Low-Molecular-Weight ß-1,3-1,6-Glucan Derived from Aureobasidium pullulans Exhibits Anticancer Activity by Inducing Apoptosis in Colorectal Cancer Cells.

ß-glucan, a plant polysaccharide, mainly exists in plant cell walls of oats, barley, and wheat. It is attracting attention due to its high potential for use as functional foods and pharmaceuticals. We have previously reported that low-molecular-weight Aureobasidium pullulans-fermented ß-D-glucan (LMW-AP-FBG) could inhibit inflammatory responses by inhibiting mitogen-activated protein kinases and nuclear factor-κB signaling pathways. Bases on previous results, the objective of the present study was to investigate the therapeutic potential of LMW-AP-FBG in BALB/c mice intracutaneously transplanted with CT-26 colon cancer cells onto their backs. Daily intraperitoneal injections of LMW-AP-FBG (5 mg/kg) for two weeks significantly suppressed tumor growth in mice bearing CT-26 tumors by reducing tumor proliferation and inducing apoptosis as compared to phosphate buffer-treated control mice. In addition, LMW-AP-FBG treatment reduced the viability of CT-26 cells in a dose-dependent manner by inducing apoptosis with loss of mitochondrial transmembrane potential and increased activated caspases. Taken together, LMW-AP-FBG exhibits anticancer properties both in vivo and in vitro.

Anti-inflammatory effects of ß-1,3-1,6-glucan derived from black yeast Aureobasidium pullulans in RAW264.7 cells.

ß-glucan derived from the black yeast Aureobasidium pullulans (A. pullulans) is one of the natural products attracting attention due to its high potential for application as a functional food and pharmaceutical. Our team of researchers obtained a highly soluble, low-molecular-weight ß-glucan from the fermentation culture medium of A. pullulans via mechanochemical ball milling method, that is, the low-molecular-weight A. pullulans-fermented ß-D-glucan (LMW-AP-FBG). We investigated the anti-inflammatory effect of LMW-AP-FBG using lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells) in the current study. LMW-AP-FBG altered LPS-stimulated inflammatory responses by reducing the release of inflammatory mediators such as nitric oxide (NO), interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α. As well, the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways were downregulated by LMW-AP-FBG. Furthermore, LMW-AP-FBG markedly reduced LPS-induced expression of cell surface molecules, CD14, CD86, and MHC class II, which mediate macrophage activation. These findings suggest that LMW-AP-FBG can be used as an effective immune modulator to attenuate the progression of inflammatory disease.

Biological Activity of High-Purity ß-1,3-1,6-Glucan Derived from the Black Yeast Aureobasidium pullulans: A Literature Review.

The black yeast Aureobasidium pullulans produces abundant soluble ß-1,3-1,6-glucan-a functional food ingredient with known health benefits. For use as a food material, soluble ß-1,3-1,6-glucan is produced via fermentation using sucrose as the carbon source. Various functionalities of ß-1,3-1,6-glucan have been reported, including its immunomodulatory effect, particularly in the intestine. It also exhibits antitumor and antimetastatic effects, alleviates influenza and food allergies, and relieves stress. Moreover, it reduces the risk of lifestyle-related diseases by protecting the intestinal mucosa, reducing fat, lowering postprandial blood glucose, promoting bone health, and healing gastric ulcers. Furthermore, it induces heat shock protein 70. Clinical studies have reported the antiallergic and triglyceride-reducing effects of ß-1,3-1,6-glucan, which are indicators of improvement in lifestyle-related diseases. The primary and higher-order structures of ß-1,3-1,6-glucan have been elucidated. Specifically, it comprises a single highly-branched glucose residue with the ß-1,6 bond (70% or more) on a backbone of glucose with 1,3-ß bonds. ß-Glucan shows a triple helical structure, and studies on its use as a drug delivery system have been actively conducted. ß-Glucan in combination with anti-inflammatory substances or fullerenes can be used to target macrophages. Based on its health functionality, ß-1,3-1,6-glucan is an interesting material as both food and medicine.

Low Molecular Weight Barley ß-Glucan Affects Glucose and Lipid Metabolism by Prebiotic Effects.

We investigated the effect of low molecular weight barley ß-glucan (LMW-BG) on cecal fermentation, glucose, and lipid metabolism through comparisons to high molecular weight ß-glucan (HMW-BG). C57BL/6J male mice were fed a moderate-fat diet for 61 days. LMW-BG or HMW-BG was added to the diet corresponding to 4% ß-glucan. We measured the apparent absorption of fat, serum biomarkers, the expression levels of genes involved in glucose and lipid metabolism in the liver and ileum, and bacterial counts of the major microbiota groups using real time PCR. The concentration of short-chain fatty acids (SCFAs) in the cecum was analyzed by GC/MS. Significant reductions in serum leptin, total- and LDL-cholesterol concentrations, and mRNA expression levels of sterol regulatory element-binding protein-1c (SREBP-1c) were observed in both BG groups. HMW-BG specific effects were observed in inhibiting fat absorption and reducing abdominal deposit fat, whereas LMW-BG specific effects were observed in increasing bacterial counts of Bifidobacterium and Bacteroides and cecal total SCFAs, acetate, and propionate. mRNA expression of neurogenin 3 was increased in the LMW-BG group. We report that LMW-BG affects glucose and lipid metabolism via a prebiotic effect, whereas the high viscosity of HMW-BG in the digestive tract is responsible for its specific effects.

