The analysis and function evaluation of bioactive polysaccharide (1,3;1,6)-β-D-glucans in mushrooms
Date Issued
2014
Date
2014
Author(s)
Wang, Chung-Huang
Abstract
The bioactive polysaccharides, (1,3)-β-D-glucans with (1,6)-β-D-glucosyl branches, are components of structural polysaccharides of fungal cell walls and have been classified as biological response modifiers (BRM). In this study, we used enzymatic-high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method to determine the amount and degree of branching (DB) of (1,3;1,6)-β-D-glucans in samples including the fruity bodies of edible mushrooms cultivated in Taiwan and the mycelium products of Ganoderma lucidum by submerged cultifation. Alkaline solution (0.5 M NaOH) extraction could increased the yield of (1,3;1,6)-β-D-glucans to 4.4 – 16.4 folds compared with the one from hot water extraction. Except the fruiting body of Ganoderma lucidum, which exhibits ligniform, the DB of edible mushrooms reveal no difference by various extraction methods. The (1,3)-β-D-glucans of G. lucidum comprised of water-soluble branching (DB 0.24) component and essential linear component that occurred in the alkaline solution extraction. Moreover, the content of insoluble dietary fiber (IDF)-(1,3;1,6)-β-D-glucans contents were 5.11 – 202.50 mg/g (dry basis), and soluble dietary fiber (SDF)-(1,3;1,6)-β-D-glucans were 0.18 – 15.36 mg/g (dry basis). The results indicated that the majority of (1,3;1,6)-β-D-glucans occurred in the insoluble dietary fiber of mushroom. We also investigated bioactivity of non-digestible polysaccharides (ND-PS) from various mushroom species by two reporter cell platforms (RAW 264.7 containing constructed plasmid, iNOS promoter-luciferase or COX-2 promoter-luciferase), and the TNF-alpha and NO concentrations in medium were determined as well. The results indicated that the immune-stimulation activities of (1,3;1,6)-β-D-glucans are mild, and some other bioactive polysaccharides may also contribute to the immune modulation activity. Moreover, we confirmed the strong positive correlations between iNOS or COX-2-directed luciferase reporter platform and the ELISA-based assay for medium TNF-alpha through this data set. This suggested that the promoter-luciferase assays successfully reflect the TNF-alpha concentration levels and the platform is applicable as a high throughput screening for the detection of mushroom polysaccharides with immune-modulatory activities.
We further analyzed twelve cultivation products of Ganoderma lucidum mycelium samples. Although the results display that the amount of (1,3;1,6)-β-D-glucans significantly varied in different fermentation conditions, the DB and molecular weight of (1,3;1,6)-β-D-glucans restrict to a narrow range. For the high aggregating tendency of (1,3;1,6)-β-D-glucans, we successfully purified (1,3;1,6)-β-D-glucans by 35% ethanol precipitation method. We further confirmed the purity of (1,3;1,6)-β-D-glucans and demonstrated its bioactivity by TNF-alpha releasing assay in RAW 264.7 cells.
Subjects
(1,3
1,6)-β-D-葡萄聚醣
分支度
可食用菇
靈芝
RAW 264.7
iNOS
COX-2
酵素水解
螢光酵素分析
Type
thesis
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