Browsing by Author "Fontes, Carlos M. G. A."
Now showing 1 - 9 of 9
Results Per Page
Sort Options
- Carcass fat partitioning and meat quality of Alentejana and Barrosã young bulls fed high or low maize silage dietsPublication . Costa, Ana S. H.; Costa, Paulo; Bessa, Rui J. B.; Lemos, José P. C.; Simões, Jorge A.; Silva, José Santos; Fontes, Carlos M. G. A.; Prates, José A. M.This study assessed the effect of breed and diet on carcass composition, particularly fat partitioning, and meat quality in young bulls. An experiment with forty young bulls from two phylogenetically distant Portuguese bovine breeds, Alentejana and Barrosã, fed two diets with different maize silage to concentrate ratios, but isoenergetic and isonitrogenous, was carried out until the animals reached 18 months of age. In the longissimus lumborum muscle, Barrosã bulls fed the low silage diet had the highest intramuscular fat (IMF) content. Bulls fed the low silage diet also had the highest IMF content in the semitendinosus muscle. Diet determined the proportions of total visceral fat and individual fat depots. Under these experimental conditions, it was shown that the genetic background is a major determinant of carcass composition and meat quality, and that the dietary differences studied had limited effect on carcass composition.
- Contents of conjugated linoleic acid isomers in ruminant-derived foods and estimation of their contribution to daily intake in PortugalPublication . Martins, Susana V.; Lopes, Paula A.; Alfaia, Cristina M.; Ribeiro, Veronica S.; Guerreiro, Teresa V.; Fontes, Carlos M. G. A.; Castro, Matilde F.; Soveral, Graca; Prates, Jose A. M.The present study provides a detailed overview of the contents of conjugated linoleic acid (CLA) isomers in the most consumed Portuguese CLA-rich foods (milk, butter, yoghurt, cheese, beef and lamb meat), by using silver ion-HPLC. In addition, the contribution of these ruminant-derived foods to the daily intake of CLA isomers was estimated based on Portuguese consumption habits. The total CLA concentration in milk and dairy products ranged from 4.00 mg/g fat in yoghurt to 7.22 mg/g fat in butter, and, regarding meats, from 4-45 mg/g fat in intensively produced beef to 11.29 mg/g fat in lamb meat. The predominant CLA isomers identified in these products were cis-9,trans-11 (59.89-79.21 %) and trans-7,cis-9 (8.04-20.20%). The average estimated total CLA intake for the Portuguese population was 73.70mg/d. Milk and cheese are probably the two products with the highest contribution to the final CLA intake, as a result of their high fat content and consumption values. The results also suggested that cis-9,trans-11 and trans-7,cis-9 are the isomers most represented, with, respectively, 76.10 and 12.56 % of the total CLA intake. Being the first detailed report on the contents of total and individual CLA isomers in Portuguese commercial ruminant-derived foods, we further discuss the implication of the results for diet characteristics and human health.
- Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for Gluco- and Galacto- Substituted mannans and Its calcium induced stabilityPublication . Ghosh, Arabinda; Luís, Ana Sofia; Brás, Joana L. A.; Pathaw, Neeta; Chrungoo, Nikhil K.; Fontes, Carlos M. G. A.; Goyal, ArunThis study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.
