Mechanochemical Phosphorylation and Solubilisation of β-D-Glucan from Yeast Saccharomyces cerevisiae and Its Biological Activities

To obtain a water-soluble β-D-glucan derivative cleanly and conveniently, a highly efficient mechanochemical method, planetary ball milling, was used to phosphorylate β-D-glucan isolated from yeast Saccharomyces cerevisiae in solid state. Soluble β-D-glucan phosphate (GP) with a high degree of substitution (0.77-2.09) and an apparent PEAK molecular weight of 6.6-10.0 kDa was produced when β-D-glucan was co-milled with sodium hexametaphosphate at 139.5-186.0 rad/s for 12-20 min. The energy transferred was 3.03-11.98 KJ/g. The phosphorylation of GPs was demonstrated by Fourier transform infrared spectroscopy and 13C and 31P Nuclear magnetic resonance spectroscopy. Three GP products with different degree of substitution (DS) and degree of polymerisation (DP) were able to upregulate the functional events mediated by activated murine macrophage RAW264.7 cells, among which GP-2 with a DS of 1.24 and DP of 30.5 exerted the highest immunostimulating activity. Our results indicate that mechanochemical processing is an efficient method for preparing water-soluble and biologically active GP with high DS.