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- Carbodiimide modification enhances activity of pig pancreatic phospholipase A2Publication . Ferreira, João Paulo M.; Sasisekharan, Ram; Louie, Otway; Langer, RobertPig phospholipase A2, pig iso-phospholipase A2 and bovine pancreatic phospholipase A2 were reacted in solution with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of N-hydroxysulfosuccinimide, at pH 7. The influence of micellar protectants was analyzed. In the presence of n-hexadecylphosphocholine, the losses of activity in micellar diheptanoyl-lecithin were 80, 35, and 10% in bovine phospholipase A2, pig iso-phospholipase A2, and pig phospholipase A2, respectively. With 1-oleoylglycerophosphocholine, the bovine enzyme lost 40% activity, but the pig enzyme was activated sevenfold. The modified pig enzyme showed pre-micellar activation on monomeric diheptanoyl-lecithin, and either reduced or increased activities on mixed micelles of bile salt with egg phosphatidylcholine, depending on the composition of the micelles. This activation is consistent with previous protein-engineering studies of pig pancreatic phospholipase A2. In this study, we present new information concerning the specificity and interfacial recognition behaviour of this enzyme in relation to this activation.
- Development of an immunoassay for ciprofloxacin using phage-displayed antibody fragmentsPublication . Gomes, Francisco Brás; Riedstra, Symon; Ferreira, A. C. Silva; Ferreira, João Paulo M.
- Changes in the pool of free fatty acids in ovine, bovine and caprine milk fats, effected by viable cells and cell-free extracts of lactococcus lactis and debaryomyces vanrijiaePublication . Regado, Mafalda A.; Cristovão, Betina M.; Tavaria, Freni K.; Ferreira, João Paulo; Moutinho, Carla G.; Balcão, Victor M.; Malcata, F. XavierLipolysis catalysed by lipases, native or released by natural microflora in milk, plays a key role in development of aroma and flavour throughout cheese ripening. This research effort was aimed at a deeper understanding of the action of two wild strains used in traditional ewe’s milk cheesemaking in Portugal, viz. Lactococcus lactis and Debaryomyces vanrijiae. They were both tested as viable cells and cellfree extracts – using bovine, ovine and caprine milk fat emulsions as model substrates. Hydrolysis reactions were carried out at 30 and 37 C, in the case of L. lactis and D. vanrijiae, respectively; the contents of short- and medium-chain fatty acids were determined by high pressure liquid chromatography. Our experimental results showed general trends, viz. preferential depletion of medium chain fatty acids throughout the whole reaction time. However, distinct patterns were observed towards different substrates, depending on the source and form of the biocatalyst at stake.
- PPO activity of pear reduced by ca storagePublication . Morais, A. M. M. B.; Ferreira, J. P. M.; Morais, R. M. S. C.
- First and second law analyses to an energetic valorization process of biogasPublication . Carolino, Cristina Guedes; Ferreira, João PauloThe limitations in the world sources of energy are being mitigated by the exploitation of renewable forms and by increases in the efficiency of energy utilization. Exergy analysis is a useful method for the design, evaluation, and improvement of energy systems, that uses conservation of mass and conservation of energy principles, together with the second law of thermodynamics. This study covers first and second law analyses of a cogeneration system run with the biogas produced in a landfill. Such plant produces useful electrical and thermal energies, while protecting the environment from greenhouse emissions. The objectives were to identify locations where major irreversibilities occur, to evaluate their magnitudes, and to assess the energy and exergy efficiencies of the global system and of its constituent units. The results show that the overall-plant first law efficiency is 37.9% and the exergy efficiency is 362%, which is far from the thermodynamic ideal limit. The internal combustion engine and one of the radiators are the most inefficient units, as judged by the parameters degree of thermodynamic perfection and exergy destruction quotient. The main potential for improvement in the plant is the harnessing of the energy in the exhaust gases.
