Bacterial recognition of thermal glycation products derived from porcine serum albumin with lactose.

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.

Conjugates of bovine serum albumin with chitin oligosaccharides prepared through the Maillard reaction.

Chitin neoglycoconjugates (BSA-CO) were obtained by the conjugation of bovine serum albumin (BSA) with chitin oligosaccharides (CO) through the Maillard reaction (nonenzymatic glycation). CO produced by acid hydrolysis of chitin were fractionated using an ultrafiltration membrane system (1-3 kDa cutoff). The Maillard reaction was carried out by heating a freeze-dried mixture containing BSA and CO at 60 °C (under 43% relative humidity for 6 and 12 h). BSA-CO were characterized by available amino groups content, intrinsic tryptophan emission spectra, gel electrophoresis, and mass spectrometry. Biological assays included interaction with wheat germ agglutinin (WGA) and with bacterial adhesins of Escherichia coli K88+ and Salmonella choleraesuis. Glycation of BSA was revealed by reduction of available amino groups and fluorescence intensity and also retarded migration through SDS-PAGE. Conjugation of BSA with chitin oligomers appeared to be time dependent and was confirmed by mass spectrometry, by which molecular mass increase for monomers and dimers was observed. Monomers were estimated to contain either one or two glycation sites (at 6 and 12 h of treatment, respectively), with one or two tetrasaccharide units attached. Consequently, dimers showed two or four glycation sites. BSA-CO presented biological recognition by WGA and E. coli K88+ and S. cholerasuis adhesins. The strategy used in this work represents a simple method to obtain glycoconjugates to study applications involving protein-carbohydrate recognition.

Actividad antibacteriana de lactoferrina bovina y lactoferrina porcina sobre Escherichia Coli K88+ / Antibacterial Activity of Bovine and Porcine Lactoferrins Against Escherichia coli K88+

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.

Evaluación de albúmina porcina como sustituto parcial de clara de huevo en panqués de chocolate / Evaluation of Porcine Albumin as a Partial Replacement for Egg White in Chocolate Cakes

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.

Biorecognition of chemically modified bovine serum albumin with lactose prepared under different conditions.

Glycoconjugates consist of glycans attached to proteins or lipids. Glycans are involved in important biological functions such as trafficking of glycoconjugates, mediation, and modulation of cell adhesion and signaling. This study was conducted to obtain neoglycoconjugates containing a large number of carbohydrates, added through the condensation of reducing sugars with protein amino groups, whose structures were recognized by lectins. Neoglycoconjugates (BSA-Lac) of bovine serum albumin (BSA) with d-lactose were obtained using two sets of glycation conditions, each previously selected by its ability to glycate proteins extensively. The conditions included dry heat at 60 degrees C (for 7, 14, 21, and 28 days) and wet heat in 43% relative humidity (RH) at 50 degrees C (for 5, 10, 15, and 20 h). Products were characterized by gel electrophoresis, tryptophan fluorescence emission spectra, mass spectrometry, free amino group analysis, and their biological recognition established by a galactose-specific lectin and Escherichia coli K88 adhesins. BSA-Lac when compared to BSA revealed an increase in monomer mass due to addition of either 13 (dry heat) or 14 (wet heat) lactoses and formation of polymers (in wet conditions). All BSA-Lac products showed reduced intensity of intrinsic fluorescence, decreased amino groups' availability, and were recognized by Ricinus communis I lectin (RCAI) and by E. coli K88 adhesins. Overall, glycation using both conditions was time-dependent, but greater biorecognition was observed with wet-heat products, due to a higher global glycation and/or to the carbohydrate accessibility. The strategy used in this work represents a simple procedure to obtain glycoconjugates that could be used for recognition studies in biological systems.

Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin.

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.

Characterization of bovine serum albumin glycated with glucose, galactose and lactose.

The non-enzymatic reaction between reducing sugars and proteins, known as glycation, has received increased attention from nutritional and medical research. In addition, there is a large interest in obtaining glycoconjugates of pure well-characterized oligosaccharides for biological research. In this study, glycation of bovine serum albumin (BSA) by d-glucose, d-galactose and d-lactose under dry-heat at 60 degrees C for 30, 60, 120, 180 or 240 min was assessed and the glycated products studied in order to establish their biological recognition by lectins. BSA glycation was monitored using gel electrophoresis, determination of available amino groups and lectin binding assays. The BSA molecular mass increase and glycation sites were investigated by mass spectrometry and through digestion with trypsin and chymotrypsin. Depending on time and type of sugar, differences in BSA conjugation were achieved. Modified BSA revealed reduction of amino groups' availability and slower migration through SDS/PAGE. d-galactose was more reactive than d-glucose or d-lactose, leading to the coupling of 10, 3 and 1 sugar residues, respectively, after 120 minutes of reaction. BSA lysines (K) were the preferred modified amino acids; both K256 and K420 appeared the most available for conjugation. Only BSA-lactose showed biological recognition by specific lectins.

Novel hydrophobic interaction chromatography matrix for specific isolation and simple elution of immunoglobulins (A, G, and M) from porcine serum.

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.