Conjugates of Bovine Serum Albumin with Chitin Oligosaccharides Prepared through the Maillard Reaction

Abstract

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.

Three-dimensional chitin rings from body segments of a pet diplopod species: Characterization and protein interaction studies

“…Therefore, their interaction with biocompatible surfaces is of fundamental importance [27]. Interactions of modified chitin or chitosan with blood proteins have been elucidated in earlier reports [28][29][30][31]. However, blood protein interaction studies of native chitin are very limited and therefore a detailed study is needed for further research in chitin-based biomimetic materials designing.…”

Maillard neoglycans as inhibitors for in vitro adhesion of F4+ enterotoxigenic Escherichia coli to piglet intestinal cells

“…The extent on glycation of the synthetized neoglycans was estimated considering the migration patterns of glycated PSA with respect to untreated PSA. The number of glycan molecules coupled to PSA was projected considering the mass of lactose, or the masses obtained for GOS and Ochit under similar conditions (Ledesma-Osuna et al, 2010;Sarabia-Sainz et al, 2013). Analysis by Image Lab software suggested PSA binding of 63, 13 and 2 molecules of Lac, GOS and Ochit, respectively.…”

“…Samples were centrifuged at 10 000 × g for 25 min at 4°C, followed by ultrafiltration of the supernatant through 3 and 1 kDa membranes in tandem to obtain Ochit (in a tetramer to heptamer range). Filtrates were lyophilized and stored at -40°C until use (Ledesma-Osuna et al, 2010).…”

“…The advantage of this type of synthesis is low cost, as the materials are readily available and are inexpensive, and thus, this process has potential in veterinary applications for preventing or reducing ETEC infections. Other carbohydrates that could potentially be recognized by F4 ETEC are prebiotic galacto-oligosaccharides (GOS), as well as chitin oligosaccharides (Ochit), derived from the controlled hydrolysis of chitin (Ledesma-Osuna et al, 2010;Sarabia-Sainz et al, 2013). The purpose of this study was to test the effectiveness of different neoglycans synthesized by non-enzymatic glycation in preventing in vitro binding of F4 + ETEC to piglet enterocytes.…”

Glycation of Animal Proteins Via Maillard Reaction and Their Bioactivity

“…Bacterial adhesion assays have been performed to assess if β-lactoglobulin protein glycated with chitin oligosaccharides (60°C for 6 and 12 h) can be recognized by bacterial adhesins from Escherichia coli K88 and Salmonella choleraesuis. Biorecognition analyses showed that glycoconjugates were in fact recognized by the adhesins of E. coli K88 and S. choleraesuis; both adhesins showed an effect similar to mucins (control) which are natural ligands for these microorganisms; that effect was not shown in the native or non-glycated protein concluding that the glycoconjugates formed could be used to investigate the interaction of protein-carbohydrate in biological systems as well as to look for alternatives to bacterial infections [74].…”