Chemical and physical characterization of bioactive soluble [1→3]- ßD-glucans [SBG] from Saccharomyces sp.
MetadataShow full item record
(1→3)-β-D-glucans are a group of important glucose polymers, some of which are used commercially for wound healing (wound closure). A branched β-1,3 glucan preparation developed by Biotec Pharmacon (Soluble β-glucan, abbreviated SBG) obtained from S. cerevisiae was studied in the work. Although being water-soluble it tends to aggregate strongly in aqueous solution, especially at low temperatures. The physical properties of SBG, including the aggregation behaviour, depend on the properties of the individual polymer molecules, which must be accurately known, both from a fundamental perspective and for regulatory approval. The thesis not only investigated different methods to obtain the unaggregated single chain state of SBG and its physical properties, but also investigated the physical and chemical properties of the aggregated states. In the first part of the study, chemical derivatization (partial O-carboxymethylation) was used to investigate the single chain state of SBG and thereby obtain the molecular weight distribution. A major problem of carboxymethylation (CM) of β-(1,3)-glucan is the possibility for degradation (β-elimination) during the chemical reaction, resulting in underestimated Mw and Mn. Our results demonstrate that degradation is avoided provided the glucan is reduced with NaBH4 prior to carboxymethylation, and hence provide easy control of the degree of substitution (DS) values. SEC-MALLS analysis of CM-SBG showed a progressive loss of aggregates with increasing DS, and for a critical value (DS = 0.53) completely aggregate-free solutions were obtained, allowing accurate determination of the average molecular weight and molecular weight distributions of SBG by SEC-MALLS. In the second part of the study, organic solvents were investigated as SEC-MALLS eluents to possibly obtain fully dissolved SBG without chemical derivatization. Although previous literature [1-5] had shown that dimethyl sulphoxide (DMSO) in the absence or presence of 0.25 LiCl could be a good solvent and SEC-MALLS eluent for several branched (SBG-like) β-1,3-glucans, this study showed that SBG dissolved apparently well in DMSO, but some aggregated material was detected by SEC-MALLS even after heating. The aggregates dissolved completely when using 0.25 M LiCl in DMSO as solvent. However, technical difficulties (SEC column) prevented us from obtaining satisfactory SEC-MALLS data. An additional problem is the large variations in the refractive index increment (dn/dc)µ which have been reported in the literature. We obtained a value of 0.062 ml/g using the 100% recovery method in SEC-MALLS. As an alternative to DMSO, 0.5% LiCl in dimethyl acetamide (DMAc) was explored for the first time as solvent and SEC-MALLS eluent for SBG. This is based on the ability of DMAc/LiCl to dissolve other polysaccharides, notably cellulose. Since heating was required to obtain rapid dissolution, the rate of depolymerisation of SBG in DMAc/LiCl at high temperatures (70-105°C) was determined. This provided a basis for defining dissolution procedures for β-1,3 glucans using elevated temperatures with a minimum of degradation. The result of the investigation is a simple and reliable protocol for preparing unaggregated, fully dissolved and undegraded SBG in DMAc/LiCl, which is well suited as a standard analysis of the molecular weight distribution of SBG-like molecules without chemical derivatization. In the last part of the study, the properties of untreated (aggregated) SBG solutions were investigated in more detail. Dynamic light scattering (DLS) experiments showed that SBG in aqueous solution consists of free chains and particles with a very wide size distribution, depending on the temperature. Oscillatory rheometry demonstrated a typical gelling/melting behavior of SBG which was influenced by its concentration (2%- 20%) and temperatures. AFM images prepared from very dilute aqueous solution (1- 5μg/ml) analysis showed that SBG consists partly of triple helical species coexisting with larger aggregates as well as single chains.