- Diet supplementation with the cis-9,trans-11 conjugated linoleic acid isomer affects the size of adipocytes in Wistar ratsPublication . Lopes, Paula A.; Martins, Susana V.; Pinho, Mario S.; Alfaia, Cristina M.; Fontes, Carlos M. G. A.; Rodrigues, Pedro O.; Morais, Graqa S. L.; Castro, Matilde F.; Pinto, Rui; Prates, Jose A. M.Previous reports have demonstrated that conjugated linoleic acid (CLA) acts on body fat accumulation in a variety of animal models. The aim of the present study was to investigate the effect of cis (c)-9,trans (t)-11 and t10,c12 CLA isomers on the number and size of adipocytes from the inguinal and retroperitoneal fats in Wistar male rats. A 5.1% palm oil-based diet was supplemented with CLA isomers as follows: 0.6% of c9,t11, 0.6% of t10,c12, 1.3% of c9,t11 and t10,c12 isomers in mixture, and a control nonsupplemented group for comparative purposes. Fat tissues were prepared on microscope slides for histologic examination using an image-analysis software to count the number of adipocytes and measure cell sizes. The results showed that CLA isomers did not affect (P .05) either final body and fat depot weights or serum lipids (with the exception of triacylglycerols) and adipocytokines (leptin and adiponectin). Animals fed the c9, t11 CLA isomer diet showed larger adipocytes when compared to other groups. Independently of the CLA dietary treatment, retroperitoneal fat showed larger adipocytes (3319 mu m(2)) and therefore a smaller number of adipocytes per unit of area, compared to inguinal fat (3055 mu m(2)) Taken together, the data suggest that a palm oil-based diet supplemented with the c9,t11 CLA isomer in Wistar rats, in contrast to the t10,c12 isomer and the mixture of both isomers, increases adipocyte dimensions in inguinal and retroperitoneal fat depots, while having a minor effect in serum lipids and adipocytokines. (c) 2008 Elsevier Inc. All rights reserved.
- Genetic background and diet impact beef fatty acid composition and stearoyl-CoA desaturase mRNA expressionPublication . Costa, Ana S. H.; Silva, Marta P.; Alfaia, Cristina P. M.; Pires, Virgínia M. R.; Fontes, Carlos M. G. A.; Bessa, Rui J. B.; Prates, José A. M.The intramuscular fat composition of ruminant meats influences the quality of the final product, which explains the increasing interest in assessing the fatty acid profile of meat from different production systems. In this study, it was hypothesized that there are breed- and diet induced variations on lipid metabolism in the muscle, which may be, at least partially, modulated by the stearoyl-CoA desaturase (SCD) gene expression levels. Forty purebred young bulls from two phylogenetically distant autochthonous cattle breeds, Alentejana and Barrosã(n = 20 for each breed), were assigned to two different diets (low vs. high silage) and slaughtered at 18 months of age. Meat fatty acid composition, including the detailed conjugated linoleic acid (CLA) isomeric profile, was determined along with the SCD mRNA levels. Meat from Barrosã bulls fed the low silage diet was richer in monounsaturated fatty acids, CLA and trans fatty acids, when compared to that from Alentejana bulls. The meat content in polyunsaturated fatty acids was similar across experimental groups. Moderate positive correlations between the SCD mRNA levels and the products of this enzyme activity were found, although they were not reflected on the calculated desaturase indices. Overall, these findings highlight the importance of taking into account the genetic background while devising feeding strategies to manipulate beef fatty acid composition.
- Irradiation effect on fatty acid composition and conjugated linoleic acid isomers in frozen lamb meatPublication . Alfaia, Cristina M. M.; Ribeiro, Paulo J. L. C.; Trigo, Maria J. P.; Alfaia, Antonio J. I.; Castro, Matilde L. F.; Fontes, Carlos M. G. A.; Bessa, Rui J. B.; Prates, Jose A. M.The effect of gamma radiation processing on the lipid content, fatty acid composition and conjugated linoleic acid (CLA) profile in frozen lamb meat was investigated. Samples of longissimus thoracis muscle from lambs fed lucerne basal diets either unsupplemented or Supplemented with polyunsaturated vegetable oils were irradiated (7 kGy) and analysed. CLA contents in lamb meat did not affect (P 0.05) the levels of lipid oxidation induced by the irradiation. No significant differences (P 0.05) were observed for fatty acid composition, related nutritional indexes (n - 6/n - 3 and PUFA/SFA), as well as for total lipid and CLA contents, between non-irradiated (control) and irradiated meat samples. In contrast, meat irradiation affected the relative proportions of total trans, trans and cis/trans CLA isomers (P 0.001), in addition to the percentage of some minor individual CLA isomers (t11,t13 and t9,t11, with P 0.05 and P 0.001, respectively). The percentage of total cis/trans CLA isomers slightly decreased in irradiated samples, while the relative proportion of total trans, trans isomers slightly increased. This observation may be explained by the higher susceptibility to autoxiclation of the cis double bond relative to the trans configuration. (c) 2007 Elsevier Ltd. All rights reserved.
