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- Wine spoilage control: impact of Saccharomycin on Brettanomyces bruxellensis and its conjugated effect with sulfur dioxidePublication . Branco, Patrícia; Coutinho, Rute; Malfeito-Ferreira, Manuel; Prista, Catarina; Albergaria, HelenaThe yeast Brettanomyces bruxellensis is one of the most dangerous wine contaminants due to the production of phenolic off-flavors such as 4-ethylphenol. This microbial hazard is regularly tackled by addition of sulfur dioxide (SO2). Nevertheless, B. bruxellensis is frequently found at low levels (ca 103 cells/mL) in finished wines. Besides, consumers health concerns regarding the use of sulfur dioxide encouraged the search for alternative biocontrol measures. Recently, we found that Saccharomyces cerevisiae secretes a natural biocide (saccharomycin) that inhibits the growth of different B. bruxellensis strains during alcoholic fermentation. Here we investigated the ability of S. cerevisiae CCMI 885 to prevent B. bruxellensis ISA 2211 growth and 4-ethylphenol production in synthetic and true grape must fermentations. Results showed that B. bruxellensis growth and 4-ethylphenol production was significantly inhibited in both media, although the effect was more pronounced in synthetic grape must. The natural biocide was added to a simulated wine inoculated with 5 x 10 2 cells/mL of B. bruxellensis, which led to loss of culturability and viability (100% dead cells at day-12). The conjugated effect of saccharomycin with SO2 was evaluated in simulated wines at 10, 12, 13 and 14% (v/v) ethanol. Results showed that B. bruxellensis proliferation in wines at 13 and 14% (v/v) ethanol was completely prevented by addition of 1.0 mg/mL of saccharomycin with 25 mg/L of SO2, thus allowing to significantly reduce the SO2 levels commonly used in wines (150–200 mg/L)
- Fermented unripe tomato paste – Development of innovative salad dressings as a contribution to circular economyPublication . Simões, Sara; Santos, Rafaela; Sousa, Isabel; Prista, Catarina; Raymundo, AnabelaAs food trends evolve towards sustainable, healthy, and mildly processed products, it is essential to consider new food sources, which contribute to positive changes in the food industry and originate interesting new products. For the tomato industry, where only completely red tomatoes are used, unripe and non-red tomatoes constitute an important by-product, with important losses in the field of a perfectly edible vegetable food source. To give value to this unripe tomato, a fermentation with a consortium of lactic acid bacteria and yeast was previously optimized, originating an acidic pulp with optimized nutritional characteristics and great potential for salad dressing development. Pulp’s texture was improved with two hydrocolloid systems: 0.5 g/100 g of xanthan gum; and a mixed system of xanthan gum and kappa-carrageenan (0.1 g/100 g each). After rheology optimization, spices and condiments were added to the thickened fermented pulp, and promising tasty sauces were obtained. These sauces were presented to a consumers’ panel, with good acceptance. The production of these new healthy sauces aims to mitigate unripe tomato waste, adding value to the tomato industry by using a major industrial by-product.
- Molecular and functional characterization of grapevine NIPs through heterologous expression in aqy-null Saccharomyces cerevisiaePublication . Farzana, Sabir; Gomes, Sara; Loureiro-Dias, Maria C.; Soveral, Graça; Prista, CatarinaPlant Nodulin 26-like Intrinsic Proteins (NIPs) are multifunctional membrane channels of the Major Intrinsic Protein (MIP) family. Unlike other homologs, they have low intrinsic water permeability. NIPs possess diverse substrate selectivity, ranging from water to glycerol and to other small solutes, depending on the group-specific amino acid composition at aromatic/Arg (ar/R) constriction. We cloned three NIPs (NIP1;1, NIP5;1, and NIP6;1) from grapevine (cv. Touriga Nacional). Their expression in the membrane of aqy-null Saccharomyces cerevisiae enabled their functional characterization for water and glycerol transport through stopped-flow spectroscopy. VvTnNIP1;1 demonstrated high water as well as glycerol permeability, whereas VvTnNIP6;1 was impermeable to water but presented high glycerol permeability. Their transport activities were declined by cytosolic acidification, implying that internal-pH can regulate NIPs gating. Furthermore, an extension of C-terminal in VvTnNIP6;1M homolog, led to improved channel activity, suggesting that NIPs gating is putatively regulated by C-terminal. Yeast growth assays in the presence of diverse substrates suggest that the transmembrane flux of metalloids (As, B, and Se) and the heavy metal (Cd) are facilitated through grapevine NIPs. This is the first molecular and functional characterization of grapevine NIPs, providing crucial insights into understanding their role for uptake and translocation of small solutes, and extrusion of toxic compounds in grapevine
- Application of the CATA methodology with children: Qualitative approach on ballot development and product characterization of innovative productsPublication . Rocha, Célia; Ribeiro, José Carlos; Lima, Rui Costa; Prista, Catarina; Raymundo, Anabela; Vaz Patto, Maria Carlota; Cunha, Luís MiguelThere is an increasing importance of the role of children and adolescents in the food market and to successfully develop food products intended for them, it is necessary to apply proper sensory evaluation methodologies. Although children can execute traditional methods for food liking and preference evaluation, traditional sensory descriptive methods may not be suitable for them and it is necessary to assess their ability to perform novel sensory profiling methods. Thus, this study aimed to assess children’s acceptance of an innovative food product – cookies incorporating fermented grass pea (Lathyrus sativus) flour – and their ability to describe a sensory profile using a Check-All-That-Apply (CATA) approach. Two different types of cookies (salty and sweet) were developed, differing in the level of substitution of wheat flour by fermented grass pea flour (between 0 and 40%). The cookies were evaluated by two sensory panels of 60 children (8–12 years), who assessed the overall liking using a 7-point facial hedonic scale and the sensory profile of the samples using a CATA ballot with 21 sensory terms previously developed through focus groups with children. Children showed the ability to discriminate the different samples with the hedonic scale and according to their sensory profile. Results revealed that the focusgroup with children is an adequate way to generate CATA ballots and that the CATA approach is adequate to evaluate how children discriminate the sensory profile of food products. Furthermore, the food neophobia level of the children negatively impacted their acceptance of the food products
- Insights into the selectivity mechanisms of grapevine NIP aquaporinsPublication . Sabir, Farzana; Di Pizzio, Antonella; Loureiro-Dias, M. C.; Casini, Angela; Soveral, Graça; Prista, CatarinaNodulin 26-like intrinsic proteins (NIPs) of the plant aquaporin family majorly facilitate the transport of physiologically relevant solutes. The present study intended to investigate how substrate selectivity in grapevine NIPs is influenced by the aromatic/arginine (ar/R) selectivity filter within the pore and the possible underlying mechanisms. A mutational approach was used to interchange the ar/R residues between grapevine NIPs (VvTnNIP1;1 withUniversidade de Lisboa, VvTnNIP6;1, and VvTnNIP2;1 with VvTnNIP5;1). Their functional characterization by stopped-flow spectroscopy in Saccharomyces cerevisiae revealed that mutations in residues of H2/H5 helices in VvTnNIP1;1 and VvTnNIP6;1 caused a general decline in membrane glycerol permeability but did not impart the expected substrate conductivity in the mutants. This result suggests that ar/R filter substitution could alter the NIP channel activity, but it was not su cient to interchange their substrate preferences. Further, homology modeling analyses evidenced that variations in the pore radius combined with the di erences in the channel’s physicochemical properties (hydrophilicity/hydrophobicity) may drive substrate selectivity. Furthermore, yeast growth assays showed that H5 residue substitution alleviated the sensitivity of VvTnNIP2;1 and VvTnNIP5;1 to As, B, and Se, implying importance of H5 sequence for substrate selection. These results contribute to the knowledge of the overall determinants of substrate selectivity in NIPs
