observation of viscous colonies in a solid medium did not yield sufficient evidence for the presence of EPS. The assessment of capsules through staining provided evidence of EPS presence only for Rahnella aquatilis ATCC 55046. The EPS yield was subsequently assessed in De-Man Rogosa and Sharpe (MRS) broth medium supplemented with 2% (w/w) fructose (MRS-f) or lactose (MRS-l), as well as in whey (Whey) and whey supplemented with 2% (w/w) lactose (Whey-l). The EPS production in the various culture media under study ranged from 194 to 1,187 mg of EPS/g of polymer dry mass (PDM). These results suggest that the culture medium and carbon sources had an impact on the EPS production of the different strains. Bifidobacterium animalis Bb12 achieved the highest EPS production in MRS-f. In the case of MRS-l, the control strain recorded the highest EPS value, along with Lactobacillus acidophilus LAC-1. Regarding Whey, Lentilactobacillus Kefir NCFB 2753 exhibited the highest EPS production, while in Whey-l, Lacticaseibacillus paracasei LCS-1 emerged as the top performer in terms of EPS production. This suggests that certain strains exhibit potential for use in the production of novel fermented EPS products, whether dairy or non-dairy.
- Bioinformatic insights into the carotenoids’ role in gut microbiota dynamicsPublication . Rocha, Helena R.; Ribeiro, Pedro; Rodrigues, Pedro Miguel; Gomes, Ana M.; Pintado, Manuela; Coelho, Marta C.Background/Objectives: Carotenoids are bioactive pigments with well-established antioxidant and immunomodulatory properties, yet their impact on gut microbiota remains poorly understood from a chemical standpoint. This study explores how carotenoid structure and gastrointestinal stability shape microbial responses combining in vitro fermentation with bioinformatic analyses. Methods: Individual carotenoids (beta (?)-carotene, lutein, lycopene) and combined carotenoids, as well as algal-derived extracts were subjected to 48 h in vitro fermentation, and microbial composition and activity were assessed through sequencing and computational analysis. Results: ?-carotene and lycopene promoted acid-tolerant taxa such as Escherichia-Shigella, whereas lutein, due to its higher polarity, supported more transient fluctuations. Mixtures and algal carotenoids exhibited synergistic effects, sustaining beneficial genera including Bifidobacterium and Bacteroides and promoting structured ecological trajectories. Conclusions: These findings provide a chemistry-driven perspective on how carotenoids act as modulators of microbial ecosystems, with direct implications for the formulation of carotenoid-enriched functional foods and dietary interventions.
- Enrichment of bread with powder from the aerial parts of purslane: effects on mineral content, phenolic compounds, antioxidant activity, sensory properties, and nutritional enhancementPublication . Aboukhalaf, Abdelghani; Lahlou, Youssef; Kalili, Adil; Rocha, João Miguel; Belahsen, RekiaAs a widely consumed staple, bread serves as an excellent vehicle for incorporating plant-based ingredients with demonstrated health-promoting properties. This study evaluated the total phenolic content, antioxidant activity, mineral composition, sensory properties, and nutrient contribution of wheat bread enriched with 0%, 5%, 10%, and 15% purslane powder. Mineral content was determined using ICP-AES, phenolic compounds using the Folin-Ciocalteu method, and antioxidant activity through the DPPH assay. Sensory evaluation was conducted using a five-point hedonic scale. Results showed that PO-enriched breads had significantly higher levels of phenolic compounds, antioxidant activity, and mineral content compared to the control (p < 0.05), with the 15% PO formulation exhibiting the highest values. Fortification enhanced the daily intake of key minerals such as iron, magnesium, manganese, and copper. Sensory analysis revealed significant differences in consumer acceptance breads with 10% and 15% PO received lower scores than the control and 5% PO, although all samples remained acceptable (overall liking score >3). Correlation and principal component analysis (PCA) demonstrated strong associations between polyphenol content, antioxidant activity, and mineral levels. The 5% PO formulation emerged as the most balanced option, offering improved nutritional value while maintaining acceptable sensory qualities. This study highlights the potential of PO as a functional ingredient to enhance the nutritional and antioxidant properties of bread, with 5% enrichment offering the best compromise between health benefits and consumer preference.
