Article pubs.acs.org/JAFC
Concentration Variation and Molecular Characteristics of Soluble (1,3;1,6)-β‑D‑Glucans in Submerged Cultivation Products of Ganoderma lucidum Mycelium Chung-Huang Wang,† Shu-Chen Hsieh,† Huei-Ju Wang,§ Miaw-Ling Chen,# Bi-Fong Lin,‡ Been-Huang Chiang,† and Ting-Jang Lu*,† †
Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan, Republic of China Department of Applied Science of Living, Chinese Culture University, Taipei, Taiwan, Republic of China # Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan City, Taiwan, Republic of China. ‡ Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, Taiwan, Republic of China §
ABSTRACT: (1,3)-β-D-Glucans with (1,6)-β-D-glucosyl branches are bioactive polysaccharides in fruiting bodies and mycelia of Ganoderma lucidum, a mushroom used in traditional Chinese medicine. Submerged cultivation of mycelium is one of the more efficient means of generating polysaccharides from this fungus. Twelve mycelium samples examined in this study demonstrated the quantitative and qualitative molecular characteristics of soluble (1,3;1,6)-β-D-glucans. It was observed that the concentration of soluble (1,3;1,6)-β-D-glucan varied substantially from 1.3 to 79.9 mg/dL. (1,3;1,6)-β-D-Glucans also preserved their molecular characteristics with degrees of branching (DB) of 0.21−0.36 and molecular masses of 105−106 g/mol for those samples with substantial quantities of β-D-glucan. Using the high aggregating tendency of these molecules, (1,3;1,6)-β-D-glucans were successfully purified via fractional precipitation with 35% (v/v) ethanol. (1,3;1,6)-β-D-Glucan was proposed as a putative bioactive marker for immunomodulation because it was the most abundant polysaccharide in G. lucidum mycelium products to stimulate macrophage RAW 264.7 cells to release TNF-α. KEYWORDS: (1,3;1,6)-β-D-glucans, Ganoderma lucidum, degree of branching, molecular weight
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INTRODUCTION Ganoderma lucidum, commonly known as Ling-Zhi and Reishi, is a medicinal mushroom that has long been a component of Oriental medicine. Polysaccharides are associated with the attributes of immunomodulating and antitumor activities of Ganoderma. Submerged fermentation of mycelium culture is one of the efficient means for producing Ganoderma polysaccharides.1−3 Recently, as fermentation strategies have improved (e.g., fed-batch, pH-shift, or dissolved oxygen tension-shift), the maximum production of soluble (extracellular) polysaccharide has risen to higher yields from the range of 10−200 mg/dL2−4 to reported yields of 259 mg/dL5 and 1500 mg/dL.6 However, not all Ganoderma polysaccharides are associated with immunoactivities. Therefore, using well-known bioactive polysaccharides as a quality marker during production is a more suitable strategy. The β-D-glucans isolated from G. lucidum displayed high growth-inhibition activities against Sarcoma 180 solid tumors in mice7 and enhanced regeneration of mouse bone marrow by protecting stem cells from irradiation damage.8 Our previous study also confirmed that (1,3;1,6)-β-D-glucan was the major active polysaccharide from G. lucidum and showed significant tumor necrosis factor-α (TNF-α) releasing stimulation activity from human mononuclear cells (MNC).9 (1,3;1,6)-β-D-Glucans have been recognized as potent bioactive polysaccharides in mushrooms and fungi.10−12 Several (1,3;1,6)-β-D-glucans and their conjugates have been commercialized for the clinical treatment of patients undergoing cancer © 2014 American Chemical Society
