Soluble Β-glucan Research Articles

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble β-Glucan

β‐Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.

Continuous Exposure to Non-Soluble β-Glucans Induces Trained Immunity in M-CSF-Differentiated Macrophages.

Beta-glucans enable functional reprogramming of innate immune cells, a process defined as “trained immunity”, which results in enhanced host responsiveness against primary (training) and/or secondary infections (resilience). Trained immunity holds great promise for promoting immune responses in groups that are at risk (e.g. elderly and patients). In this study, we modified an existing in vitro model for trained immunity by actively inducing monocyte-to-macrophage differentiation using M-CSF and applying continuous exposure. This model reflects mucosal exposure to β-glucans and was used to study the training effects of a variety of soluble or non-soluble β-glucans derived from different sources including oat, mushrooms and yeast. In addition, trained immunity effects were related to pattern recognition receptor usage, to which end, we analyzed β-glucan-mediated Dectin-1 activation. We demonstrated that β-glucans, with different sources and solubilities, induced training and/or resilience effects. Notably, trained immunity significantly correlated with Dectin-1 receptor activation, yet Dectin-1 receptor activation did not perform as a sole predictor for β-glucan-mediated trained immunity. The model, as validated in this study, adds on to the existing in vitro model by specifically investigating macrophage responses and can be applied to select non-digestible dietary polysaccharides and other components for their potential to induce trained immunity.

Consumption of barley ameliorates the diabetic steatohepatitis and reduces the high transforming growth factor β expression in mice grown in α-minimum essential medium in vitro as embryos

Non-alcoholic fatty liver disease (NAFLD), which includes the subtype non-alcoholic steatohepatitis (NASH), is a major complication of type 2 diabetic mellitus (T2DM), even among non-obese patients. However, the exact cause of NAFLD/NASH in non-obese patients with T2DM is unclear. We studied a non-obese mouse model of T2DM created through the malnourishment of embryos by culture in vitro for 48 h in α-minimum essential medium (MEM) at the two-cell stage. We compared the development of steatohepatitis in these MEM mice with control mice that were similarly cultured in standard potassium simplex-optimized medium (KSOM). We also studied the effects of 10 weeks of consumption of barley, which contains large amounts of the soluble fiber β-glucan, on the steatohepatitis of the adult MEM mice. The size of lipid droplets, the area of fibrosis, and the mRNA expression of the transforming growth factor beta (Tgfb) gene in the liver were higher in adult MEM mice fed a rice-based diet than in KSOM mice fed the same diet. However, barley consumption reduced the area of fibrosis and TGFB expression in MEM mice. In conclusion, adult mice that are cultured in MEM at the two-cell embryo stage develop steatohepatitis and T2DM, accompanied by higher hepatic TGFB expression, than KSOM controls. Furthermore, the consumption of barley during adulthood ameliorates the steatohepatitis and reduces the TGFB expression.

Dectin-1-Mediated Production of Pro-Inflammatory Cytokines Induced by Yeast β-Glucans in Bovine Monocytes.

Yeast-derived products containing β-glucans have long been used as feed supplements in domesticated animals in an attempt to increase immunity. β-glucans are mainly recognized by the cell surface receptor CLEC7A, also designated Dectin-1. Although the immune mechanisms elicited through Dectin-1 activation have been studied in detail in mice and humans, they are poorly understood in other species. Here, we evaluated the response of bovine monocytes to soluble and particulate purified β-glucans, and also to Zymosan. Our results show that particulate, but not soluble β-glucans, can upregulate the surface expression of costimulatory molecules CD80 and CD86 on bovine monocytes. In addition, stimulated cells increased production of IL-8 and of TNF, IL1B, and IL6 mRNA expression, in a dose-dependent manner, which correlated positively with CLEC7A gene expression. Production of IL-8 and TNF expression decreased significantly after CLEC7A knockdown using two different pairs of siRNAs. Overall, we demonstrated here that bovine monocytes respond to particulate β-glucans, through Dectin-1, by increasing the expression of pro-inflammatory cytokines. Our data support further studies in cattle on the induction of trained immunity using dietary β-glucans.

Effects of exogenous β-glucanase on ileal digesta soluble β-glucan molecular weight, digestive tract characteristics, and performance of coccidiosis vaccinated broiler chickens fed hulless barley-based diets with and without medication.

Limited use of medication in poultry feed led to the investigation of exogenous enzymes as antibiotic alternatives for controlling enteric disease. The objective of this study was to evaluate the effects of diet β-glucanase (BGase) and medication on β-glucan depolymerization, digestive tract characteristics, and growth performance of broilers. Broilers were fed hulless barley (HB) based diets with BGase (Econase GT 200P from AB Vista; 0 and 0.1%) and medication (Bacitracin and Salinomycin Na; with and without) arranged as a 2 × 2 factorial. In Experiment 1, 160 broilers were housed in cages from d 0 to 28. Each treatment was assigned to 10 cages. In Experiment 2, broilers (2376) were housed in floor pens and vaccinated for coccidiosis on d 5. Each treatment was assigned to one floor pen in each of nine rooms. In Experiment 1, the soluble β-glucan weighted average molecular weight (Mw) in the ileal digesta was lower with medication in the 0% BGase treatments. Peak molecular weight (Mp) and Mw were lower with BGase regardless of medication. The maximum molecular weight for the smallest 10% β-glucan (MW-10%) was lower with BGase addition. In Experiment 2, Mp was lower with medication in 0% BGase treatments. Beta-glucanase resulted in lower Mp regardless of medication, and the degree of response was lower with medication. The MW-10% was lower with BGase despite antibiotic addition. Body weight gain and feed efficiency were higher with medication regardless of BGase use through-out the trial (except d 11-22 feed efficiency). Beta-glucanase resulted in higher body weight gain after d 11 and worsened and improved feed efficiency before and after d 11, respectively, in unmedicated treatments. BGase and medication caused the depolymerization of soluble ileal β-glucan. Beta-glucanase acted as a partial replacement for diet medication by increasing growth performance in coccidiosis vaccinated broilers.

