Recovery and upgrading bovine rumen protein by extrusion: effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9 per cent or 3.8 per cent) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9 per cent residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8 per cent in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end

Influence of thermoplastic extrusion on the nutritive value of bovine rumen protein

This Study investigated the impact of thermoplastic extrusion on the nutritive quality of bovine rumen protein. Proximal composition, amino acid profile and in vivo true protein digestibility among rats were determined in raw (RBR) and extruded (EBR) rumen. Raw and extruded bovine rumen presented high percentages of protein (more than 95% on dry basis). Neither raw nor extruded proteins had any limiting amino acid, and the RBR and EBR amino acid scores were, respectively, 1.28 (leucine) and 1.25 (methionine plus cystine). Extrusion reduced significantly true protein digestibility from 97.7% to 93.1% (p < 0.001), but protein digestibility-corrected amino acid scores for both proteins (RBR and EBR) were 100%. Animal growth presented comparable profiles using raw and extruded rumen. In conclusion, thermoplastic extrusion did not affect the protein quality of bovine rumen, and this does not hinder the use of this material as a food ingredient. (C) 2009 Elsevier Ltd. Ail rights reserved.

Recovery and upgrading bovine rumen protein by extrusion: Effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9% or 3.8%) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein Solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9% residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8% in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end. (C) 2009 Elsevier Ltd. All rights reserved.

Sensory acceptability of raw and extruded bovine rumen protein in processed meat products

The use of bovine rumen protein (raw and extruded) as a replacement for extruded soy protein concentrate in three meat products (pork sausage, chicken hamburger, and kibbe) was investigated. Similarity between rumen and soy protein meat products was assessed using triangle tests and sensory acceptability evaluated by consumer panelists using a nine-point hedonic scale. The addition of raw rumen protein was detected in all meat product types tested, while extruded rumen protein was only detected in kibbe. The addition of raw rumen protein decreased the acceptability of pork sausage aroma and flavor, but improved kibbe appearance, texture and overall acceptability. The addition of extruded rumen protein reduced the acceptability of chicken hamburger texture, but improved pork sausage flavor. Replacement of soy protein by bovine rumen protein is feasible based upon sensory results, but depended upon its form and the type of meat product to which it was added. (C) 2011 Elsevier Ltd. All rights reserved.

Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

OBJECTIVES: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. MATERIAL AND METHODS: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized pre-surgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). RESULTS: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm² in the test group and 2 mm² in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). CONCLUSIONS: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions.

Volatile compounds in the thermoplastic extrusion of bovine rumen

The volatile compounds of raw and extruded bovine rumen, extracted by dynamic headspace, were separated by gas chromatography and analyzed by GC-MS. Raw and extruded materials presented thirty-two volatile compounds. The following compounds were identified in raw bovine rumen: heptane, 1-heptene, 4-methyl-2-pentanone, toluene, hexanal, ethyl butyrate, o-xylene, m-xylene, p-xylene, heptanal, limonene, nonanal, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane and octadecane. The following compounds were identified in the extruded material: 1-heptene, 2,4-dimethylhexane, toluene, limonene, undecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane and nonadecane. Mass spectra of some unidentified compounds indicated the presence of hydrocarbons with branched chains or cyclic structure.

Effect of an energy-deficient diet on populations of ciliate protozoans in bovine rumen

Ten young rumen-cannulated crossbred steers were randomly divided into two groups: a control group (C; n=4), which was fed a balanced diet for daily weight gain of 900g; and a pronounced energy-deprived group (PED; n=6), receiving 30% less of the required energy for maintenance. After 140 days of these alimentary regimes, rumen fluid and urine samples were collected for biochemical and functional tests, before feeding and at 1, 3, 6, and 9 hours after feeding. The energy-deprivation diet caused a significant reduction in the number of Entodinium, Eodinium, Isotricha, Dasytricha, Eremoplastron, Eudiplodinium, Metadinium, Charonina, Ostracodinium, and Epidinium protozoa. There was no effect of the time of sampling in both groups on the total number of ciliates in rumen fluid. A higher number of protozoan forms in binary division were recorded in the control group, at the 6th and 9th hours after feeding (P<0.019). There was a high positive correlation between the total count of protozoans in rumen fluid and glucose fermentation, ammonia, and urinary allantoin excretion index; and a negative correlation between the total count of protozoa and metilene blue reduction, and a medium correlation between the total count of protozoa and total volatile fatty acids concentration. The determination of the protozoa populations does not imply in the use of complex and hard-to-execute techniques, although it is time consuming and needs practice. This exam particularly helps in clinical expected diagnosis.

Effects of Extrusion on the Emulsifying Properties of Rumen and Soy Protein

Defatted rumen protein and soy protein concentrate were extruded in a 15.5:1 L/D single-screw extruder at the optimum conditions for their expansion (150A degrees C and 35% moisture, and 130A degrees C and 35% moisture, respectively). Emulsions were produced with these proteins and studied by rheology and time domain low-resolution (1)H nuclear magnetic resonance (TD-NMR). Extrusion increased storage modulus of rumen protein emulsions. The opposite was observed for soy protein. Mechanical relaxation showed the existence of three relaxing components in the emulsions whose relative contributions were changed by extrusion. Likewise, spin-spin relaxation time constants (T (2)) measured by TD-NMR also showed three major distinct populations of protons in respect to their mobility that were also altered by extrusion. Extrusion increased surface hydrophobicity of both rumen and soy protein. Solubility of rumen protein increased with extrusion whereas soy protein had its solubility decreased after processing. Extrusion promoted molecular reorganization of protein, increasing its superficial hydrophobicity, affecting its interfacial properties and improving its emulsifying behavior. The results show that extrusion can promote the use of rumen, a by-product waste from the meat industry, in human nutrition by replacing soy protein in food emulsions.