- Small angle X-ray scattering analysis of Clostridium thermocellum cellulosome N-terminal complexes reveals a highly dynamic structurePublication . Currie, Mark A.; Cameron, Kate; Dias, Fernando M. V.; Spencer, Holly L.; Bayer, Edward A.; Fontes, Carlos M. G. A.; Smith, Steven P.; Jia, ZongchaoClostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.
- The family 6 carbohydrate-binding module (CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca2+ ionsPublication . Ahmed, Shadab; Luis, Ana Sofia; Brás, Joana L. A.; Fontes, Carlos M. G. A.; Goyal, ArunThe gene encoding CtCBM6B of Clostridium thermocellum α-L-arabinofuranosidase (Ct43Araf) was cloned in pET-21a(+) vector, over-expressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography (IMAC). The recombinant CtCBM6B showed a molecular size close to 15 kDa by SDS-PAGE analysis, which was close to the expected size of 14.74 kDa. The ligand-binding affinity of CtCBM6B was assessed against ligands for which the catalytic enzyme, Ct43Araf showed maximum activity. The affinity-gel electrophoresis of CtCBM6B with rye arabinoxylan showed lower equilibrium association constant (Ka, 4.0% C− 1), whereas, it exhibited higher affinity (Ka, 19.6% C− 1) with oat spelt xylan. The ligand-binding analysis of CtCBM6B by fluorescence spectroscopy also revealed similar results with low Ka (3.26% C− 1) with rye arabinoxylan and higher affinity for oat spelt xylan (Ka, 17.9% C− 1) which was corroborated by greater blue-shift in case of oat spelt xylan binding. The CtCBM6B binding with insoluble wheat arabinoxylan by adsorption isotherm analysis showed significant binding affinity as reflected by the equilibrium association constant (Ka), 9.4 × 103 M− 1. The qualitative analysis by SDS-PAGE also corroborated the CtCBM6B binding with insoluble wheat arabinoxylan. The protein-melting curve of CtCBM6B displayed the peak shift from 53°C to 59°C in the presence of Ca2+ ions indicating that Ca2+ ions impart thermal stability to the CtCBM6B structure.
- Thermostable Recombinant β‑(1→4)-Mannanase from C. thermocellum: biochemical characterization and manno-oligosaccharides productionPublication . Ghosh, Arabinda; Luís, Ana Sofia; Brás, Joana L. A.; Fontes, Carlos M. G. A.; Goyal, ArunFunctional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum ATCC 27405. Its sequence comparison the exhibited highest similarity with Man26B of C. thermocellum F1. The full length CtManf and truncated CtManT were cloned in the pET28a(+) vector and expressed in E. coli BL21(DE3) cells, exhibiting 53 kDa and 38 kDa proteins, respectively. On the basis of the substrate specificity and hydrolyzed product profile, CtManf and CtManT were classified as β-(1→4)-mannanase. A 1.5 fold higher activity of both enzymes was observed by Ca2+ and Mg2+ salts. Plausible mannanase activity of CtManf was revealed by the classical hydrolysis pattern of carob galactomannan and the release of manno-oligosaccharides. Notably highest protein concentrations of CtManf and CtManT were achieved in tryptone yeast extract (TY) medium, as compared with other defined media. Both CtManf and CtManT displayed stability at 60 and 50 °C, respectively, and Ca2+ ions imparted higher thermostability, resisting their melting up to 100 °C.