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- Mead production: tradition versus modernityPublication . Ramalhosa, Elsa; Gomes, Teresa; Pereira, Ana Paula; Dias, Teresa; Estevinho, Leticia M.Honey is a natural product with recognized physical and chemical properties, which contribute to its biological activity. However, honey is currently being sold at low prices, making it imperative to find alternatives to make apiculture a viable national enterprise. One of these alternatives could be mead production. Despite the excellent properties of honey, mead production faces several problems, namely, delays and “pouts” fermentations, lack of product uniformity, and production of yeast off-flavors. Many factors might be related with these problems, such as honey variety, temperature, medium composition (vitamin and nitrogen content), fermentative yeast, and pH. Due to all these factors, mead production has decreased over the years. To overcome this situation, more research is needed to optimize the production of this beverage. This chapter presents a comprehensive review of previous research on mead production. It will focus on honey characterization and mead production. The first section covers honey composition and the way this affects honey properties, as well as important parameters that are indicators of honey quality. The second section discusses mead production, including fermentative microorganisms, fermentation conditions, and required postfermentation adjustments and maturation conditions. The final section focuses on the problems that must be surpassed and what the future holds for mead production.
- Mead production: comparison of different production scales (preliminary results)Publication . Gomes, Teresa; Barradas, Carla; Dias, Teresa; Andrade, João Verdial; Morais, Jorge Sá; Ramalhosa, Elsa; Estevinho, Leticia M.Mead production represents a possible economic alternative to honey producers that intend to obtain honey products with surplus value. From that the present work aims to study the influence of using different production scales on the quality of the final mead obtained and on the process performance. Increasing the production scale almost ten times (1.5 to 20 L), some differences were observed. Maximum specific growth rates equal to 0.045 and 0.038 h-1 were obtained for fermentations carried out at 1.5 and 20 L, respectively. The time course of glucose and glycerol were similar for both production scales. Nevertheless, slight differences at the end of the fermentations were observed for fructose and acetic acid. In relation to ethanol, a higher final concentration was found in the pilot-scale, resulting in a higher ethanol yield. In conclusion, these preliminary results are a good promise to local honey producers who intent to obtain large-productions of mead.
- Isolation and sequence analysis of alfa-tubulin gene from Phytophora cinnamomiPublication . Dias, Teresa; Andrade, Maria; Jorge, Lurdes; Vaz, Madalena; Martins, Fátima; Dominguez, Ángel; Choupina, AltinoPhytophthora diseases cause widespread economic and environmental losses worldwide. Thousands of plant species are susceptible. In Portugal, Phytophthora cinnamomi is responsible for chestnut ink disease. Despite the differences there are a number of key steps common to most infection strategies, including adhesion to the plant surface, plant penetration through the secretion of a diverse range of cell wall-degrading enzymes and hyphal growth. The cell cytoskeleton plays a critical role in these processes. Microtubules are a major constituent of the cell cytoskeleton. They participate in a wide range of cellular functions, such as motility, division, maintenance of cell shape, and intracellular transport. However, microtubule role is variable depending on the organism, cell type and other factors. Tubulin is the major constituent of microtubules and is composed of a heterodimer of two closely related proteins, alpha and beta tubulin. In S. cerevisiae cells, the essential TUB1 gene is the major gene, while the nonessential gene TUB3 is a minor gene, encoding α-tubulin. The β-tubulin subunit is encoded by the TUB2 gene. In Magnaporthe grisea both α-and β-tubulins are found as single-copy genes. The Oomycetes are, however, phylogenetically quite distinct from the fungi. Analysis of structural, biochemical and molecular characteristics have led to the Oomycetes being grouped with the chromophyte algae. In order to elucidated the role of cytoskeleton in pathogenicity mechanisms of Phytophthora cinnamomi, was cloned a gene encoding alpha-tubulin from P. cinnamomi. To isolated this gene, the existing Tub1 nucleotide sequences were retrieved from the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank). These sequences were aligned in Clustal and degenerate primers Tub1 and Tub2 were designed. A 1200bp fragment was generated from genomic DNA by PCR and subsequently cloned into pGEM-T vector. To complete the open reading frame it was used the HE-TAIL PCR. The complete ORF was sequenced and submitted in EMBL databases (Accession number AM412177.1). Based on the computational analysis through BioEdit software, TUB1 has a 1362 bp ORF and encodes a 453 a.a protein with a molecular weight of 49,911kDa. Phylogenetic analysis of deduced amino acid sequence using FASTA programs from EMBL databases revealed that Tub1 revealed 99.6% identity with alpha-tubulin of P. infestans T30.4 and 98.9% identity with P. capsici, but only 68,1 % with alpha-tubulin of S. cerevisiae. Key Words: Phytophthora cinnamomi; Ink disease; Alpha-tubulin; microtubules Acknowledgements: The Project COMBATINTA/SP2.P11/02 Interreg IIIA – Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund, and the Project “Identification, characterization and role of molecular factors associated with the mechanisms of infection of Fagaceae species by Phytophthora cinnamomi” (PTDC/AGR-AAM/67628/2006) FCT, supported this work.