- The importance, prevalence and determination of vitamins B6 and B12 in food matrices: A reviewPublication . Santos, A.J.M.; Khemiri, S.; Simões, Sara; Prista, Catarina; Sousa, Isabel; Raymundo, AnabelaVitamins are a vast group of fundamental organic compounds, which are not produced by the human body but are essential for the living organisms’ good health. Vitamins B6 and B12 belong to the same group of hydrophilic vitamins. Structurally unrelated, they share the same purpose as essential components for normal cellular operation, growth and development. Vitamin B6 is an enzymatic co-factor that is vital for countless biochemical reactions, and is also important in sugar and fatty acid metabolization. It encompasses three natural and inter-convertible pyridine-derivatives: pyridoxine, pyridoxal and pyridoxamine. Vitamin B12 is a cobalt organometallic complex also indispensable in numerous human physiological func- tions. It has four bioactive forms: cyanocobalamin, methylcobalamin, hydroxocobalamin and 5′-deoxy- adenosylcobalamin, and only a few prokaryotes have the ability to biosynthesize cobalamin. This work reviews the significant aspects of vitamins B6 and B12: their vital roles, consequences of deficit; food sources; and methods of determination and respective matrices, with heavy emphasis on chromatographic techniques developed within the last two decades
- Clean Label “Rocha” Pear (Pyrus communis L.) Snack Containing Juice By-Products and Euglena gracilis MicroalgaePublication . Lomba-Viana, Xavier; Raymundo, Anabela; Prista, Catarina; Alegria, Maria João; Sousa, Isabel“Rocha do Oeste” pear is a Portuguese Protected Designation of Origin variety and one of the country’s most relevant fruits for its nutritional value, production area, and exportation amounts. The recent integration of a pilot-scale juice production line brought to SUMOL+COMPAL company the need to characterize the new resulting fractions and value the new by-products. The objective of this work was to value the juice clarification by-products, producing a clean label and fiber-rich snack, in a circular economy rationale, where the secondary products are upcycled back into the food value chain, by creating another food product that includes those by-products. For the above to be possible, the laboratory conditions to produce pear fractions were optimized. After optimizing the puree centrifugation, using response surface methodology (RSM), and optimizing the turbid juice crossflow filtration, the different fractions were characterized in rheological, nutritional, and physical aspects. Comparison to the pulps revealed an increase in the viscosity of the pomace; an enriching effect on the fructose, glucose, and dietary fiber levels in the pomace, and maintenance of the vitamin C levels after centrifugation; and with no effect on the contents of total phenols during the filtration step. A thick pear snack was developed, incorporating retained fraction, inulin, and Euglena gracilis in the pomace, and optimized regarding its firmness and dietary fiber content. The snack characterization revealed an interesting total phenols content (which was maintained from the raw materials). Compared to the snack without microalgae and a commercial fruit snack, the pear snack with E. gracilis was well-accepted by the sensory panel, mainly in texture and appearance, and can be further improved in aroma and flavor. The snack without microalgae was the favorite among the three samples, in most sensory parameters, and never got the answer “I’m sure I wouldn’t buy it.” Therefore, an innovative, clean label and plant-based snack was developed, in a circular economy rationale, which was relatively well-appreciated by the panel. This snack is rich in dietary fiber, having the possibility of presenting various nutritional claims, and the potential for easy sensory optimization
