Strain Screening And Fermentation Condition Study For Higher Immunomodulatory Activity Ganoderam Polysaccharide

Twenty-five Ganoderma strains belonging to 5 different species, including G. lucidum, G. tsugae, G. oerstedii, G. resinaceum and G. subamboinens were cultured in liquid medium. They were screened according to an integrated index, which consisted total polysaccharide andβ-(1, 3)-D-glucan contents and the stimulation activities of splenocyte proliferation and macrophage phagocytosis and NO production in vitro. These strains were cultivated in an identical condition. Their mycelial polysaccharides were extracted with hot-water following with ethanol precipitation. The polysaccharide andβ-(1, 3)-D-glucan contents were measured by using phenol-sulfuric acid method and limulus G test assay respectively. Their stimulation activities on NO production and phagocytosis by mouse Raw264.7 cell line were measured using Griess reaction and neural red method. MTT assay was used to detect the extract activity on mouse splenocyte proliferation in vitro, with or without the presence of Con A.The results showed that among all the extracts, the polysaccharide content in G. tsugae T1 strain extract was the highest (65.96%) and theβ-(1, 3)-D-glucan of G. subamboinens S1 strain was the highest (2.04%). All the polysaccharide extracts could significantly stimulate nitric oxide production and phagocytosis, and stimulate splenocyte proliferation or inhibit proliferation with the presence of Con A. Among these extract, the one from G. subamboinens strain S1 showed the highest immunomodulatory activities.Fermentation condition study revealed that 1% PEG added to adjust media osmolality could promote intracellular polysaccharide production, while adding 0.5% NaCl and 1% PEG could significantly increase polysaccharide andβ-(1,3)-D-glucan contents in extracts. Adding trehalose at 2.5% or PEG at 1% could enhance the activity of NO production stimulated by polysaccharide extracts, 0.5% NaCl could elevate the extracts activity on splenocyte proliferation. However, the extract from that with 1% PEG showed a reversed activity on splenocyte proliferation, while the remaining conditions didn't show such effects. Shake-flask culture mode was found to benefit the production of intracellular polysaccharide (1.86g/L), while such effects were failed being observed in still culture mode and two-stage culture mode, which combined Shake-flask and still culture successively. However, the later two modes could significantly increase the polysaccharide andβ-(1, 3)-D-glucan contents by 1.76 and 1.63 times (still) or 8.44 and 1.73 times (two-stage) higher respectively compared to the shake-flask culture mode. The immunomodulatory experiment results showed that the polysaccharide produced in two-stage mode had much higher immune activities than shake-flask culture, and the stimulation activity on NO production and splenocyte proliferation were 1.65 and 1.10 times higher, while phagocytosis stimulation activity remained unchanged. Stillness culture mode could enhance the activities of polysaccharide extracts on NO production, phagocysis and splenocyte proliferation by 4.00, 1.09 and 1.56 times higher than shake-flask culture mode.Squalene, a precursor for triterpenoid synthesis, showed different actions on polysaccharide content at different concentrations and cultivation modes. Low concentration squalene elevated theβ-(1,3)-D-glucan content in polysaccharide extracts, but at higher concentrations it showed an adverse effect dose-dependently. Squalene didn't show a markedly effect to increase immune-enhancement activity of polysaccharide extracts in shake-flask culture, but it did in two-stage culture. It could increase the accumulation of polysaccharide and glucan, and stimulate the NO production and splenocyte proliferation by polysaccharide extracts.This study further investigated the correlation of total polysaccharide content andβ-(1, 3)-D-glucan content in Ganoderma mycelial polysaccharide extracts with their immunomodulatory activities. The data revealed that the immunological activity was not associated with the total polysaccharide content but with theβ-(1, 3)--D-glucan content (p <0.01,p <0.05). Thes results suggest that theβ-(1, 3)-D-glucan content but not polysaccharide content is more appropriate for the evaluation of Ganoderma polysaccharide extract's immune-enhance activities.