Browsing by Author "Albergaria, Helena"
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- Admissibility Grid to Support the Decision for the Preferential Routing of Portuguese Endogenous Waste Biomass for the Production of Biogas, Advanced Biofuels, Electricity and HeatPublication . Crujeira, Teresa; Trancoso, Maria Ascensão; Eusebio, Ana; Oliveira, Ana Cristina; Passarinho, Paula; Abreu, Mariana; Marques, Isabel Paula; Marques, Paula; Marques, Susana; Albergaria, Helena; Pinto, Filomena; Costa, Paula; Andre, Rui N.; Girio, Francisco; Moura, PatríciaABSTRACT: A methodology was developed to assess the allocation of different types of endogenous waste biomass to eight technologies for producing electricity, heat, biogas and advanced biofuels. It was based on the identification of key physicochemical parameters for each conversion process and the definition of limit values for each parameter, applied to two different matrices of waste biomass. This enabled the creation of one Admissibility Grid with target values per type of waste biomass and conversion technology, applicable to a decision process in the routing to energy production. The construction of the grid was based on the evaluation of 24 types of waste biomass, corresponding to 48 sets of samples tested, for which a detailed physicochemical characterization and an admissibility assessment were made. The samples were collected from Municipal Solid Waste treatment facilities, sewage sludges, agro-industrial companies, poultry farms, and pulp and paper industries. The conversion technologies and energy products considered were (trans)esterification to fatty acid methyl esters, anaerobic digestion to methane, fermentation to bioethanol, dark fermentation to biohydrogen, combustion to electricity and heat, gasification to syngas, and pyrolysis and hydrothermal liquefaction to bio-oils. The validation of the Admissibility Grid was based on the determination of conversion rates and product yields over 23 case studies that were selected according to the best combinations of waste biomass type versus technological solution and energy product.
- Antimicrobial peptides from Saccharomyces cerevisiae induce physiological changes in Hanseniaspora guilliermondiiPublication . Branco, Patricia; Albergaria, Helena; Arneborg, NilsSaccharomyces cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against other wine-related yeasts (e.g. Hanseniaspora guilliermondii) (Albergaria et al., 2010) and bacteria (e.g. Oenococcus oeni) (Osborne and Edwards, 2007). In the present study we assessed the physiological changes induced by those AMPs on sensitive yeast cells of Hanseniaspora guilliermondii, namely membrane permeability and intracellular pH (pHi) alterations. Membrane permeability was evaluated by staining cells with propidium iodide (PI) and pHi by the fluorescence ratio imaging microscopy (FRIM) technique (Guldfeldt and Arneborg, 1998). Results showed that after 20 min of incubation with inhibitory concentrations of AMPs, the average pHi of cells dropped from 6.5 to 5.4. After 8 h of incubation, 32% of the cells had lost their ĢpH (=pHi-pHext) and after 24 h that percentage rose to 77%. The culturability (plating) and viability (PI staining) of the sensitive yeast cells also decreased in the presence of the AMPs. After 24 h of exposure to AMPs, 61% of the cells were dead (PI-stained) and the number of viable cells fell from 1 ~105 to 1.5 CFU/ml, which means that virtually all cells (99.999%) became unculturable but a sub-population of 39% of cells remained in a viable but non-culturable (VBNC) state. However, those VBNC cells were able to recover their culturability after incubation at optimal growth conditions. Our study revealed that the mode of action of these AMPs seems to be primarily targeted to the cell membranes, reducing their permeability and preventing cells to maintain pH homeostasis.
- Dominance of Saccharomyces cerevisiae in wine fermentations: secretion of antimicrobial peptides and microbial interactionsPublication . Albergaria, Helena; Branco, Patricia; Francisco, Diana; Coutinho, Rute; Monteiro, Margarida; Malfeito-Ferreira, Manuel; Arneborg, Nils; Almeida, M. Gabriela; Caldeira, JorgeIn present work we investigated the antagonistic effect by S. cerevisiae against several wine-related microbial species vis-à-vis the secretion of antimicrobial peptides (AMPs).
- Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux ratesPublication . Branco, Patricia; Albergaria, Helena; Arneborg, Nils; Prista, CatarinaABSTRACT: Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.
- Effect of saccharomycin, a natural Saccharomyces cerevisiae biocide, on Hanseniaspora guilliermondii cells surfacePublication . Calvário, Joana; Silva, Nelly; Almeida, M. Gabriela; Albergaria, Helena; Eaton, Peter; Macedo, Anjos L.; Caldeira, JorgeABSTRACT: During spontaneous wine fermentations, most of the non-Saccharomyces yeasts present in grape musts show an early decline in their population. It was traditionally assumed that Saccharomyces cerevisiae (S.c.) prevalence was due to the higher resistance of this species to ethanol. However, wine fermentations performed with single cultures of non-Saccharomyces strains showed that those strains could withstand much higher ethanol levels [1]. It was then found that S.c. (strain CCMI 885) produced antimicrobial peptides (AMPs) that are responsible for the early death of the non Saccharomyces yeasts [2]. In previous work, we isolated, purified and sequenced those ntimicrobial peptides (AMPs) and found that they derive from the glyceraldehyde 3-phosphate dehydrogenase enzyme [3]. These GAPDH-derived AMPs compose the natural biocide secreted by S.c., which we named saccharomycin, and are effective against sensitive yeasts both in its natural/isolated and synthetic form.
- Evaluation of Biotechnological Active Peptides Secreted by Saccharomyces cerevisiae with Potential Skin BenefitsPublication . Mauricio, Elisabete; Branco, Patricia; Araújo, Ana Luiza Barros; Roma-Rodrigues, Catarina; Lima, Katelene; Duarte, Maria Paula; Fernandes, Alexandra; Albergaria, HelenaABSTRACT:Biotechnological active peptides are gaining interest in the cosmetics industry due to their antimicrobial, anti-inflammatory, antioxidant, and anti-collagenase (ACE) effects, as well as wound healing properties, making them suitable for cosmetic formulations. The antimicrobial activity of peptides (2–10 kDa) secreted by Saccharomyces cerevisiae Ethanol-Red was evaluated against dermal pathogens using broth microdilution and challenge tests. ACE was assessed using a collagenase activity colorimetric assay, antioxidant activity via spectrophotometric monitoring of nitrotetrazolium blue chloride (NBT) reduction, and anti-inflammatory effects by quantifying TNF-α mRNA in lipopolysaccharides (LPS)-exposed dermal fibroblasts. Wound healing assays involved human fibroblasts, endothelial cells, and dermal keratinocytes. The peptides (2–10 kDa) exhibited antimicrobial activity against 10 dermal pathogens, with the Minimum Inhibitory Concentrations (MICs) ranging from 125 µg/mL for Staphylococcus aureus to 1000 µg/mL for Candida albicans and Streptococcus pyogenes. In the challenge test, peptides at their MICs reduced microbial counts significantly, fulfilling ISO 11930:2019 standards, except against Aspergillus brasiliensis. The peptides combined with MicrocareⓇ SB showed synergy, particularly against C. albicans and A. brasilensis. In vitro, the peptides inhibited collagenase activity by 41.8% and 94.5% at 250 and 1000 µg/mL, respectively, and demonstrated antioxidant capacity. Pre-incubation with peptides decreased TNF-α expression in fibroblasts, indicating anti-inflammatory effects. The peptides do not show to promote or inhibit the angiogenesis of endothelial cells, but are able to attenuate fibrosis, scar formation, and chronic inflammation during the final phases of the wound healing process. The peptides showed antimicrobial, antioxidant, ACE, and anti-inflammatory properties, highlighting their potential as multifunctional bioactive ingredients in skincare, warranting further optimization and exploration in cosmetic applications.
- Evaluation of the biocontrol potential of a commercial yeast starter against fuel-ethanol fermentation contaminantsPublication . Branco, Patricia; Diniz, Mário; Albergaria, HelenaABSTRACT: Lactic acid bacteria (LAB) and Brettanomyces bruxellensis are the main contaminants of bioethanol fermentations. Those contaminations affect Saccharomyces cerevisiae performance and reduce ethanol yields and productivity, leading to important economic losses. Currently, chemical treatments such as acid washing and/or antibiotics are used to control those contaminants. However, these control measures carry environmental risks, and more environmentally friendly methods are required. Several S. cerevisiae wine strains were found to secrete antimicrobial peptides (AMPs) during alcoholic fermentation that are active against LAB and B. bruxellensis strains. Thus, in the present study, we investigated if the fuel-ethanol commercial starter S. cerevisiae Ethanol Red (ER) also secretes those AMPs and evaluated its biocontrol potential by performing alcoholic fermentations with mixed-cultures of ER and B. bruxellensis strains and growth assays of LAB in ER pre-fermented supernatants. Results showed that all B. bruxellensis strains were significantly inhibited by the presence of ER, although LAB strains were less sensitive to ER fermentation metabolites. Peptides secreted by ER during alcoholic fermentation were purified by gel-filtration chromatography, and a bioactive fraction was analyzed by ELISA and mass spectrometry. Results confirmed that ER secretes the AMPs previously identified. That bioactive fraction was used to determine minimal inhibitory concentrations (MICs) against several LAB and B. bruxellensis strains. MICs of 1-2 mg/mL were found for B. bruxellensis strains and above 2 mg/mL for LAB. Our study demonstrates that the AMPs secreted by ER can be used as a natural preservative in fuel-ethanol fermentations.
- Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactionsPublication . Branco, Patricia; Francisco, Diana; Chambon, Christophe; Hébraud, Michel; Arneborg, Nils; Gabriela Almeida, M.; Caldeira, Jorge; Albergaria, HelenaSaccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vastworldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.
- Interactions between Saccharomyces cerevisiae and Hanseniaspora guilliermondii: cell-cell contact mechanismPublication . Branco, Patricia; Kemsawasd, Varongsiri; Arneborg, Nils; Albergaria, HelenaSeveral studies have shown that the early death of non-Saccharomyces during wine fermentations are due to yeast-yeast interactions induced by Saccharomyces cerevisiae (Sc) through different mechanisms such as growth arrest mediated by a cell-cell contact mechanism (Nissen et al. 2003) and death mediated by killer-like toxins (Pérez-Nevado et al 2006; Albergaria et al. 2010). Besides, previous work also showed that death of non-Saccharomyces in co-cultivation with Sc is always triggered at the end of exponential growth phase (Pérez-Nevado et al 2006). In order to investigate the role of cell-cell contact in the early death of non-Saccharomyces, we performed assays in which Sc cells pre-grown at enological growth conditions for 12 and 48 h, respectively, were in direct contact with Hanseniaspora guilliermondii (Hg) cells at high cellular density (107-108 cells/ml) in a carbon-free medium. As a negative control we performed similar assays in which Sc and Hg cells were separated by a dialysis tube (pore cut-off of 1000 kDa) and as a positive control a single Hg culture. Results showed that Hg cell density decreased by 2 orders of magnitude (i.e. from 108-106 cells/ml) in contact with 48 h-grown Sc cells, while its viability remained unchanged (108cfu/ml) in the presence of 12 h-grown Sc cells. Moreover, Hg viability was not affected both in the dialysis tube experiments and single culture, which confirmed the death-induced cell-cell contact phenomenon.
- Proteomics insights into the responses of Saccharomyces cerevisiae during mixed-culture alcoholic fermentation with Lachancea thermotoleransPublication . Peng, Chuantao; Andersen, Birgit; Arshid, Samina; Larsen, Martin R.; Albergaria, Helena; Lametsch, Rene; Arneborg, NilsABSTRACT: The response of Saccharomyces cerevisiae to cocultivation with Lachancea thermotolerans during alcoholic fermentations has been investigated using tandem mass tag (TMT)-based proteomics. At two key time-points, S. cerevisiae was sorted from single S. cerevisiae fermentations and from mixed fermentations using flow cytometry sorting. Results showed that the purity of sorted S. cerevisiae was above 96% throughout the whole mixed-culture fermentation, thereby validating our sorting methodology. By comparing protein expression of S. cerevisiae with and without L. thermotolerans, 26 proteins were identified as significantly regulated proteins at the early death phase (T1), and 32 significantly regulated proteins were identified at the late death phase (T2) of L. thermotolerans in mixed cultures. At T1, proteins involved in endocytosis, increasing nutrient availability, cell rescue and resistance to stresses were upregulated, and proteins involved in proline synthesis and apoptosis were downregulated. At T2, proteins involved in protein synthesis and stress responses were up- and downregulated, respectively. These data indicate that S. cerevisiae was stressed by the presence of L. thermotolerans at T1, using both defensive and fighting strategies to keep itself in a dominant position, and that it at T2 was relieved from stress, perhaps increasing its enzymatic machinery to ensure better survival.