- Levels of hygiene and safety quality indicators along the processing stages of a Portuguese traditional dry-fermented sausage (Linguiça)Publication . Gonzales-Barron, Ursula; Pereira, Ana Paula; Gomes, Aline; Lopes-da-Silva, M.F.; Rodrigues, Paula; Estevinho, Leticia M.; Cadavez, Vasco; Dias, TeresaWhile Portuguese fermented sausages are highly appreciated, research has shown that their microbiological quality is inconsistent due to the high variability in the production process and insufficient hygiene quality. Linguica is a dryfermented sausage, which has been found to harbour food-borne pathogens. Hence, the objective of this study was to investigate the changes in the levels of total viable counts (TVC), Enterobacteriaceae, L. monocytogenes and S. aureus at the key production stages of Linguica (raw meat, maceration, stuffing and smoking/drying). A microbiological survey followed a batch throughout production, extracting samples of raw meat (n=3), batter before maceration (n=3), batter after 3- or 4-day maceration (n=3) and sausages after curing/drying (n=5). Samples were taken from a total of three batches from each of the two factories under examination. Overall, TVC counts did not change considerably from raw meat to stuffing (5.5-5.9log CFU/g, 95% Cl: 5.15-5.82 log CFU/g), yet it increased significantly up to the end of drying (7.83 log CFU/g, 95% Cl: 7.57- 8.09 log CFU/g)- seemingly, because of the multiplication of lactic acid bacteria (LAB). Unlike the Enterobacteriaceae counts, which decreased (p<0.001) from batter before maceration (3.23 log CFU/g, 95% Cl: 2.80-3.66 log CFU/g) to the end of drying (1.56 log CFU, 95 percent Cl: 1.23-1.90 log CFU/g), S. aureus increased significantly between these two processing stages (2.58 log CFU/g; 95% Cl: 2.20-2.93 log CFU/g in the finished product). L. monocytogenes was detected in the products of one of the factories although their counts decreased after batter maceration. Because Linguica is a product that can be consumed without cooking, there is a need to further investigate the risk factors that determine their presence along processing. The presence of these two pathogens should also prompt industries to reinforce good hygiene practices in the processing of traditional sausages.
- Mechanisms underlying the effect of commercial starter cultures and a native yeast on ochratoxin A production in meat productsPublication . Meftah, Sana; Abid, Salwa; Dias, Teresa; Rodrigues, PaulaProcessed meat products are of worldwide importance, but they are highly prone to fungal and ochratoxin A (OTA) contamination. In previous studies, several Lactic Acid Bacteria (LAB) and yeasts have been tested as biocontrol agents against P. nordicum growth and OTA production in meat products, with promising results. However, A. westerdijkiae has been poorly studied for this matrix. The aim of this work was to evaluate in vitro the mechanisms underlying the effects of a commercial starter culture and of a meat-native Candida zeylanoides strain on the growth and OTA production of P. nordicum and A. westerdijkiae, by co-culture in ham and sausage-based media under different conditions. In ham medium, C. zeylanoides live cells, cell broth and diffused compounds significantly inhibited OTA production by P. nordicum, but live cells of the starter culture significantly increased it. For A. westerdijkiae strong and significant stimulation was observed by direct contact in both media. In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way. This study sheds some light on the mechanisms behind the different effects of microorganisms.
- Bee propolis effect on protection of RBCs membrane integrityPublication . Moreira, Leandro; Rogão, Mónica; Pereira, Ana Paula; Morais, Margarida; Costa, Bruno; Dias, L.G.; Dias, Teresa; Estevinho, Leticia M.Propolis is a resinous substance collected from plants by bees. The propolis composition depends on the surrounding vegetation, the season, and the area from which derive. This hive product usually contains a variety of chemical compounds such as polyphenols (flavonoids, phenolic acids and esters), steroids, and amino acids. The hereditary spherocytosis (HS) is a congenital hemolytic anemia, with origin in the modification of membrane proteins of erythrocytes, which leads to increased susceptibility to hemolysis and a decrease of the cell over-life.
