Rui Barros | Universidade Católica Portuguesa (original) (raw)

Papers by Rui Barros

International Journal of Polymeric Materials and Polymeric Biomaterials, 2015

In the last years, the number of products for oral care has been expanded to adapt to consumer ne... more 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.

Enzyme and Microbial Technology, 2001

Blends of cardosins A and B, enzymes present in aqueous extracts of the flowers of the thistle (C... more Blends 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 stigmae 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 ␤-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 ␣-lactalbumin, one of the major proteins in bovine whey.

Enzyme and Microbial Technology, 2001

Hydrolysis of whey proteins may produce peptide mixtures with better functional properties than t... more Hydrolysis 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. ␣-lactalbumin (␣-La), ␤-lactoglobulin (␤-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, ␣-La was substantially depleted, whereas ␤-Lg was very poorly hydrolyzed and BSA was only slightly hydrolyzed. The native proteins were converted to lower and lower molecular weight peptides.

Food Chemistry, 2004

The enzymatic hydrolysis of the major whey proteins, namely β-lactoglobulin (β-Lg) and α-lactalbu... more The enzymatic hydrolysis of the major whey proteins, namely β-lactoglobulin (β-Lg) and α-lactalbumin (α-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

Journal of Agricultural and Food Chemistry, 2002

The purpose of this research work was to study the proteolytic activity of aqueous crude extracts... more The purpose of this research work was to study the proteolytic activity of aqueous crude extracts of flowers of the plant Cynara cardunculus on the major whey proteins, namely, beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-La). These extracts, containing a mixture of cardosins A and B (i.e., two distinct aspartic proteases), have been employed for many years in traditional cheese-making in Portugal and Spain. Cow's milk sweet whey was incubated for up to 24 h at various ratios of addition of crude enzyme extract, under controlled pH (5.2 and 6.0) and temperature (55 degrees C). The samples collected were assayed by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A mechanistic model was proposed for the kinetics of the hydrolysis process, which is basically a double-substrate, double-enzyme Michaelis-Menten rate expression; the kinetic parameters were estimated by multiresponse, nonlinear regression analysis. The best estimates obtained for the specificity ratio (i.e., k(cat)/K(m)) of each cardosin within the mixture toward each whey protein indicated that said aspartic proteases possess a higher catalytic efficiency for alpha-La (0.42-4.2 mM(-1).s(-1)) than for beta-Lg (0-0.064 mM(-1).s(-1)), at least under the experimental conditions used. These ratios are below those previously reported for caseins and a synthetic hexapeptide. Cardosins are more active at pH 5.2 than at pH 6.0 and (as expected) at higher enzyme-to-substrate ratios.

Enzyme and Microbial Technology, 2003

In the present research effort, production of derivatives of cardosin A (a plant protease) encomp... more In 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 ␤-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 ␣-lactalbumin (␣-La), one of the major proteins in bovine whey. Hydrolysates of ␣-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 ␣-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.

International Journal of Polymeric Materials and Polymeric Biomaterials, 2015

In the last years, the number of products for oral care has been expanded to adapt to consumer ne... more 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.

Enzyme and Microbial Technology, 2001

Blends of cardosins A and B, enzymes present in aqueous extracts of the flowers of the thistle (C... more Blends 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 stigmae 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 ␤-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 ␣-lactalbumin, one of the major proteins in bovine whey.

Enzyme and Microbial Technology, 2001

Hydrolysis of whey proteins may produce peptide mixtures with better functional properties than t... more Hydrolysis 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. ␣-lactalbumin (␣-La), ␤-lactoglobulin (␤-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, ␣-La was substantially depleted, whereas ␤-Lg was very poorly hydrolyzed and BSA was only slightly hydrolyzed. The native proteins were converted to lower and lower molecular weight peptides.

Food Chemistry, 2004

The enzymatic hydrolysis of the major whey proteins, namely β-lactoglobulin (β-Lg) and α-lactalbu... more The enzymatic hydrolysis of the major whey proteins, namely β-lactoglobulin (β-Lg) and α-lactalbumin (α-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

Journal of Agricultural and Food Chemistry, 2002

The purpose of this research work was to study the proteolytic activity of aqueous crude extracts... more The purpose of this research work was to study the proteolytic activity of aqueous crude extracts of flowers of the plant Cynara cardunculus on the major whey proteins, namely, beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-La). These extracts, containing a mixture of cardosins A and B (i.e., two distinct aspartic proteases), have been employed for many years in traditional cheese-making in Portugal and Spain. Cow's milk sweet whey was incubated for up to 24 h at various ratios of addition of crude enzyme extract, under controlled pH (5.2 and 6.0) and temperature (55 degrees C). The samples collected were assayed by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A mechanistic model was proposed for the kinetics of the hydrolysis process, which is basically a double-substrate, double-enzyme Michaelis-Menten rate expression; the kinetic parameters were estimated by multiresponse, nonlinear regression analysis. The best estimates obtained for the specificity ratio (i.e., k(cat)/K(m)) of each cardosin within the mixture toward each whey protein indicated that said aspartic proteases possess a higher catalytic efficiency for alpha-La (0.42-4.2 mM(-1).s(-1)) than for beta-Lg (0-0.064 mM(-1).s(-1)), at least under the experimental conditions used. These ratios are below those previously reported for caseins and a synthetic hexapeptide. Cardosins are more active at pH 5.2 than at pH 6.0 and (as expected) at higher enzyme-to-substrate ratios.

Enzyme and Microbial Technology, 2003

In the present research effort, production of derivatives of cardosin A (a plant protease) encomp... more In 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 ␤-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 ␣-lactalbumin (␣-La), one of the major proteins in bovine whey. Hydrolysates of ␣-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 ␣-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.