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- Interesterification and acidolysis of butterfat with oleic acid by Mucor javanicus lipase: changes in the pool of fatty acid residuesPublication . Balcão, Victor M.; Malcata, F. X.Lipases have become powerful tools in the manufacture of structured fats either via randomization of their glyceride composition or incorporation of externally supplied fatty acid residues in such glycerides. The present communication reports on changes that occurred in the fatty acid pool of anhydrous butterfat subject to interesterification and to acidolysis with oleic acid catalyzed by a commercial lipase immobilized by plain physical adsorption onto hydrophobic hollow fibers at 40°C under controlled water activity. The main goal of this research effort was to engineer butterfat so as to increase its level of unsaturated fatty acid residues and concomitantly decrease its level of medium- and long-chain saturated fatty acid residues (viz. lauric and myristic acids). Although a certain degree of net hydrolysis of butterfat was observed, the triacylglycerols of butterfat subject to acidolysis were found to possess more (approximately 30% w/w) oleic acid and significantly less (8% w/w) lauric acid and less (2% w/w) myristic acid than those of the original butterfat.
- Hydrolysis of whey proteins by proteases extracted from Cynara cardunculus and immobilized onto highly activated supportsPublication . Lamasa, Estela M.; Barrosa, Rui M.; Balcão, Victor M.; Malcata, F. XavierBlends of cardosins A and B, enzymes present in aqueous extracts of the flowers of the thistle (Cynara cardunculus L.), have for long been used as rennets by the cheesemaking industry in the Iberian Peninsula. These dimeric proteases are present in the stigmæ and stylets of said flowers, and are thought to play a role in sexual reproduction of the plant. In the present research effort, production of cardosin derivatives (starting from a crude extract), encompassing full stabilization of their dimeric structure, has been attempted via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and β-mercaptoethanol did not lead to leaching of enzyme, thus proving the effectiveness of the attachment procedure. Furthermore, derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at lab-scale trials to perform (selective) hydrolysis of α-lactalbumin, one of the major proteins in bovine whey.
- Lipase-catalyzed acidolysis of butterfat with oleic acid: characterization of process and productPublication . Balcão, Victor M.; Kemppinen, Asmo; Malcata, F. Xavier; Kalo, Paavo J.The modification of anhydrous butterfat via interesterification reactions with oleic acid catalyzed by a lipase from Mucor circinelloides immobilized by adsorption onto hydrophobic hollow fibers is described. A reasonable degree of incorporation of free (externally added) oleic acid into the triacylglycerols of butterfat has been achieved while short-chain fatty acid residues remained virtually unaffected. Total saturated triacylglycerols decreased by 27%, and triacylglycerols with 32–44 acyl carbons (which contained two or three lauric, myristic, or palmitic acid residues) decreased by 33%. Total monoene and polyene triacylglycerols increased by 21% and 17%, respectively. The triacylglycerols (TAG) of interesterified butterfat had approximately 27% more oleic acid residues and approximately 8% less lauric, 6% less myristic, and 6% less palmitic acid residues than those of the original butterfat; the fraction of low-melting TAG peak increased by 19% whereas that of high-melting TAG decreased by 83%. Although a certain degree of butterfat hydrolysis was observed, enzymatic acidolysis was technically feasible and able to produce a modified butterfat with a stronger nutraceutical character.
- 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.
- Nanoencapsulation of bovine lactoferrin for food and biopharmaceutical applicationsPublication . Balcão, Victor M.; Costa, Carla I.; Matos, Carla M.; Moutinho, Carla G.; Amorim, Manuela; Pintado, Manuela E.; Gomes, Ana M. P.; Vila, Marta M.; Teixeira, Jose A.Lactoferrin has for long captured the interest of many researchers as a natural compound with a wide variety of uses. Lactoferrin is a monomeric, iron-binding 80 kDa glycoprotein, and appears to be the subfraction of whey with the best documented antiviral, antimicrobial, anticancer and immune modulating/enhancing effects. It belongs to the family of transferrin proteins, and serves to control iron levels in body fluids by sequestering and solubilizing ferric iron. In the present research effort, production of lactoferrin derivatives (starting from a purified commercial extract), encompassing full stabilization of its three-dimensional structure, has been attempted via nanoencapsulation within lipid nanovesicles, integrating a multiple water-in-oil-in-water emulsion. Long-term storage of the multiple nanoemulsions produced did not lead to leaching of protein, thus proving the effectiveness of the encapsulation procedure. Furthermore, lactoferrin nanovesicle derivatives prepared under optimal conditions were successfully employed at lab-scale antimicrobial trials.
