| Summary: | 博士 === 國立臺灣大學 === 食品科技研究所 === 91 === Several polysaccharides of Ganoderma lucidum have been associated with its immuno-modulating activities. The dissertation has demonstrated that (1→6) branched (1→3)-β-D-glucans were the major polysaccharide component with immuno-modulating activities in boiling-water extracts of the fruiting bodies using high-performance size-exclusion chromatography and quantitative separation techniques. A new fluorometric and a new enzyme-chromatographic method have developed for determining the (1→3)-β-D-glucans.
The neutral polysaccharides composed of glucose were the major constituents of the ethanol precipitate of boiling-water extracts of G. lucidum fruiting bodies. The yield of partially purified polysaccharides from the ethanol precipitate was 90% (w/w) through a fractionation by precipitation with cetylpyridinium chloride for removing acidic polysaccharides and digestion of amylase and protease for removing (1→4)-α-D-glucans and protein, respectively. The (1→3)-β-D-glucans with (1→6)-β-D-glucosyl branches composed 31% of the partially purified polysaccharides and had 2.08 × 106 daltons weight-average molecular weight determined using high-performance size-exclusion chromatography with multi-angle laser light scattering detection. The glucans were obtained using differential precipitation with ethanol at 20% (v/v) and were subjected to bioactivity and chemical property study. The stimulating activity of the β-D-glucans on human mononuclear cells for producing tumor necrosis factor-α (TNF-α) was confirmed in an in vitro study. The chemical structure and its degree of branching (0.35) were elucidated by sugar composition analysis, aniline blue selective staining, and 1H- and 13C-NMR spectroscopy. There were two hetero-polysaccharides containing D-glucose, D-galactose and D-mannose at different ratios in the polysaccharide fraction.
The fluorometric method was based on the specific interaction of (1→3)-β-D-glucans with sirofluor, a weakly fluorescent component in aniline blue dye. The characteristic fluorescence of complexes that had excitation and emission maximum wavelength at 395 and 495 nm, respectively was used to quantify the content of (1→3)-β-D-glucans in G. lucidum extract and commercial products. The purified G. lucidum (1→3)-β-D-glucans were used as standard. The glucan concentration and the fluorescence intensity had shown a linear relationship in the range of 0~50 μg/mL of glucan concentration. Due to poor solubility and high aggregation tendency of (1→3)-β-D-glucans, the method involved dissolving samples in 0.3 N NaOH which enhanced the intensity and carrying out reaction in a pH 11.5 buffer containing 0.5 N NaCl for 2 hours. Commercial (1→3)-β-D-glucans, such as curdlan, laminarin, Lentinan and pachyman, all showed similar linear relationship at different glucan concentration ranges and slopes. The common components in fermentation medium, glucose, peptone, potato dextrose broth, malt extract and yeast extract did not significantly affected the fluorescence intensity at concentration of 1 mg/mL, but reduced the intensity at concentration of 5 mg/mL, except glucose. The dark color substances in the fruiting bodies extracts concealed the fluorescent emission. The interference could be removed using 5% hydrogen peroxide as bleaching agent that reacted at 90°C for 20 minutes.
The difficulty of completely converted branched (1→3)-β-D-glucans to its backbone monomer, glucose, and branching dimer, gentiobiose (O-6-β-D-glucosyl- D-glucopyranose) by (1→3)-β-D-glucanase, was overcome. Alkaline pretreatment (0.3 N NaOH) increased the solubility and stability of (1→3)-β-D-glucans in the enzymatic digestion buffer. The simultaneous usage of endo- and exo-(1→3)- β-D-glucanase (EC 3.2.1.39 and EC 3.2.1.58, respectively) by-passed the steric obstacles of branching points. The content and degree of branching of the (1→3, 1→6)-β-D-glucans could be calculated according the amount of glucose and gentiobiose determined by using high-performance anion-exchange chromatography coupled with pulsed amperometric detection and its molar ratio in the enzymatic hydrolysate. The validation of this method had conducted using commercial purified (1→3)-β-D-glucans, such as curdlan, laminarin, Lentinan and pachyman.
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