- Isolation and phylogenetic analysis of two actin genes from Phytophthora cinnamomiPublication . Jorge, Lurdes; Dias, Teresa; Andrade, Maria; Vaz, Madalena; Dominguez, Ángel; Choupina, AltinoActins, as the essential component of cellular microfilament, are ubiquitous and highly conserved proteins that play key roles in several basic functions of organism such as cytoskeleton morphology, cell division, cell motility, cellular signal transduction, cellular interaction and organelle movements, as well as locomotion, phagocytosis, endocytosis and exocytosis . Actins are highly conserved structural proteins, found in all eukaryotes. So, actin gene sequences are used as tools in scientific research, for example, for phylogenetic analysis. Actin in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and filamentous fungi (Candida albicans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Filobasidiella neoformans) where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. Phytophthora cinnamomi is a host-nonspecific, soilborne, pathogen of many plant species. In Portugal it is most important as a pathogen of chestnut trees. The purpose of this study was to clone and determine the phylogenetic retionships evidencided by Phytophthora cinnamomi actins. In order to isolate the actin genes, P. cinnamomi was grown in cellophane-PDA medium and genomic DNA was used as a template in PCR amplification reactions combining degenerate primers Act1, Act2, Act3 and Act4. PCR fragments were purified, cloned into pGEM-T vector and transformants were selected. Complete open reading frames (ORFs) of act1 and act2 genes were achieved by HE-TAIL PCR, and submitted to EMBL databases (Accession numbers AM412175.1 and AM412176.1). Act1 has an 1128bp ORF, encoding a deduced protein of 375aa and 41,972kDa. Act2 ORF has 1083bp and encodes a deduced protein of 360aa and 40,237kDa. Deduced amino acid sequences were analyzed using FASTA programs from EMBL databases. Act1 showed a 98.9% identity with P. melonis actB, 94.4% with P. megasperma actin and 96.0% with P. infestans actin2. Act2 showed a 98.9% identity with Pythium splendens actin and 98.6% with P. brassicae actinA.
- Mead production: comparison of different production scales (preliminary results)Publication . Gomes, Teresa; Barradas, Carla; Dias, Teresa; Andrade, João Verdial; Morais, Jorge Sá; Ramalhosa, Elsa; Estevinho, Leticia M.Mead production represents a possible economic alternative to honey producers that intend to obtain honey products with surplus value. From that the present work aims to study the influence of using different production scales on the quality of the final mead obtained and on the process performance. Increasing the production scale almost ten times (1.5 to 20 L), some differences were observed. Maximum specific growth rates equal to 0.045 and 0.038 h-1 were obtained for fermentations carried out at 1.5 and 20 L, respectively. The time course of glucose and glycerol were similar for both production scales. Nevertheless, slight differences at the end of the fermentations were observed for fructose and acetic acid. In relation to ethanol, a higher final concentration was found in the pilot-scale, resulting in a higher ethanol yield. In conclusion, these preliminary results are a good promise to local honey producers who intent to obtain large-productions of mead.
- Antioxidant and chealting activities of fermentation broths containing fructooligossacharidesPublication . Lama, Aelina; Dias, Teresa; Peres, António M.Fructooligossacharides are present in plants and fruits at low concentrations and with varying individual relative proportions. So, for industrial application, fructooligossacharides extraction from natural sources may not be economically viable. Recently, several works have been published within this field of research usually aiming to establish the best experimental conditions to maximizing fructooligossacharides yield/synthesis [1-3]. However, although it is known that these compounds may have a great health impact, it is also reported that beneficial health effects may depend on the relative fructooligossacharides composition, seeming that nystose-rich diet is preferable compared to a kestoserich preparation, exhibiting a higher anti-hydroxyl radical activity [4,5]. So, in this work it is reported the results regarding a screening study concerning the bioactivity activity of the fermentation broth extracts obtained using fungi with potential for producing fructooligossacharides. The potential use of five fungi (i.e., A. aculeatus, A. japonicus, A. opinicosulum, P. thoumii and P. cornyphipum) was evaluated. Batch fermentation were carried out during 96 hours, at constant temperature (27 ºC) and agitation (100 rpm), being the initial sucrose concentration fixed at 30 g/L. Samples were taken at 24-h intervals and the radical scavenging activity as well as the iron binding ability of chelators, of the different broth extracts, were determined using the DPPH and ferrozine. The overall results obtained showed that although the extracts possessed relatively low bioactive activity; it was possible to set some preliminary insights that allowed selecting the most promissory(s) mold(s), which could enable achieving, in a near future, a final fructooligossacharides formulation with the greatest antioxidant capacity.
- Influence of starter culture and ripening temperature on survival of L. monocytognes in traditional Portuguese dry-feremnted sausagePublication . Cadavez, Vasco; Pereira, Ana Paula; Gonzales-Barron, Ursula; Dias, TeresaLinguiça is a Portuguese traditional dry-fermented sausage whose microbiological quality and safety have been reported to be highly variable. The present study evaluated the influence of the addition of a commercial starter culture and ripening temperature (10° C and 18°C) on the survival of L monocytogenes in linguiça