β-Glucan Assay Kit (Yeast & Mushroom) 

A robust and reliable method has been developed for the measurement of β-glucan in mushroom and mycelial products. Total glucan (plus free glucose and glucose from sucrose) was measured using controlled acid hydrolysis with H₂SO₄ and the glucose released specifically was measured using glucose oxidase/peroxidase reagent. α-Glucan (starch/glycogen) plus free glucose and glucose from sucrose were specifically measured after hydrolysis of starch/glycogen to glucose with glucoamylase and sucrose to glucose plus fructose with invertase and the glucose specifically measured with GOPOD reagent. β-Glucan was determined by the difference. Several acid and enzyme-based methods for the hydrolysis of the β-glucan were compared, and the best option was the method using H₂SO₄. For most samples, similar β-glucan values were obtained with both the optimized HCl and H₂SO₄ procedures. However, in the case of certain samples, specifically Ganoderma lucidum and Poria cocus, the H₂SO₄ procedure resulted in significantly higher values. Hydrolysis with 2 N trifluoroacetic acid at 120°C was found to be much less effective than either of the other two acids evaluated. Assays based totally on enzymatic hydrolysis, in general, yielded much lower values than those obtained with the H₂SO₄ procedure.

Mushrooms have unique sensory properties and nutritional values as well as health benefits due to their bioactive compounds, especially beta-glucans. Well-known edible and medicinal mushroom species as well as uncommon or unknown species representing interesting sources of bioactive beta-glucans have been widely studied. Commercially cultivated and wild growing mushrooms were analysed for their beta-glucan contents. Enzymatic determinations of all glucans, alpha-glucans and beta-glucans in 39 mushrooms species were performed, leading to very remarkable results. Many wild growing species present high beta-glucan contents, especially Bracket fungi. The well-known cultivated species Agaricus bisporus, Lentinula edodes and Cantharellus cibarius as well as most screened wild growing species show higher glucan contents in their stipes than caps.

β-glucans from Basidiomycetes like Grifola frondosa (the maitake mushroom) are well known for their health benefits. Polysaccharide preparations from medicinal mushrooms such as G. frondosa have been successfully tested in a vast number of studies. Many mushroom extracts have been developed and today are merchandized for use medicinally and commercially. Studies could show that, in particular, chemical structural features such as the molecular size of β-glucans significantly influence their bioactivity. Thus it is highly important to explore the composition and structural properties of β-glucans extracted from medicinal mushrooms and their effects on human tumor cell viability. Our study focuses on the molecular weight cutoff distribution of β-glucans in hot water-based extracts from maitake mushrooms. Cross-flow ultrafiltration was applied to obtain 5 fractions of different molecular size. β-glucan content was quantified using an enzyme-based test kit, specialized to 1,3-1,6-β-glucans. Here we show that only small amounts of β-glucans with a high molecular weight (>100 kDa) could be detected from an aqueous extract of G. frondosa. The main compounds encompass substances with a low molecular weight (<5 kDa), composing about 35% of the whole extract. In addition, tumor cell viability studies demonstrate significant cytotoxic potential in 2 different solid cancer cell types for the fraction with a high molecular weight (>100 kDa) and for 1 fraction with a low molecular weight (5-10 kDa). In summary, our experiments prove that cross-flow ultrafiltration serves as a quick and easy method for dividing crude aqueous mushroom extracts into different molecular-weight fractions that inhibit tumor cell viability in vitro.

Fruit bodies (separately pilei and stems) of mushrooms Pleurotus ostreatus (four strains) and Pleurotus eryngii were characterised as a source of polysaccharides. The contents of glucans and dietary fibres were determined with using the respective Megazyme enzymatic kits. Enzymatic analysis of the fruit bodies confirmed significant differences in the contents of these components among the species and strains. The stems contained more insoluble dietary fibres than the pilei in all the cases and more β-glucans in most cases. However, relatively high contents of β-glucan (20–50% of dry matter) could be a result of incomplete enzymatic hydrolysis of insoluble α-1,3-glucans. Nevertheless, low food quality stems of mushrooms Pleurotus sp. could be a valuable source of cell wall glucans for the preparation of food supplements.

Cultivated oyster mushrooms (genus Pleurotus) are interesting as a source of biologically active glucans. Partially, β-glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. Specific glucans were isolated from stems of Pleurotus ostreatus and Pleurotus eryngii by subsequent boiling water and alkali extraction. Obtained water soluble (L1), alkali soluble (L2) and insoluble (S) fractions were characterised by various analytical methods. Spectroscopic analysis detected glucans in all the fractions: branched 1,3-1,6-β-D-glucan predominated in L1 and S, while linear 1,3-α-D-glucan in L2. Fractions L1 also contained marked amount of proteins partially in complex with glucans; protein content in L2 was insignificant. Effective deproteinisation of L1 and separation of α- and β-glucans in L2 was achieved by the treatment with phenolic reagent. Small amount of chitin was found in S as a component of cell wall chitin–glucan complex. Potential prebiotic activity of extracts L1 and L2 was testing using nine probiotic strains of Lactobacillus, Bifidobacterium and Enterococcus. These probiotics showed different growth characteristics dependently on used extract and strain specificity due to the presence of structurally diverse compounds. The extracts L1 and L2 can be applied to synbiotic construction only for carefully selected probiotic strains. This exploitation of fruit body extracts extends the use of mushrooms P. ostreatus and P. eryngii for human health.

β-Glucan is a cell wall component that is one of the most plentiful cell polysaccharides. Moreover, it has been found to have several beneficial effects on the immune system. In yeast, β-glucan is mainly contained in the yeast cell wall, and thus it is important to produce high levels of cell mass for the mass production of yeast β-glucan. Response surface methodology (RSM) offers a potential means of optimizing process factors and medium components; it has been used to estimate the effects of medium components on cell mass production. In the present study, the optimal concentrations of molasses and corn steep liquor (CSL) in the medium were determined to be 6.4% (v/v) and 17% (v/v). The cell mass predicted by statistical analysis was 9.76 g/L after 20 h of cultivation. In a 2.5-L stirred tank reactor (STR), the cell mass produced in a batch culture was 36.5∼39.3 g/L. The maximum cell mass in the fed-batch cultures of Saccharomyces cerevisiae JUL3 was 95.7 g/L using 50% molasses solution and a feed rate of 10 mL/h. The cell mass obtained in the fed-batch culture was 2.4-fold higher than that obtained in the batch culture.

Three aloe fermentation products from Ganoderma lucidum (AG), Hericium erinaceum (AH), and Phellinus linteus (AP) were obtained by fermenting mushroom mycelia using aloe as a substrate. When AG, AH, and AP were added to sterilized aloe and fermented for 5 days, the color of the aloe fermentation product changed from pink to beige, which is aloe’s natural color, through the fermentation time. The pH of the aloe fermentation products ranged 4.32–4.36 shortly after inoculation and then 4.62–4.68 during the 5 days of fermentation. pH increased by 7% during the total fermentation time. The solid content had increased 1.28–1.40 times. The contents of aloin A and B increased with fermentation time. β-Glucan content decreased with fermentation time. The urease inhibition activity (%) were remarkable in AG-4 96.70, AH-4 92.30, and AP-4 66.40%, indicating these products had growth inhibition effects against Helicobacter pylori. Moreover, AG and AH were most effective as anti-H. pylori treatments.

The extraction procedures for β-glucans from cauliflower mushrooms (Sparassis crispa) were optimized by response surface methodology. Experimental design was used to investigate the effect of 3 extraction parameters (pH, extraction time, and ratio of water to raw material) on β-glucan content. The parameter ranges investigated were 6–10 for extraction pH (X₁), 5-15 h for extraction time (X₂), and 10–30 for water to raw material ratio (X₃). The experimental results were in good agreement with a polynomial regression model by a multiple regression analysis (R²=0.95, p=0.0074) for β-glucan content extracted from cauliflower mushrooms. The optimal conditions for β-glucan extraction from cauliflower mushrooms were determined as extraction pH of 6.05, extraction time of 8 h 55 min, and ratio of water to raw material of 19.74, showing 60.76% of the predicted content of β-glucan.

Allergic inflammatory disease such as food allergy, asthma and atopic dermatitis are increasing worldwide. In this study, we investigated the effect of water extract of Sparassis crispa (WESC) Fr. (Aphyllophoromycetideae) on mast cell-mediated allergic inflammation and the possible mechanisms of action. WESC inhibited compound 48/80-induced systemic anaphylaxis and serum histamine release in mice. WESC decreased immunoglobulin E (IgE)-mediated passive cutaneous anaphylaxis. Additionally, WESC reduced histamine release and intracellular calcium in human mast cells activated by phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. WESC decreased PMA and A23187-stimulated expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)- α, inlerleukin (IL)-6 and IL-1β. The inhibitory effect of WESC on pro-inflammatory cytokines was nuclear factor-κB, extracellular signal-regulated kinase and p38 mitogen-activated protein kinase-dependent. Our results suggest potential therapeutic application of WESC in allergic inflammatory diseases.

Partially purified polysaccharides were obtained from four medicinal mushroom species, Agaricus bisporus, Agaricus brasiliensis, Phellinus linteus and Ganoderma lucidum by hot water extraction, followed by ethanol precipitation. The four samples contained varying amounts of both α- and β-glucans as determined by FT-IR and by quantitative estimation after prior partial hydrolysis (Megazyme β-glucan assay kit). EC₅₀ values of the DPPH scavenging activity of the polysaccharides from G. lucidum spores and P.linteus fruiting bodies were found to be particularly low, i.e. EC₅₀ < 0.1 mg/ml. For A. brasiliensis and A. bisporus, EC₅₀ values were 0.27 and 2.0 mg/ml. EC₅₀ values of the antioxidant activity were 7.07 mg/ml for G. lucidum, 13.25 mg/ml for A. brasiliensis and >20 mg/ml for A. bisporus polysaccharide, respectively. EC₅₀ values of the chelating activity of ferrous ions ranged from 0.59 mg/ml for G. lucidumto 7.80 mg/ml for A. bisporus. The EC₅₀ values of the extracts in the reducing power assay ranged from 0.47 to 14.83 mg/ml. A correlation was found between EC₅₀ values of the chelating and reducing power abilities and the amount of total glucans content in the extracts. In vitro measurements of immunomodulatory capacity of polysaccharide extracts showed that A. bisporus, A. brasiliensis fruiting bodies and G. lucidum spores extracts express an immunostimulating effect on activated human PBMCs and induce synthesis of IFN-γ. The polysaccharide extract of P. linteus fruiting bodies showed an immunosuppressive effect.

Sparassis crispa and Phellinus linteus are edible/medicinal mushrooms that have remarkably high contents of β-(1→3)-D-glucan, which acts as a biological response modifier, but difficulty in cultivating the fruiting bodies and extraction of β-D-glucan have restricted detailed studies. Therefore, a novel process for nanoparticle extraction of Sparan, the β-D-glucan from Sparassis crispa, and Phellin, the β-D-glucan from Phellinus linteus, has been investigated using insoluble tungsten carbide as a model for nanoknife technology. This is the first report showing that the nanoknife method results in high yields of Sparan (70.2%) and Phellin (65.2%) with an average particle size of 150 and 390 nm, respectively. The extracted Sparan with β-(1→3) linkages showed a remarkably high water solubility of 90% even after 10 min of incubation at room temperature. Therefore, it is likely that this nanoknife method could be used to produce β-D-glucan for food, cosmetic, and pharmaceutical industries.

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L^-1 xylose and 37 g L^-1 corn steep liquor, high yields (39.2 g L^-1) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g^-1 mycelium dry weight, respectively.

Five types of macroalgae from the southern hemisphere were analysed for the presence of β-1,3/1,6-glucan and its immunostimulant properties. We were able to extract soluble β-1,3/1,6-D-glucan from Durvillaea antarctica (Chamisso) Hariot (DA). The morphology of the brown algae influenced extraction, and the highest percentage of β-glucan was found in the fronds. The content of β-glucan in the stipes and holdfast was on average 33% and <5%, respectively, of that in the fronds. A simple laboratory extraction process was developed. A highly pure water-soluble polysaccharide, mainly composed of glucose residues, was obtained with a dominant average molecular weight of 6.9 kDa. NMR spectroscopy confirmed the polysaccharide structure to be of β-1,3/1,6-glucan type, comprising a β-1,3-glucan backbone and 21% degree of branching of β-1,6-glucan side chains. Mouse cells were exposed to four DA extract concentrations in water (50, 100, 250 and 500 μg/mL) and no adverse effects on survival were noted. Remarkably, the β-glucan induced a 16.9% increase in activated CD19+ B lymphocytes compared with the control sample. The optimal concentration for maximum activity was 100 μg DA extract/mL.