- Salt reduction and temperature abuse implications on Listeria monocytogenes behaviour in chouriçãoPublication . Barbosa, Joana; Carvalho, Teresa Bento de; Komora, Norton; Azevedo, Maria A.; Silva, Beatriz Nunes; Teixeira, PaulaReducing sodium in cured meats, like chourição, is essential to address health concerns related to excessive salt intake. However, reformulation presents challenges, as salt reduction may facilitate the growth of undesirable contaminants. n this study, the main objective was to evaluate the impact of a new formulation of reduced salt chourição on the survival of Listeria monocytogenes. For that, slices of traditional and 25% (w/w) salt-reduced chourição were inoculated (n = 3) with a cocktail of seven L. monocytogenes strains (~3 log CFU/g) and stored for 60 days at 4 and 10 °C (temperature abuse). Listeria monocytogenes counts were determined, and the Baranyi and Roberts model was used to describe pathogen growth. In traditional chourição, the pathogen maximum growth rate (?max ; log CFU/g day-1) at 10 °C (0.21 ± 0.03) was 1.5 times higher than ?max at 4 °C (0.14 ± 0.04). In salt-reduced chourição, ?max at 10 °C (0.40 ± 0.24) was approximately 3 times ?max at 4 °C (0.13 ± 0.02), highlighting a greater impact of temperature on microbial behaviour when salt is reduced. At 4 °C, pathogen ?max in traditional (0.14 ± 0.04) and in salt-reduced chourição (0.13 ± 0.02) were similar. However, at 10 °C, ?max was higher in salt-reduced chourição (0.40 ± 0.24), compared to traditional (0.21 ± 0.03), probably due to the combined effect of higher temperature and lower salt content. In salt-reduced chourição, storage at 4 and 10 °C led to shorten lag phases (18 and 12 days) in comparison to that of traditional chourição (23 days at 4 °C). Product reformulation requires careful consideration so that food safety is not compromised. Various factors, including temperature, are important to hinder pathogen proliferation and guarantee the microbiological safety of foods when reducing the preservative effect of one hurdle (in this case, salt content), as illustrated by this study.
- Plant-derived bioactive compounds: one health perspectivePublication . Gonçalves, Ana C.; Pinto, Ana R.; Cima, André; Olo-Fontinha, Eva; Martins, Joana C. L.; Garcia, Juliana; Lemos, André; Saavedra, Maria José; Pintado, Maria Manuela; Alves, Maria JoséThis review, within the One Health framework, compiles information on plant-derived bioactive compounds and emphasises their multifunctional role in improving environmental, animal, and human health. These compounds support sustainable health and ecological stability by influencing biological and environmental processes. Data from literature research are combined to explain the mechanisms and potential uses of different key bioactive compounds. Mechanistic insights focus on their capacity to regulate oxidative stress, inflammation, and microbial balance, linking these effects to therapeutic benefits in human health, enhanced animal productivity, and environmental sustainability. These compounds show antioxidant, anti-inflammatory, antimicrobial, and metabolic activities, helping prevent chronic diseases, strengthen immunity, and reduce reliance on antibiotics and pollution. Examples like quercetin, resveratrol, and curcumin demonstrate their roles in modulating inflammatory and metabolic pathways to foster sustainable health and ecological balance. Bioactive compounds are linked to the One Health strategy, providing benefits across biological systems. Nonetheless, challenges such as variability, bioavailability, and standardization remain. Future directions should aim to develop sustainable extraction and formulation methods, leverage omics technologies and artificial intelligence for discovery and characterization, and foster industry partnerships to validate these compounds and secure global regulatory approval.
- Time-dependent accumulation of biogenic amines and microbial succession during dry-aging of beef: safety implicationsPublication . Ribeiro, Ana J.; Milheiro, Juliana; Nunes, Fernando M.; Carvalho, Teresa B. de; Barbosa, Joana B.; Silva, Filipe; Teixeira, Paula; Saraiva, Cristina M.Dry-aging of beef under controlled temperature, humidity and airflow reshapes the surface microbiota and may influence biogenic amine (BA) accumulation. In this study, culture-based enumeration, 16 S rRNA gene profiling of combined crust + inner meat, and validated HPLC quantification of BAs were integrated to track safety- and quality-relevant changes over 60 days. Sequencing showed Pseudomonadota and Bacillota consistently >95 % of reads; communities were dominated by Pseudomonas, Brochothrix and Psychrobacter, with Acinetobacter rising at mid-aging. Alpha diversity peaked at day 35 (Shannon 1.33???2.12; overall P = 0.0225; day 35 vs day 1, adjusted P = 0.0069) and became heterogeneous by day 60. Culture confirmed a surface-led, aerobic succession: crust counts increased and Pseudomonas reached 5.6 log CFU/g at day 60, whereas inner-muscle counts declined across groups; pathogens were not detected. In the inner meat, cadaverine rose from non-detectable to 31 ± 37 mg/kg at day 60 P < 0.001), spermine peaked at day 35 (52 ± 14 mg/kg; P < 0.001), while histamine remained <5 mg/kg and other BAs showed no significant change. All inner-meat BAs remained below commonly cited concern ranges, though late-stage variance indicates sporadic hot spots, likely reflecting diffusion from the crust and proteolysis-enabled precursor supply. Integrating microbes and metabolites identified two ecological–metabolic tendencies linking psychrotrophic genera with polyamines or diamines. In practice, day 35 emerges as a quality “sweet spot,” whereas approaching day 60 warrants tighter surface management and targeted monitoring of pseudomonads, Enterobacteriaceae and cadaverine/putrescine.