- Antimicrobial effects of a microemulsion and a nanoemulsion on enteric and other pathogens and biofilmsPublication . Teixeira, Paula C.; Leite, Gonçalo M.; Domingues, Ricardo J.; Silva, Joana; Gibbs, Paul A.; Ferreira, João PauloSome microemulsions and nanoemulsions may have antimicrobial properties and be effective anti-biofilm agents. We examined the abilities of two fine emulsions, designated BCTP and TEOP, to inactivate suspensions of vegetative cells of Salmonella spp. Escherichia coli 0157:H7 (VT-), Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. BCTP is an O/W nanoemulsion of soybean oil and tri-n-butyl phosphate emulsified with Triton X-100, while TEOP is an O/W microemulsion of ethyl oleate with Tween 80 as emulsifier and n-pentanol as a co-emulsifier. BCTP was effective in reducing the cell numbers of L. monocytogenes, while TEOP was effective against all five organisms investigated. The abilities of these emulsions to reduce preformed biofilms of the five bacteria were also investigated. With the exception of the biofilm formed by L. monocytogenes, which surprisingly was not significantly affected by BCTP, all biofilms were inhibited by both BCTP and TEOP.
- Development of an immunoassay for ciprofloxacin based on phage-displayed antibody fragmentsPublication . Gomes, Francisco Bruno Moreira Brás; Riedstraa, Symon; Ferreira, João Paulo MedeirosThe widespread use of ciprofloxacin in human, animal and plant health has raised an environmental problem, paralleled by several other antibiotics. The aim of this work is the development of a rapid and sensitive ELISA assay for ciprofloxacin, which can constitute an alternative to time-consuming HPLC methods. For this purpose, we worked with antibody fragments, instead of whole antibodies, and used magnetic beads as solid support. Ciprofloxacin was successfully immobilized onto this support with a carbodiimide-mediated reaction. A library of phage particles that express human single-chain antibodies at their surface was then screened with an optimized protocol. Several positive fragments were isolated and identified as being VL fragments. These were then fully characterized. A reproducible competitive ELISA was developed using the magnetic beads — ciprofloxacin as support and the phages displaying the VL fragment as recognition entity. This assay showed limits of detection and quantification of 9.3 nM and 33 nM, respectively. Also, competitive ELISAs with ciprofloxacin homologues and other molecules showed cross-reactivities lower than 12%.
- Understanding the thermodynamics of equilibria across semipermeable membranesPublication . Ferreira, João Paulo MedeirosOsmotic equilibrium is ubiquitous in biological systems and it also underlies a number of technological processes, such as reverse osmosis. Quite often, students know how to calculate osmotic pressures in ideal diluted solutions, but they do not have a deep understanding of the driving forces for transport and how these relate to thermodynamic variables. This article derives, in is simple and straightforward way, the conditions for equilibria through selective membranes, starting with ideal gas mixtures. Chang with physical conditions of several thermodynamic properties are evaluated.Then, the analysis is extended to ideal liquid mixtures, pointing out the similarities and difterences with the previous situation. an expression for the reversible work associated with a change in concentration is derived, and it is applied to the process of desalination of seawater
- Flavour development via lipolysis of milkfats: changes in free fatty acid poolPublication . Regado, Mafalda A.; Cristóvão, Betina M.; Moutinho, Carla G.; Balcão, Victor M.; Aires-Barros, Raquel; Ferreira, João Paulo M.; Malcata, F. XavierUnder the trade name lipolysed milkfat (LMF), concentrated cheese flavours obtained via enzymatic treatments of cheese (or other dairy substrates) have been made available in increasing numbers. In this research effort, (anhydrous) milkfats from cows, ewes and goats were used as substrates for production of cheesy flavours. Those milkfats were subjected to modifications brought about by ten different (commercial) lipases and one cutinase, and the free fatty acids released were analysed by high performance liquid chromatography. Both the degrees of hydrolysis and the free fatty acid profiles of the final products were similar to those reported for several LMF products, although specific keynotes could be pinpointed. Consequently, those milkfats may represent alternative raw materials for manufacture of cheesy flavours, while contributing to alleviate the problem derived from the increasing surplus of milkfat in Western countries.