- On the performance of a hollow-fiber bioreactor for acidolysis catalyzed by immobilized lipasePublication . Balcão, Victor M.; Malcata, F. XavierThe present communication describes the chemical modification of anhydrous butterfat by interesterification with oleic acid catalyzed by a lipase of Mucor javanicus. Two reactor configurations were tested, a batch-stirred tank reactor containing suspended lipase and a batch-stirred tank reactor in combination with a hollow-fiber membrane module containing adsorbed lipase. The goal of this research was to assess the advantage of using a (hydrophobic) porous support to immobilize the lipase in attempts to engineer butterfat with increased levels of unsaturated fatty acid residues (oleic acid) at the expense of medium-to-long chain saturated fatty acids (myristic and palmitic acids). Reactions were carried out at 40°C in the absence of solvent under controlled water activity, and were monitored by chromatographic assays for free fatty acids. The results obtained indicate that the rate of interesterification using the proposed reactor configuration is enhanced by a factor above 100 relative to that using suspended lipase, for the same protein mass basis. Although hydrolysis of butterfat occurred to some degree, the enzymatic process that uses the hollow-fiber reactor was technically superior to the stirred tank system.
- Stability of a commercial lipase from Mucor javanicus: kinetic modeling of pH and temperature dependenciesPublication . Balcão, Victor M.; Oliveira, Teresa A.; Malcata, F. XavierThe present communication reports experimental and modelling work pertaining to the independent roles of pH and temperature on deactivation of a crude lipase from Mucor javanicus. Experimental data oflipolytic activities were generated by a classic pH-stat assay on a triolein emulsion following incubation at several pH values for a fixed time, or at several temperatures for various times; postulated models were then fitted by nonlinear fitting to such data. The pH-dependence data were best fit by assumption of three forms of enzyme with increasing states of protonation, with pKa values of 6.2 and 11.3, respectively, where only the intermediate form is stable within the time frame considered. The thermal-dependence data were best fit by assumption of parallel steps of deactivation and rearrangement, with activation energies of 228.8 and 221.7 kJ mol~l, respectively.
- Coimmobilization of L-asparaginase and glutamate dehydrogenase onto highly activated supportsPublication . Balcão, Victor M.; Mateo, Cesar; Fernández-Lafuente, R.; Malcata, F. Xavier; Guisán, José M.In the present research work, production of coimmobilized derivatives of L-asparaginase and glutamate dehydrogenase was attempted. Comparison of immobilization of each enzyme independently with coimmobilization of the two enzymes unfolded important advantages of the latter, namely a decrease in the induction period (time before the maximum reaction rate is virtually achieved) and an increase in the maximum reaction rate. The effectiveness of the independent enzyme derivatives was low; however, it was enhanced by three-fold when the enzymes were coimmobilized onto the same agarose-glutaraldehyde support. Each supporting agarose bead may in fact be viewed as a nano-reactor with in situ reaction and separation (i.e. elimination of the ammonia formed), with the nanoenvironment surrounding each enzyme molecule being essentially devoid of steric hindrance.
- Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supportsPublication . Balcão, Vitor M.; Mateo, Cesar; Fernández-Lafuente, R.; Malcata, F. Xavier; Guisán, José M.A new protocol for the stabilization of the quaternary structure of multimeric enzymes has been attempted using as model enzyme (tetrameric) L-asparaginase from Escherichia coli. Such strategy is based upon multisubunit covalent immobilization of the enzyme onto activated supports (agarose-glutaraldehyde). Supports activated with different densities of reactive groups were used; the higher the density of groups, the higher the stabilization attained. However, because of the complexity of that enzyme, even the use of the highest densities of reactive groups was not enough to encompass all four subunits in the immobilization process. Therefore, a further chemical intersubunit cross-linking with aldehyde-dextran was pursued; these derivatives displayed a fully stabilized multimeric structure. In fact, boiling the modified enzyme derivative in the presence of sodium dodecyl sulfate and â-mercaptoethanol did not lead to release of any enzyme subunit into the medium. Such a derivative, prepared under optimal conditions, retained ca. 40% of the intrinsic activity of the free enzyme and was also functionally stabilized, with thermostabilization enhancements of ca. 3 orders of magnitude when compared with its soluble counterpart. This type of derivative may be appropriate for extracorporeal devices in the clinical treatment of acute leukemia and might thus bring about inherent advantages in that all subunits are covalently bound to the support, with a longer half-life and a virtually nil risk of subunit release into the circulating blood stream.
- Lipase catalyzed modification of milkfatPublication . Balcão, Victor M.; Malcata, F. XavierDecreasing consumption of high fat milk and dairy products is driving the dairy industry to seek other uses for increasing surplus of milkfat. Enzyme catalyzed modification of milkfat using lipases is receiving particular attention. This review examines lipase-mediated modification of milkfat. Especial attention is given to industrial applications of lipases for producing structured and modified milkfat for improved physical properties and digestibility, reduced caloric value, and flavor enhancement. Features associated with reactions such as hydrolysis, transesterification, alcoholysis and acidolysis are presented with emphasis on industrial feasibility, marketability and environmental concerns. Future prospects for enzyme catalyzed modification of milk fat are discussed.