- Chemical and nutritional characterization of loengo (Anisophyllea boehmii) fruits as a source of important bioactive with impact on healthPublication . Lofa, Alcides Morais; Mourato, Miguel; Prista, Catarina; Sousa, Isabel; Ferreira, Ricardo BoavidaIntroduction: Fruits are vital for human health, providing nutrients and other compounds linked to protection against different diseases. Loengo (Anisophyllea boehmii Engl.) produces an edible stone fruit, plum-shaped (drupe), dark blue and maroon or carmine in the ripening stage, which is generally, consumed fresh in the production areas. Characterization of this fruit will contribute to estimate its nutritional and pharmaceutical potential impact. The present work aimed at the physic-chemical, nutritional and antioxidant activity characterization of loengo fruit. Methods: Soluble solid contents (SSC) in the pulp were assessed in juice, at room temperature. Total protein was determined using the Dumas method. In what concerns the mineral composition were determined by ICP-OES. Organic acids identification and quantification was accomplished using a high-performance liquid chromatography (HPLC) system. The total polyphenol contents (TPC) were determined in 96-well microplate assays. Antioxidant activity were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the ferric reducing antioxidant power (FRAP) assay Results and discussion: Composition, in terms of fresh matter is as follows: pulp soluble sugar content (refractometer) is 19.5% ± 0.02%, and pulp titratable acidity 1.2% ± 0.1% expressed in citric acid; total protein content is 7.4% ± 0.02%, 8.0% ± 0.1%, 16.9% ± 0.2%, for the peel, pulp and seed, respectively. In what concerns the mineral composition, the results obtained show that it is a good source of minerals, mainly potassium and phosphorus, but also calcium, sulfur, and magnesium, with the pulp richer in potassium, the seed in phosphorus, and the peel in calcium. As expected, the pulp showed the highest total soluble sugar content, whereas the seed had the lowest. The main sugars found in the pulp were glucose and fructose. In the peel citric acid was the main organic acid found, together with malic, oxalic, and ascorbic acids. Most antioxidant activity was found in the seed. The results of this work provide important information to recommend the consumption of this fruit for its sweet taste, chemical and nutritional composition, and, most of all, for its richness in total phenolic compounds for it potential to formulate nutraceutical foods.
- Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensisPublication . Chandra, Mahesh; Branco, Patricia; Prista, Catarina; Malfeito-Ferreira, ManuelSulfite is a common preservative in wine, but the spoilage yeast Brettanomyces bruxellensis can produce volatile phenols even with the recommended sulfite dose. The purpose of this study was to examine how wine components, p-coumaric acid (a precursor of volatile phenols), and micronutrients influence culturability, viability, and volatile phenols production by B. bruxellensis under sulfite stress. In red wine, a high sulfite dose (potassium metabisulfite, 100 mg L1) led to an immediate death phase followed by growth recovery after two weeks. However, 4-ethylphenol (4-EP) was continuously produced by dead or nonculturable cells. Nonetheless, an event of growth recovery could not be observed in the case of the model wine. However, when the model wine was supplemented with minerals and vitamins, both growth recovery and 4-EP production were noticed, suggesting that the minerals and vitamins played an important role in maintaining the viability of cells under the sulfite stress. The yeast could also utilize the p-coumaric acid (p-CA) as an energy source, showing a specific growth rate of 0.0142 h1 with 1 mM of p-CA in model wine. Furthermore, the sulfite-stressed cells exhibited ATP production by means of proton efflux while utilizing the p-CA. This work highlights the novel finding that the conversion of p-CA into 4-EP provides sufficient energy for the cell to remain metabolically active under the sulfite stress.
- Grapevine aquaporins: Diversity, cellular functions, and ecophysiological perspectivesPublication . Sabir, Farzana; Zarrouk, Olfa; Noronha, Henrique; Loureiro-Dias, M. C.; Soveral, Graça; Gerós, Hernâni; Prista, CatarinaHigh-scored premium wines are typically produced under moderate drought stress, suggesting that the water status of grapevine is crucial for wine quality. Aquaporins greatly influence the plant water status by facilitating water diffusion across the plasma membrane in a tightly regulated manner. They adjust the hydraulic conductance of the plasma membrane rapidly and reversibly, which is essential in specific physiological events, including adaptation to soil water scarcity. The comprehension of the sophisticated plant-water relations at the molecular level are thus important to optimize agricultural practices or to assist plant breeding programs. This review explores the recent progresses in understanding the water transport in grapevine at the cellular level through aquaporins and its regulation. Important aspects, including aquaporin structure, diversity, cellular localization, transport properties, and regulation at the cellular and whole plant level are addressed. An ecophysiological perspective about the roles of grapevine aquaporins in plant response to drought stress is also provided
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