Action of an endo-ß-1,3(4)-glucanase on cellobiosyl unit structure in barley ß-1,3:1,4-glucan.

ß-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ß-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-ß-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ß-1,3:1,4-glucan. To find the minor structure on which the endo-ß-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ß-1,3:1,4-glucan by endo-ß-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-ß-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ß-Glc-1,3-ß-Glc-1,4-ß-Glc-1,3-ß-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-ß-1,3(4)-glucanase acts on the cellobiosyl units of barley ß-1,3:1,4-glucan in an endo-manner.

Barley low molecular weight ß-glucan potently induces maturation of mouse dendritic cells.

BACKGROUND: Accumulating evidence indicates that non-toxic immunostimulants with strong differentiation/maturation-inducing activity for dendritic cells (DCs) might be useful for preventing or even curing cancer. MATERIALS AND METHODS: Mouse bone marrow (BM) cells were cultured in the presence of various glucans and their differentiation/maturation-inducing activities were compared by measuring cytokines secreted in the culture medium. RESULTS: Barley-derived ß-glucan with an average molecular weight of 2 kDa (BBG-Low) remarkably stimulated the formation of mature DCs from immature mouse DCs. The amount of interleukin-6 produced by sequential treatment of BM cells with granulocyte-macrophage colony-stimulating factor and 10 µg/mL of BBG-Low was approximately 30 times higher than that obtained by a similar sequential treatment using barley ß-glucan of 40-70 kDa instead of BBG-Low. CONCLUSION: BBG-Low induces the formation of mature DCs from immature DCs and suggests that BBG-Low will be useful as a potent nontoxic immunostimulator.

Induction of IFN-γ by a highly branched 1,3-ß-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes via dectin-1-independent pathways.

We have previously elucidated the precise structure of a unique type of 1,3-ß-D-glucan, AP-FBG (Aureobasidium pullulans-fermented ß-D-glucan), from the fungus A. pullulans and found that AP-FBG strongly induced the production of various cytokines in DBA/2 mouse-derived splenocytes in vitro. However, the mechanism(s) of action of AP-FBG on in vitro mouse primary cells have not been characterized in detail. Herein, we report that the production of IFN-γ in DBA/2 mouse-derived splenocytes by AP-FBG was not inhibited following treatment with an anti-dectin-1 neutralizing antibody. In addition, AP-FBG not only failed to activate dectin-1-mediated signaling pathways, examined by a reporter gene assay but also failed to bind to dectin-1, a pivotal receptor for 1,3-ß-D-glucan. Taken together, AP-FBG induced cell activation via dectin-1-independent pathways.

Granulocyte macrophage colony-stimulating factor is required for cytokine induction by a highly 6-branched 1,3-ß-D-glucan from Aureobasidium pullulans in mouse-derived splenocytes.

We have previously obtained and elucidated the precise structure of a highly branched 1,3-ß-D-glucan (with 6-monoglucopyranosyl side chains), Aureobasidium pullulans-fermented ß-D-glucan (AP-FBG), from the fungus A. pullulans. However, the mechanism(s) of the effects of AP-FBG on in vitro mouse primary cells have not been analyzed in detail. Herein, we report that the induction of cytokines by AP-FBG was dependent on the existence of a granulocyte macrophage colony-stimulating factor (GM-CSF); this is similar way to be a typical 1,3-ß-D-glucan from Sparassis crispa (SCG), which is a 1,3-ß-D-glucopyranosyl backbone with single 1,6-ß-D-glucopyranosyl side branching units every three residues. In other words, the production of cytokines in DBA/2-mouse-derived splenocytes by AP-FBG was completely hampered by an anti-GM-CSF neutralizing monoclonal antibody. Furthermore, the addition of exogenous GM-CSF to C57BL/6-derived splenocytes, which are less sensitive to AP-FBG, induced the production of cytokines by AP-FBG. Therefore, GM-CSF is indispensable for the induction of cytokines by AP-FBG in mouse-derived splenocytes. This finding has provided a new insight into our understanding of the actions of ß-D-glucan but will also aid in the design and development of more effective ß-D-glucan agents.

A highly branched 1,3-beta-D-glucan extracted from Aureobasidium pullulans induces cytokine production in DBA/2 mouse-derived splenocytes.

We recently elucidated the structure of a highly branched 1,3-beta-D-glucan with 6-monoglucopyranosyl side chains, extracted from Aureobasidium pullulans (AP-FBG). Although the biological effects of beta-D-glucans are known to depend on their structures, the effects of a highly branched 1,3-beta-D-glucan on the production of cytokines by leukocytes in mice have not yet been elucidated. In this study, we found that AP-FBG strongly induced the production of various cytokines, especially Th1 cytokines (e.g., IFN-gamma and IL-12p70) and Th17 cytokines (e.g., IL-17A), but did not induce the production of IL-4, IL-10, and TNF-alpha in DBA/2 mouse-derived splenocytes in vitro.

Identification of a putative vacuolar serine protease gene in the rice blast fungus, Magnaporthe grisea.

We identified and cloned a gene designated SPM1, encoding a serine protease from the rice blast fungus Magnaporthe grisea. SPM1 is a single-copy gene, encoding a subtilisin-like serine protease with 536 amino acids. Analyses of the deduced amino acid sequence of SPM1 suggested that SPM1 would be localized in a vacuole, an important organelle in pathogenicity.