- Automated high-throughput microscopy screening unveiled new Listeria monocytogenes genes involved in cell infectionPublication . Alves, Ângela; Meireles, Diana; Suriano, Chiara; Monteiro, Ricardo; Oliveira, Rute; Bernardes, Beatriz G.; Sousa, Sandra; Pombinho, Rita; Cabanes, DidierTo uncover novel genetic factors required for Listeria monocytogenes cell infection, we developed an automated high-throughput microscopy screening pipeline that integrates GFP-expressing bacteria with machine learning-based image analysis. Using this approach, we screened a mariner transposon library comprising 4,224 L. monocytogenes EGDe mutants and identified 58 with significantly reduced numbers of intracellular bacteria. Sequencing revealed 24 unique insertion sites corresponding to 14 genes, including previously known virulence factors and nine novel candidates not previously implicated in cell infection. These genes encode the protease chaperone ClpX, the ferric uptake regulator Fur, the sensor histidine kinase LisK, the peptide chain release factor 2 PrfB, proteins involved in proline and purine biosynthesis (ProAB, PurAB), and Lmo2217, a protein of unknown function. Among these, the targeted deletion of the adenylosuccinate synthetase gene, purA, resulted in impaired growth in minimal medium, severely reduced proliferation in epithelial and macrophage cell lines, and attenuated virulence in mice. Unexpectedly, PurA was also essential for bacterial internalization into cells. Supplementation with AMP or adenine, but not ATP, rescued the invasion capacity of the ?purA mutant. Mechanistically, purA deletion induced a reduction in the levels of surface-associated GAPDH, a putative plasminogen-binding protein, likely contributing to the observed invasion defect. Overall, these findings highlight the power of automated high-throughput microscopy screening to dissect host–pathogen interactions, identify novel L. monocytogenes genes required for cell infection, and uncover an unexpected role for PurA in maintaining GAPDH surface localization and promoting bacterial entry into host cells.
- Microbial contamination in a chicken slaughterhouse: insights from sampling throughout productionPublication . Silva, Beatriz Nunes; Moen, Birgitte; Jensen, Merete Rusås; Langsrud, Solveig; Teixeira, PaulaMotivation: In some slaughterhouses, the high volume of chicken processed (~190k chicks/day) creates a challenging environment where contamination of the carcasses may be difficult to avoid, even with proper hygienic measures. Goal: To evaluate microbial contamination at various locations in the slaughterhouse throughout the production day. Results: Between the first and the last production batch, the microbial load of the chicken cages and of the scalding water decreased. Microbial load of blades remain stable throughout production. No differences between Belts_early vs. Belts_late. This suggests that cutting blades and each conveyor belt do not become increasingly contaminated throughout production. Belt_early is the most different group, especially compared to Cages and Scalding water. The biggest and most significant difference was Belt_early vs. Cages_early. Scalding water_early also differs considerably from the Belt groups. Conclusions: Large variability in microbial load at specific sampling points. Hygiene samples with higher microbial levels than samples during production ? must be investigated! High volume of carcasses processed and lack of time to stop production and sanitize the production line pose a hindrance to reducing microbial levels in the final product.
- Biotechnological valorisation of rice okara for high-protein fruit purées: from protein hydrolysis to functional food prototypesPublication . Brassesco, María Emilia; Cassoni, Ana C.; Calixto, João D.; Duarte, Tiago; Correia, Daniela; Figueiredo, Bruna; Alegria, Maria João; Pintado, ManuelaThis study describes a multi-actor biotechnological approach for the valorisation of rice okara into stable, high-protein fruit purées. Fresh okara supplied by White&Green was stabilised by SimplYeast through dehydration (OAD) and hydrolysis (thermal, OAT; enzymatic, OAE), generating protein-rich flours containing 68.7 ± 0.2 to 69.8 ± 0.5% protein, 1.7 ± 0.0 to 2.8 ± 0.0% ash, and 1.3 ± 0.0 to 2.2 ± 0.1% lipids. Characterization showed that both hydrolysis treatments improved flour properties, increasing solubility from 24.8 ± 3.2 (OAT) to 49.7 ± 1.8% (OAE2) and degree of hydrolysis from 0.50 ± 0.10 (OAE1) to 12.66 ± 0.56% (OAE2). Enzymatic hydrolysis markedly enhanced bioactive potential, raising total phenolics from 114.9 ± 6.7 to 453.8 ± 65.9 mg GAE g-1 and antioxidant capacity from 400.5 ± 37.9 to 27.090 ± 2.046 ?mol TE g-1 (ORAC), confirming strong functional improvement for food use. Based on these results, fruit purées combining apple pomace (Compal) and banana with 5% okara flour were developed. Two stages were followed: (i) exploratory testing with all flours, including physicochemical and sensory analyses, and (ii) optimisation using OAD and OAT, adjusting the recipe and standardising the protocol—pre-hydration of okara flour with water and xanthan gum (5 min at 100 °C) followed by stepwise mixing of fruit components and acidifiers. Pilot-scale trials at Yogan confirmed process reproducibility, and SONAE supports market integration. Optimized purées reached 3.5 g protein 100 g-1, with 21% of total energy from protein, fulfilling EU “high-protein” standards. Improved texture, colour and sensory performance, particularly for OAT-based purées, were observed. Protein solubility, emulsifying capacity and antioxidant activity confirmed the positive effect of hydrolysis on structure–function behaviour. This work exemplifies a circular bioeconomy model, showing how controlled protein hydrolysis transforms underutilised co-products into sustainable, value-added ingredients.