Acurácia, precisão e robustez das estimativas da digestibilidade aparente da matéria seca determinada com o uso de indicadores em ovinos

O objetivo foi avaliar a acurácia, precisão e robustez das estimativas da digestibilidade aparente da matéria seca obtidas utilizando-se como indicadores fibra em detergente ácido indigestível (FDAi), fibra em detergente neutro (FDNi) indigestível, lignina em detergente ácido (LDA), LDA indigestível (LDAi) e óxido crômico em comparação ao método de coleta total de fezes. Dezoito ovinos (56,5 ± 4,6 kg PV) foram designados aleatoriamente a dietas compostas de 25, 50 ou 75% de concentrado e feno de Coast cross por 25 dias. As fezes foram coletadas por cinco dias para determinação da digestibilidade aparente da MS. As amostras de alimentos e fezes foram incubadas no rúmen de três bovinos por 144 horas, para obtenção das frações indigestíveis. Óxido crômico foi administrado a 4,0 g/animal/dia. A acurácia foi avaliada pela comparação do viés médio (DAMS predito - DAMS observado) entre os indicadores; a precisão, por meio da raiz quadrada do erro de predição e do erro residual; e a robustez, pelo estudo da regressão entre o viés e o consumo de matéria seca, o nível de concentrado e o peso vivo. A recuperação fecal e a acurácia das estimativas da digestibilidade aparente da MS foram maiores para FDAi, seguida pela FDNi, LDAi, pelo óxido crômico e depois pela lignina em detergente ácido. O viés linear foi significativo apenas para FDAi, FDNi e LDAi. O uso de óxido crômico permitiu estimativas mais precisas da digestibilidade aparente da MS. Todos os indicadores foram robustos quanto à variação no consumo de matéria seca e apenas LDAi e óxido crômico foram robustos quanto aos níveis de concentrado na dieta. O óxido crômico não foi robusto quando houve variação no peso vivo animal. Assim, a FDAi é o indicador mais recomendado na estimativa da digestibilidade aparente da MS em ovinos quando o objetivo é comparar aos dados da literatura, enquanto o óxido crômico é mais recomendado quando o objetivo é comparar tratamentos dentro de um mesmo experimento.

The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of similar to 35-45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0-9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(l), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some refolding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.

On the interaction of bovine serum albumin with ionic surfactants: Temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility

The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations. HPS presented a smaller effect at concentrations above 1.5 mM. At 10 mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant-BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue. (C) 2011 Elsevier B.V. All rights reserved.

Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Effect of the kappa-casein gene polymorphism, breed and seasonality on physicochemical characteristics, composition and stability of bovine milk

The objective of this study was to evaluate the effect of genetic polymorphism of kappa-casein, breed and seasonality on the physicochemical characteristics, composition and stability of milk in commercial dairy herds. A total of 879 milk and blood samples were collected from 603 Holstein and 276 Girolando cows, obtained during rainy and dry seasons. Milk samples were analyzed to determine the physicochemical characteristics, composition and ethanol stability, while blood samples were subjected to polymerase chain reaction to identify the kappa-casein genotype. The frequencies of genotypes AA, AB and BB of k-casein were respectively, 66.83, 31.84 and 1.33% for Holstein, and 71.38, 27.90 and 0.72% for the Girolando cows, respectively. The A allele was more frequent than the B allele, both for Holstein (0.827 and 0.173) and Girolando cows (0.853 and 0.147), respectively. Cows of AB and BB genotypes showed a higher milk fat content compared to the AA genotype. There was an interaction between breed and seasonality on the concentration of milk urea with higher values for Holstein and Girolando cows in the rainy and dry season, respectively. The levels of lactose, total solids, crude protein, true protein, casein and the casein:true protein ratio were higher during the dry season, while during the rainy season, the somatic cell count and milk urea concentration were higher. There was no association between milk stability and k-casein genotypes, but Holstein cows showed higher milk stability than Girolando cows, and milk was more stable during the rainy season than during the dry season.

Polyelectrolyte-protein complexation driven by charge regulation

The interplay between the biocolloidal characteristics (especially size and charge), pH, salt concentration and the thermal energy results in a unique collection of mesoscopic forces of importance to the molecular organization and function in biological systems. By means of Monte Carlo simulations and semi-quantitative analysis in terms of perturbation theory, we describe a general electrostatic mechanism that gives attraction at low electrolyte concentrations. This charge regulation mechanism due to titrating amino acid residues is discussed in a purely electrostatic framework. The complexation data reported here for interaction between a polyelectrolyte chain and the proteins albumin, goat and bovine alpha-lactalbumin, beta-lactoglobulin, insulin, k-casein, lysozyme and pectin methylesterase illustrate the importance of the charge regulation mechanism. Special attention is given to pH congruent to pI where ion-dipole and charge regulation interactions could overcome the repulsive ion-ion interaction. By means of protein mutations, we confirm the importance of the charge regulation mechanism, and quantify when the complexation is dominated either by charge regulation or by the ion-dipole term.

Report of a chimeric origin of transposable elements in a bovine-coding gene

Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is similar to 85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage.