RESUMO
Recently, glyco-therapy is proposed to prevent the interaction of bacterial lectins with host ligands (glycoconjugates). This interaction represents the first step in infection. Neoglycans referred to as PSA-Lac (PSA-Glu (ß1-4) Gal) were obtained by conjugation of porcine serum albumin (PSA) with lactose at 80 °C, 100 °C and 120 ºC. Characterization studies of the products showed that PSA could contain 1, 38 or 41 added lactoses, depending on the reaction temperature. These neoglycans were approximately 10 times more glycated than PSA-Lac obtained in previous work. Lactose conjugation occurred only at lysines and PSA-Lac contained terminal galactoses as confirmed by Ricinus communis lectin recognition. Furthermore, Escherichia coli K88+, K88ab, K88ac and K88ad adhesins showed affinity toward all PSA-Lac neoglycans, and the most effective was the PSA-Lac obtained after 100 ºC treatment. In vitro, this neoglycan partially inhibited the adhesion of E. coli K88+ to piglet mucin (its natural ligand). These results provide support for the hypothesis that glycated proteins can be used as an alternative for bioactive compounds for disease prevention.
Assuntos
Glicoconjugados/metabolismo , Lactose/química , Albumina Sérica/química , Albumina Sérica/metabolismo , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/metabolismo , Animais , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/fisiologia , Infecções Bacterianas/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Escherichia coli/química , Escherichia coli/metabolismo , Glicoconjugados/química , Glicoconjugados/uso terapêutico , Glicosilação , Mucinas/química , Lectinas de Plantas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , TemperaturaRESUMO
Se comparó el efecto antibacteriano de las lactoferrinas (Lfs) bovina y porcina sobre Escherichia coli K88+ (E. coli K88+), uno de los principales agentes etiológicos de las diarreas en lechones en el hemisferio norte. Las Lfs se purificaron por cromatografía de intercambio iónico, confirmando su pureza por electroforesis en condiciones desnaturalizantes y reductoras (SDS-PAGE) en geles al 8% y por inmuno-detección con anticuerpos anti- lactoferrina. La actividad bacteriostática se probó utilizando concentraciones de 0,5 y 1,0 mg/mL de Holo (saturada de hierro) y Apo-Lf (libre de hierro). En todos los casos la actividad bacteriostática de las Holo-Lfs fue insignificante, mientras que en ambas concentraciones, la Apo-Lf bovina mostró un mayor efecto en la inhibición del crecimiento de la E. coli K88+ que la Apo-Lf porcina. La actividad bactericida se ensayó utilizando concentraciones de 2,0; 4,0; 6,0 y 8,0 mg/mL de Lfs bovina o porcina. La Lf bovina mostró efecto bactericida a una concentración de 8 mg/mL, mientras que la Lf porcina no presentó este efecto. Los resultados indican que la fuente bovina puede ser útil en la prevención de diarreas en lechones.
The antibacterial effect of bovine lactoferrin (Lf) towards Escherichia coli K88+ (E. coli K88+) was compared with that of porcine Lf. E. coli K88+ is one of the main etiological agents of piglet diarrhea in the northern hemisphere. Lactoferrins (Lfs) were purified by ion exchange chromatography and further analyzed by electrophoresis using 8% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and immuno detection with anti-lactoferrin antibodies. Bacteriostatic effect was assayed using 0.5 and 1.0 mg/mL of Holo-Lf (iron saturated) or Apo-Lf (iron free). In all test the holo-LFs showed negligible bacteriostatic activity while for Apo-Lfs the bovine protein had the highest activity. Bactericide activity was assayed using bovine or porcine Lf concentrations of 2.0, 4.0, 6.0 y 8.0 mg/mL. Bovine Lf had bactericide activity at 8.0 mg/mL while no growth inhibition was observed with porcine Lf. These results suggest that bovine Lf may serve in the prophylaxis of piglets diarrhea.
RESUMO
Se analizó el efecto de la sustitución de clara de huevo por albúmina sérica porcina (ASP) en panqués de chocolate. La ASP se obtuvo mediante un método escalado de aislamiento por cromatografía de interacción hidrofóbica. En la formulación del panqué se reemplazó el 50 y 100% de la clara de huevo con ASP. Todos los panqués presentaron valores similares (P >0.05) de los parámetros de color en la miga: L (25,7-26,2), a* (9,8-10,1) y b* (14,5-15,0) y en la costra: L (25,7-26,2), a* (9,8-10,1) y b* (14,5-15,0). La textura (2,9 N) y el volumen (148,9 ± 1,8 cm ³) de los panqués con 50% de ASP fueron similares (P> 0,05) a los de los controles. El análisis sensorial indicó que los panqués en los que se reemplazó 50% de la clara por ASP, gustaron tanto como los controles. Los panqués con un reemplazo del 100%, gustaron menos. La excelente calidad microbiológica de los panqués muestra las óptimas condiciones sanitarias durante la obtención de la ASP y su elaboración.
The effect of porcine serum albumin (PSA) as a substitute for egg white (EW) in chocolate cakes was examined. PSA was obtained by a lab-scaled method of Hydrophobic Interaction Chromatography. 50 and 100% of the normal level of EW was replaced with PSA in cake formulation. All cakes had similar (P > 0.05) crumb L (25.7-26.2), a* (9.8-10.1) y b* (14.5-15.0) and crust: L (25.7-26.2), a* (9.8-10.1) y b* (14.5-15.0) color values. Texture (2.9 N) and volume (148.9 1.8 cm ³) of cakes with 50% PSA replacing EW were similar (P > 0.05) to those of the controls. Sensory analysis indicated that cakes replaced with 50% EW for ASP were as well liked as control cakes. The excellent microbiological quality of formulated cakes points out the optimal sanitary conditions in the PSA isolation and in the cake elaboration process.
RESUMO
Escherichia coli (E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc beta (1-4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets' diarrhea.
Assuntos
Adesinas de Escherichia coli/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Albumina Sérica/metabolismo , Adesinas de Escherichia coli/química , Aminas , Animais , Antígenos de Bactérias/química , Aderência Bacteriana/efeitos dos fármacos , Eletroforese , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/química , Glicosilação/efeitos dos fármacos , Lactose/metabolismo , Peso Molecular , Lectinas de Plantas/farmacologia , Albumina Sérica/química , Albumina Sérica/isolamento & purificação , Albumina Sérica/farmacologia , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sus scrofa , Triptofano/metabolismoRESUMO
A new, highly acetylated agarose matrix (HA-Sepharose) was synthesized and used as a hydrophobic interaction chromatography (HIC) medium to specifically isolate immunoglobulins (Igs) from porcine serum. Recovery of Igs was in a single step and under mild conditions. HA-Sepharose adsorption was studied in terms of salt, gel acetylation time, flow rate, and protein concentration on the loading buffer. At 0.5 M Na2SO4, control with unmodified Sepharose retained a small fraction (0.70 mg/mL of matrix) of serum albumin. On the contrary HA-Sepharose retained primary Igs (IgA, IgG, and 53% of IgM) as revealed by sodium dodecyl sulphate 10% polyacrylamide gel electrophoresis (SDS-PAGE), quantitative radial immunodiffusion and immunodetection. At a flow rate of 1 mL/min, the HA-Sepharose column capacity (3.9 mg/mL of matrix) was similar to the reported capacity for the commercial thiophilic T-gel. However, HA-Sepharose showed higher recovery of IgA and IgM than the T-gel in the same salt conditions, clearly an advantage in terms of immunoglobulin recovery strategies. Acetylation changed the matrix adsorption from albumin to immunoglobulins; thus, the highly acetylated gel rendered recoveries of Igs from unprocessed porcine serum practically free of albumin.