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- Immune responses and gut morphology of Senegalese sole (Solea senegalensis, Kaup 1858) fed monospecies and multispecies probioticsPublication . Batista, S.; Ramos, M. A.; Cunha, S.; Barros, Rui; Cristóvão, B.; Rema, P.; Pires, M. A.; Valente, L. M. P.; Ozório, R. O. A.The current study aimed to determine the effects of dietary probiotic supplementation on growth, gut morphology and non-specific immune parameters in Senegalese sole (Solea senegalensis) juveniles during a 1-month trial. Fish were fed for 1-month two diets with 1.0 or 4.6 × 106 CFU kg−1) of probiotic A (Bacillus sp., Pediococcus sp., Enterococcus sp. and Lactobacillus sp.) and two diets with 3.5 or 8.6 × 105 CFU kg−1 of probiotic B (Pediococcus acidilactici) and tested against an unsupplemented diet (control). Growth performance, as well as respiratory burst activity, nitric oxide (NO), alternative complement pathway (ACH50), lysozyme and peroxidase activities, was not affected by the dietary treatments. Probiotic supplementation tended to increased growth homogeneity between tanks having diet A1 the best possible alternative to decrease costs associated to size grading. Villous length and number of goblet cells of the anterior intestine did not vary among treatments. Muscle duodenal layer was significantly thicker in fish fed probiotic A compared to probiotic B, when included at the lowest level (A2 versus B2). The current study indicate that the use of the multispecies probiotic at 1.0 × 106 CFU kg−1 might enhance protection against pathogen outbreak and increase nutrient absorption, whereas at the highest concentration could reduced size dispersion among tanks.
- A kinetic model for hydrolysis of whey proteins by cardosin a extracted from Cynara CardunculusPublication . Barros, Rui M.; Malcata, F. XavierThe enzymatic hydrolysis of the major whey proteins, namely b-lactoglobulin (b-Lg) and a-lactalbumin (a-La), was experimentally studied using whey as substrate; an aspartic protease (cardosin A), previously extracted from the flowers of Cynara cardunculus and purified by gel filtration and ion exchange chromatographies, was used for this purpose. Sweet whey was incubated for 24 h at various enzyme:substrate ratios, at controlled pH (5.2 and 6.0) and temperature (55 C); the hydrolyzates were assayed by gel permeation chromatography and electrophoresis. A mechanistic model was proposed for the kinetics, which basically leads to a double-substrate, single-enzyme Michaelis–Menten rate expression containing four adjustable parameters; these parameters were estimated by applying multiresponse, nonlinear regression analysis to the experimental data, so that the model would yield good fits. The best estimates obtained for Km were markedly lower for a-La than for b-Lg, so cardosin A shows a higher affinity for a-La than for b-Lg. The experimental results also suggest that b-Lg is rather resistant to enzyme-mediated hydrolysis under all experimental conditions tested. The highest activity (measured by kcat) of cardosin A was recorded toward a-La (i.e. 0.013 s 1) at pH 5.2. Furthermore, the specificity ratio (kcat=Km), obtained toward each whey protein, indicated that cardosin A possesses a higher catalytic efficiency for hydrolysis of a-La than of b-Lg; the highest value for this ratio was recorded for a-La at pH 5.2, and was close to that reported elsewhere for cardosin A acting on caseins and casein-like substrates.
- Molecular characterization of peptides released from beta-lactoglobulin and alpha-lactalbumin via cardosins A and BPublication . Barros, R. M.; Malcata, F. X.Crude mixtures of aspartic proteases from flowers of the plant Cynara cardunculus have been studied frequently, as have activities of such enzymes (in pure form) on caseins from bovine, ovine, and caprine sources. This research study addressed pure bovine whey protein as substrates; that is, α-lactalbumin (α-LA) and β-lactoglobulin (α-LG), submitted to hydrolysis by 1 of 2 aspartic proteases (cardosins A and B), previously extracted and purified from C. cardunculus. Samples collected, following incubation at 55°C and pH 5.2, were assayed by fast protein liquid chromatography, reversed phase-high performance liquid chromatography, and tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis; the major peptides released were then collected and sequenced by Edman degradation. Cardosin B and, to a lesser degree, cardosin A showed proteolytic activity toward α-LA, but the hydrolyzates produced were characterized by distinct peptide profiles. Cardosin B possesses a broad specificity, and produces several hydrophobic peptides (at least 5, with molecular mass in the range 2 to 8 kDa) in the early stages, which eventually become more hydrophilic (with molecular mass below 2 kDa) at later stages of hydrolysis. Cardosin A was found to cleave α-LA at the peptide bonds Phe28-Arg29, Gly54-Tyr55, Ala59-Ile60, Leu71-Phe72, and Leu105-Thr106, whereas cardosin B cleaved Ala19-Glu20, Phe28-Arg29, Glu30-Leu31, Tyr37-Gly38, Trp45-Val46, Phe50-His51, Ala59-Ile60, Ser66-Thr67, Leu71-Phe72, Phe72-Gln73, Gln73-Ile74, Ile78-Trp79, Leul15-Asp116, and Leu124-Ala125. Conversely, cardosins A and B are apparently not active on β-LG.
- Hydrolysis of caseins by extracts of Cynara Cardunculus precipitated by ammonium sulfatePublication . Silva, S. V.; Barros, R. M.; Malcata, F. XavierPolyacrylamide gel electrophoresis (in the presence of urea) and gel permeation chromatography were employed to assess the profile of hydrolysis of caseins and the activity of enzymes contributed by the flowers of the plant Cynara cardunculus on bovine caseins, after previous precipitation with ammonium sulfate or in a plain crude aqueous extract. Results indicated that the qualitative degradation profile of bovine caseins by plant enzymes (cardosins) remains essentially unchanged upon extraction, and quantitative analysis showed that the precipitated fractions had a higher coagulant-to-proteolytic activity ratio; hence, the results showed that inexpensive precipitation with ammonium sulfate can successfully be used as a purification method in the production of that plant coagulant in standardized form.
- Development of oral strips containing chitosan as active ingredient: a product for buccal healthPublication . Cardelle-Cobas, Alejandra; Madureira, Ana Raquel; Costa, Eduardo; Barros, Rui; Tavaria, Freni K.; Pintado, Manuela E.In the last years, the number of products for oral care has been expanded to adapt to consumer needs. Thus, in addition to conventional products, new pocket products such as sugar-free chewing gums and oral strips (OS) have been developed for oral care. In the present study, OS were formulated using chitosan as the film-forming polymer in adequate concentrations to also be used as antimicrobial agent. Other strip components, such as the type of plasticizer, were also optimized. Mechanical properties of the optimal OS were evaluated and, due to chitosan's characteristic astringency, the strips were also sensorially evaluated.
- Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfatePublication . Barros, Rui M.; Ferreira, Carla A. Ferreira; Silva, Sofia V. Silva; Malcata, F. XavierHydrolysis of whey proteins may produce peptide mixtures with better functional properties than the original protein mixture, viz. higher solubilites and lower allergenic effects. Cynara cardunculus is a wild plant that possesses (aspartic) proteases in its flower cells; those enzymes exhibit general proteolytic and specific milk clotting activities, which are rather useful in traditional cheesemaking. This study was thus aimed at characterizing the enzymatic action of crude extracts of said plant after preliminary purification by salting out with ammonium sulfate at two different concentration levels, viz. 30% and 70% saturation. The coagulant activity on milk, and the proteolytic activity using casein and azocasein as substrates, of the crude extract and of each precipitated fraction were measured at 37°C and pH 5.2. The profile of hydrolysis of the major whey proteins, i.e. α-lactalbumin (α-La), β-lactoglobulin (β-Lg) and bovine serum albumin (BSA) was characterized by gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate. The 30% and 70% saturation fractions exhibited lower coagulant and proteolytic specific activities than the crude extract. However, the relative ratio of coagulant to proteolytic activity, which is a useful indicator of appropriateness for cheesemaking, was higher for the partially purified fractions. The extents of hydrolysis of whey proteins brought about by the partially purified extracts were above those by their crude counterpart, but qualitative hydrolysis patterns were essentially identical to each other; by 24 h, α-La was substantially depleted, whereas β-Lg was very poorly hydrolyzed and BSA was only slightly hydrolyzed. The native proteins were converted to lower and lower molecular weight peptides.
- Hydrolysis of α-lactalbumin by cardosin A immobilized on highly activated supportsPublication . Barros, Rui M.; Extremina, Clara I.; Gonçalves, Inês C.; Braga, Beatriz O.; Balcão, Victor M.; Malcata, F. XavierIn the present research effort, production of derivatives of cardosin A (a plant protease) encompassing full stabilization of its dimeric structure has been achieved, 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 providing evidence for the effectiveness of the attachment procedure. Furthermore, the cardosin A derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at laboratory-scale trials to perform (selective) hydrolysis of α-lactalbumin (α-La), one of the major proteins in bovine whey. Hydrolysates of α-La were assayed for by the OPA method, as well as by FPLC, SDS–PAGE and HPLC. Thermal inactivation of the immobilized cardosin A was also assessed at 40, 50 and 55 °C; at these temperatures, no thermal denaturation took place during incubation for 48 h. The highest degree of hydrolysis was attained by 5 h reaction, at 55 °C and pH 5.2. SDS–PAGE of α-La hydrolysates displayed bands corresponding to low molecular weight peptides. Our results suggest that cardosin A in immobilized form is a good candidate to bring about proteolysis in the dairy industry, namely in whey processing.
- Bacterial diversity and probiotic activity in sole aquaculturePublication . Cunha, Sofia; Carvalho, Ana; Barros, Rui; Gomes, Ana; Manaia, C. M.
- Decreases in molecularity promote conversion when reactions are catalyzed by enzymes immobilized in slab-shaped beadsPublication . Barros, R.M.; Malcata, F. XavierApplication of Fick's first law to substrate undergoing chemical reaction catalyzed by an enzyme immobilized in a porous slab-shaped bead leads to substrate concentration profiles that are flatter when the ratio of stoichiometric coefficients of product to reactant (x) is lower. Since the actual effectiveness factor decreases when x increases (at approximately the same rate irrespective of the value taken by the Michaelis-Menten parameter), then considerable overprediction of the conversion levels obtained within a given reaction timeframe will occur if the simplistic (and more easily modelled) situation of equimolar counterdiffusion is taken for modelling purposes when x>1.
- Peach polyphenol and carotenoid content as affected by frozen storage and pasteurizationPublication . Oliveira, Ana L.; Alexandre, Elisabete M. C.; Coelho, Marta; Barros, Rui M.; Almeida, Domingos P. F.; Pintado, ManuelaThe profile of polyphenol and carotenoid of peach were analysed on fresh fruits, immediately after freezing or after pasteurization, and during frozen storage at -20 ºC for 360 days. Freezing did not affect the concentration of polyphenols or carotenoids. Pasteurization of fresh and frozen samples induced an increase in the extractability of chlorogenic acid (38% and 24%) and of zeaxanthin (336% and 127%) and a decrease of lutein (22% and 56%) and b-cryptoxanthin (32 and 51%). Total antioxidant capacity and total phenolic content changed by less than 20% after frozen storage for 360 d. However, total carotenoid content decreased 41% and 48% in unpasteurized and pasteurized samples, after the same storage period. Long term storage induced an increase in the extractability of (+)-catechin (47%) and zeaxanthin (90%) and a decrease in neochlorogenic acid (59%), chlorogenic acid (39%), quercetin-3-glucoside (21%), lutein (24%) and b-cryptoxanthin (40%). Pasteurization of frozen samples promoted a decrease in the concentration of neochlorogenic acid (91%), chlorogenic acid (87%) and quercetin-3-rutinoside (71%) and increase in lutein (16%), b-cryptoxanthin (42%) and b-carotene (31%). Peach should be stored for periods no longer than 230 d, since pasteurization after that, induced a higher decrease in neochlorogenic, chlorogenic acids and quercetin-3-rutinoside content.