Quantitative Analysis of (1→3)-β-D-glucans in Ganoderma Products by Fluorometry

摘要

A simple fluorometric method is introduced to determine (1→3)-β-D-glucans in Ganoderma products. The method is based on the specific interaction of (1→3)-β-D-glucans with Sirofluor, a weakly fluorescent component of aniline blue dye. The interacted complexes of (1→3)-β-D-glucans with Sirofluor showed a strong fluorescence with an excitation maximum of 395nm and an emission maximum of 495nm. The intensity of fluorescence varied with the amount of fluorophore existed in aniline blue, the amount and molecular size of (1→3)-β-D-glucans, and the pH and ionic strength of solution. The method involved solubilizing sample in 0.3N NaOH and carrying out reaction in a pH 11.5 buffer contained 0.5N NaCl. Five selected (1→3)-β-D- glucans were used as standards included glucan isolated from Ganoderma lucidum, curdlan, laminarin, lentinan and pachyman. All tested glucans had shown a good linear correlation between glucan concentration and fluorescence intensity, whereas the linear range and the slope of the standard curve varied with the different glucan. It is recommended to use laminarin, a known and commercial available (1→3)-β-D-glucan, as standard to quantify the amount of the glucans in samples, when the glucan from selected material are not available. If laminarin is used as standard, the results should present as laminarin equivalents (LE). This paper also studied the possible interference of carbohydrates and polypeptides commonly used as carbon and nitrogen sources in mycelium culture. Amino acids, peptides, glucose, maltose, maltodextrin and starch did not interfere the fluorescence of the assay but media with brown color such as yeast extracts [may contain (1→3)-β-D-glucan], malt extracts, peptone, potato dextrose broth did reduce the fluorescence of the assay. Brown substances, from fruiting body extracts of Ganoderma or from Maillard reaction products produced in autoclaving, concealed the fluorescence of the reaction. It was because the brown substances absorbed the emission of 495nm. Five percent of hydrogen peroxide, as bleaching agent, with appropriate heating could effectively reduce the inference of brown substances existed in the samples. Spiking techniques were applied to validate this fluorometric method. For surveying the quality difference of commercial Ganoderma products, contents of (1→3)-β-D-glucan in some selected products were determined by using this method and contents of crude polysaccharides of them were determined by using solvent precipitation with phenol-sulfuric acid method.