therapy, such as those treated with schizophyllan, lentinan, grifolan, and Krestin (PSK, polysaccharide−protein complex).13,14 In Japan, lentinan is prescribed to patients with gastric cancer, showing encouraging results in enhancing survival rates of patients.15 (1,3)-β-D-Glucans are components of the fungal cell wall,16,17 which can be recognized by specific cell-surface receptors, for example, dectin-1 in animal immunocytes18,19 due to its pathogen-associated molecular pattern.20 The interaction of (1,3)-β-D-glucans with immune cell receptors triggers a variety of immunological responses, leading to immunomodulation and antitumor activity. The distinct chemical structures of β-D-glucans provide various affinities toward these receptors.21−23 It has been reported that the presence of (1,6)-linked side-chain branches on (1,3)-β-Dglucan increases the affinity of pattern recognition receptors23 and dectin-1.21 The activity plateau of these (1,3;1,6)-β-Dglucans showed a degree of branching (DB) ranging from 0.2 to 0.33.10,24,25 Moreover, glucans with larger molecular weight may be required to cross-link spatially separated receptors and alter immunocyte function.26 There is limited information concerning the quantitative and qualitative molecular characteristics of soluble (1,3;1,6)-β-DReceived: Revised: Accepted: Published: 634
October 9, 2013 December 30, 2013 January 4, 2014 January 5, 2014 dx.doi.org/10.1021/jf404533b | J. Agric. Food Chem. 2014, 62, 634−641
Journal of Agricultural and Food Chemistry
Article
generated during methanolysis were further hydrolyzed with 2 M TFA at 100 °C for 1.5 h. After removal of TFA by repeated evaporation under vacuum with HPLC grade distilled water, the sugars in the hydrolysate were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD). The HPAEC-PAD consisted of a Bioscan 817 Metrohm IC system (Metrohm, Herisau, Switzerland) including an IC pump 709, injection valve unit 812 with a 20 μL loop, and an electrochemical detector with a gold working electrode (E1 = 0.05 V, 0.48 s; E2 = 0.80 V, 0.18 s; E3 = −0.30 V, 0.36 s). A CarboPac PA1 (4 mm × 250 mm) analytical column (Dionex Corp., Sunnyvale, CA, USA) with a guard column (4 mm × 50 mm) was used. For separation of neutral monosaccharides, the eluent consisted of 19 mM NaOH containing 1 mM barium acetate applied at a flow rate of 0.5 mL/min. For separation of acidic monosaccharides (galacturonic acid and glucuronic acid), the eluent consisted of 75 mM NaOH, 150 mM sodium acetate, and 1 mM barium acetate applied at a flow rate of 1 mL/min. Data were collected and analyzed using Metrodata IC Net 2.1 software (Metrohm). Concentration and Degree of Branching of (1,3;1,6)-β-D-Glucans. The concentration and the ratio of (1,6)-β-D-glucosyl branches of (1,3;1,6)-β-D-glucans were determined according to an enzymatic HPAEC-PAD method developed by Chang.28 Samples (5 mg) were dissolved in 0.5 M NaOH and stirred at ambient temperature overnight. After addition of arabinose (Sigma) as an internal standard, the pH of the sample solution was adjusted to 4.5 by the addition of 1 M HCl, and the volume was filled to 5 mL with 50 mM sodium acetate buffer (pH 4.5) for use as the stock for enzymatic hydrolysis. A 1 mL sample aliquot was reacted with exo-(1,3)-β-D-glucanase (1 U; Megazyme, Wicklow, Ireland) and endo-(1,3)-β-D-glucanase (0.1 U, Megazyme) for 3 h at 40 °C, followed by boiling in water for 15 min to stop the enzymatic reaction. A 4 mL volume of ethanol (95%, v/v) was added to precipitate undigested polysaccharides. The supernatant was dried by vacuum evaporation and redissolved in distilled water. The glucose and gentiobiose released from the enzyme digestion were quantified by using HPAEC-PAD. The system conditions applied were identical to those described in identifying the sugar composition of polysaccharides above, except that isocratic elution was performed at 1 mL/min with 10 mM NaOH eluent containing 2 mM barium acetate. The concentration and the DB of (1,3;1,6)-β-D-glucan in sample were calculated as follows:
glucans in submerged cultivation products of G. lucidum. Our preliminary study indicated that the concentration of soluble (1,3;1,6)-β-D-glucans varied substantially even under the same cultivating parameters. Twelve samples with different concentrations of (1,3;1,6)-β-D-glucans were selected to demonstrate this variance, and their DB and molecular weight data were also examined to understand their molecular characteristics.
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MATERIALS AND METHODS
Submerged Cultivation Products of Ganoderma lucidum Mycelium. Twelve G. lucidum mycelium culture products from BCRC 36123 (Bioresource Collection and Research Center, Taiwan) kindly provided by Professor B. H. Chiang, Graduate Institute of Food Science and Technology, National Taiwan University, were used as samples in this study. The whole fermentation cultures were autoclaved and the mycelia removed by centrifugation. Only broth was used for analysis and coded S1−S2 and C1−C10, according to (1,3;1,6)-β-D-glucan concentration (in descending order). Samples S1 and S2 were products of static mycelium culturing with synthetic chemically defined medium (200 mL of medium consisted of 3% glucose, 2% malt extract, and 0.1% peptone with 10% mycelium inoculation) cultivated in customized 600 mL glass flasks (the inside diameters both of the bottom and of the broth surface were 14 cm) in a 28 °C incubator without aeration, illumination, and shaking. The harvest time was set when the mycelium mat fully covered the broth surface, which were 22 and 17 days for S1 and S2, respectively. Broth from three flasks was pooled for each sample. For comparison of the concentration variation of (1,3;1,6)-β-D-glucans in different samples, we designated S1 and S2 as references because they were cultured at the conditions (medium and temperature) suggested by BCRC and the cultivating conditions can be easily controlled. Samples C1−C10 were products from a commercial fermentor with leguminous composite medium [consisting of 2% glucose, 5% soybean (Glycine max L.), and 2% Astragalus membranaceus]. The fermentation was conducted in the dark, and the aeration rate, agitation speed, and inoculum size were 0.75 vvm, 50 rpm, and 10%, respectively, for 12 days. The fermentation temperature was at 30 °C for all of the Csamples except C6 and C7, for which 24 °C was used. Samples C1, C2, C6, and C7 were products of a 200 L customized pilot-scale manufacture fermentor (Grape King Inc., Taoyuan County, Taiwan) and others were of a 5 L fermentor (FB-6S, Firstek Scientific Co., Ltd., New Taipei City, Taiwan). No culture medium containted a detectable amount of (1.,3;1,6)-β-D-glucan (data not shown). Preparation and Fractionation of Polysaccharides. The polysaccharides in the samples were obtained by precipitating polysaccharides in 4 volumes of 95% ethanol (Taiwan Tobacco and Wine Corp., Taipei, Taiwan) in a fermentation broth. In this study, C2 was used to isolate (1,3;1,6)-β-D-glucans by using repeating fractional precipitation with ethanol. The polysaccharides of C2 were further separated into two fractions: (1,3;1,6)-β-D-glucans and C2-GRF (glucan-removed fraction). Ethanol was dripped into 3% (w/v) C2 polysaccharide aqueous solution with stirring; ethanol concentrations required for (1,3;1,6)-β-D-glucans and C2-GRF precipitation were 35 and 76% (v/v), respectively. After settling in a refrigerator overnight, the polysaccharide-precipitated fractions were collected by centrifugation (1500g for 15 min). The (1,3;1,6)-β-Dglucan fraction was precipitated three times to increase its purity. Analytical Methods. Content of Polysaccharides. Polysaccharides were dispersed in 0.5 M NaOH and stirred at ambient temperature overnight until completely dissolved. The polysaccharide content of the sample was determined using a phenol−sulfuric acid method,27 and glucose (Sigma Chemicals, St. Louis, MO, USA) was used as a standard (10−100 μg/mL). Sugar Composition of Polysaccharides. The hydrolysis procedure used consisted of a combination of methanolysis and trifluoroacetic acid (TFA) hydrolysis. The polysaccharide sample (1−5 mg) was methanolyzed under vacuum in 1 mL of anhydrous 2 M HCl in absolute methanol in a sealed hydrolytic tube at 80 °C for 12 h. Next, the reagent was removed by evaporation, and the methyl glycosides
content of (1,3;1,6)‐β‐D‐glucan in polysaccharide (mg/mg) = (Wt‐Glc × 0.9 + Wt‐Gen × 0.95)/Wt‐Poly Wt-Glc and Wt-Gen are the calculated weights of glucose and gentiobiose in samples, respectively; 0.9 and 0.95 are factors to account for differences in molecular weight of a glucosyl monomer and gentiobiosyl (a dimer) bound in a polysaccharide, respectively, compared to the free form; and Wt-Poly is the weight of polysaccharide present in the stock solution for enzymatic hydrolysis. The weight of polysaccharide was determined by phenol−sulfuric acid.
Wt‐Glc = PA‐Glc/PA‐Is × RFGlc × Wt‐Is
Wt‐Gen = PA‐Gen/PA‐Is × RFGen × Wt‐Is PA-Glc, PA-Gen, and PA-Is denote peak areas of glucose, gentiobiose, and internal standard, respectively; RFGlc and RFGen denote response factor of glucose and gentiobiose in chromatography, respectively; WtIs denotes the weight of internal standard present in the stock solution for enzymatic hydrolysis.
DB (degree of branching) = Gen/(Glc + Gen) Gen denotes mmol of gentiobiose = Wt-Gen/342.3, and Glc denotes mmol of glucose = Wt-Glc/180.2 (both denominators denote the formula for the weight of the compound). Molecular Weight of (1,3)-β-D-Glucans and Their Polydispersity. Polysaccharides were dissolved in 0.5 M NaOH (2 mg/mL) overnight and then filtered through Whatman no. 5 filter paper (GE Healthcare, Florham Park, NJ, USA). Ascending gel filtration chromatography was 635
dx.doi.org/10.1021/jf404533b | J. Agric. Food Chem. 2014, 62, 634−641
Journal of Agricultural and Food Chemistry
Article
Table 1. Concentrations of Soluble (1,3;1,6)-β-D-Glucans and Polysaccharides and the Molecular Characteristics of (1,3;1,6)-βa D-Glucans in Submerged Cultivation Products of Ganoderma lucidum Mycelium molecular characteristics of (1,3;1,6)-β-D-glucans code
(1,3;1,6)-β-D-glucanb (mg/dL) ± ± ± ± ±
C1 C2 C3 C4 C5
79.9 56.3 33.0 32.4 17.5
S1 S2
12.8 ± 0.4 e 8.2 ± 0.5 f
C6 C7 C8 C9 C10
6.7 5.2 2.6 1.9 1.3
± ± ± ± ±
3.9 2.4 0.9 0.4 0.8
0.8 0.2 0.1 0.2 0.1
a b c c d
fg g h h h
polysaccharides (mg/dL) 322.6 235.0 154.2 116.2 101.3
± ± ± ± ±
2.8a 13.5b 13.5d 14.9 ef 4.0 fg
26.4 ± 5.2i 24.2 ± 5.1i 117.4 126.5 70.4 170.1 100.1
± ± ± ± ±
6.5e 6.2e 8.8h 9.9c 5.9g
(1,3;1,6)-β-D-glucanb/ polysaccharides (%) 24.8 24.0 21.4 27.8 17.2
± ± ± ± ±
1.2 1.0 0.6 0.3 0.8
d d e c f
± ± ± ± ±
0.7 0.2 0.2 0.1 0.1
Mw × 104 (g/mol)
polydispersity (Mw/Mn)
e cd bc c e
47.0 38.0 29.2 79.1 11.0
69.1 57.8 64.9 101.8 42.1
1.5 1.5 2.2 1.3 3.8
0.36 ± 0.01 a 0.35 ± 0.03 ab
22.6 14.3
41.3 32.3
1.8 2.3
0.2 0.1 21.8 0.1 1.6
0.8 0.3 46.0 0.1 1.8
3.9 3.6 2.1 2.1 1.1
0.23 0.31 0.33 0.32 0.21
48.4 ± 1.5 a 33.9 ± 2.2 b 5.7 4.1 3.7 1.1 1.3
Mn × 104 (g/mol)
degree of branchingb
g gh h i i
0.01 0.08 0.29 0.10 0.08
± ± ± ± ±
± ± ± ± ±
0.01 0.01 0.02 0.01 0.01