Role of β-glucan content, molecular weight and phytate in the bile acid binding of oat β-glucan

There is controversy about the role of viscosity and co-migrating molecules on the bile acid binding of beta-glucan. Thus, this study aimed to investigate the impact of β-glucan molecular weight and the content of both β-glucan and phytate on the mobility of bile acids by modelling intestinal conditions in vitro. Two approaches were used to evaluate factors underlying this binding effect. The first studied bile acid binding capacity of soluble β-glucan using purified compounds. Viscosity of the β-glucan solution governed mainly the mobility of bile acid since both a decrease in β-glucan concentration and degradation of β-glucan by enzyme hydrolysis resulted in decreased binding. The second approach investigated the trapping of bile acids in the oat bran matrix. Results suggested trapping of bile acids by the β-glucan gel network. Additionally, hydrolysis of phytate was shown to increase bile acid binding, probably due to better extractability of β-glucan in this sample.

Split Enzyme-Based Biosensors for Structural Characterization of Soluble and Insoluble β-Glucans.

β-Glucan is widely distributed in various plants and microorganisms and is composed of β-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with β-1,6- or β-1,4-linkage. Numerous studies have investigated different β-glucans and revealed their bioactivities. To understand the structure-function relationship of β-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain β-1,6-branched β-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived β-1,3-glucan recognition protein and fungal endo-β-1,6-glucanase (Neg1)-derived β-1,6-glucan recognition protein, respectively. In this approach, two β-glucan recognition proteins bound to β-glucan molecules come into close proximity, resulting in the assembly of the full-length reporter enzyme and induction of transient luciferase activity, indicative of the structure of β-glucan. To test the applicability of this assay, β-glucan and two β-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a β-1,3-glucan with a long polymer of β-1,6-glucan in a dose-dependent manner. This simple test also allows the monitoring of real-time changes in the side chain structure and serves as a convenient method to distinguish between β-1,3-glucan and long-chain β-1,6-branched β-1,3-glucan in various soluble and insoluble β-glucans.

Industrial applications of selected JFS articles

Industrial applications of selected <i>JFS</i> articles

Effects of hulless barley and exogenous beta-glucanase levels on ileal digesta soluble beta-glucan molecular weight, digestive tract characteristics, and performance of broiler chickens

The reduced use of antibiotics in poultry feed has led to the investigation of alternatives to antibiotics, and one such substitution is fermentable carbohydrates. Exogenous β-glucanase (BGase) is commonly used in poultry fed barley-based diets to reduce digesta viscosity. The effects of hulless barley (HB) and BGase levels on ileal digesta soluble β-glucan molecular weight, digestive tract characteristics, and performance of broiler chickens were determined. A total of 360 day-old broilers were housed in battery cages (4 birds per cage) and fed graded levels of high β-glucan HB (CDC Fibar; 0, 30, and 60% replacing wheat) and BGase (Econase GT 200 P; 0, 0.01, and 0.1%) in a 3 × 3 factorial arrangement. Beta-glucan peak molecular weight in the ileal digesta was lower with 30 and 60 than 0% HB, whereas the peak decreased with increasing BGase. The weight average molecular weight was lower at 0.1 than 0% BGase in wheat diets, whereas in HB diets, it was lower at 0.01 and 0.1 than 0% BGase. The maximum molecular weight was lower with 0.01 and 0.1 than 0% BGase regardless of the HB level. The maximum molecular weight was lower with HB than wheat at 0 or 0.01% BGase. Overall, empty weights and lengths of digestive tract sections increased with increasing HB, but there was no BGase effect. Hulless barley decreased the duodenum and jejunum contents, whereas increasing the gizzard (diets with BGase), ileum, and colon contents. The jejunum and small intestine contents decreased with increasing BGase. Ileal and colon pH increased with increasing HB, but there was no BGase effect. Treatment effects were minor on short-chain fatty acids levels and performance. In conclusion, exogenous BGase depolymerized the ileal digesta soluble β-glucan in broiler chickens in a dose-dependent manner. Overall, feed efficiency was impaired by increasing HB levels. However, HB and BGase did not affect carbohydrate fermentation in the ileum and ceca, although BGase decreased ileal viscosity and improved feed efficiency at the 0.1% dietary level.

A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

Characterisation of Wheat–Oat Flour Extrudate: Physicochemical and Β-Glucan Attributes

The objective of this study was to evaluate the effect of wheat–oat flour ratio on the physical properties and β-glucan characteristics of extrudates. Results showed that increasing the wheat–oat flour ratio resulted in a decrease in the water solubility index (r2=0.8567) and hardness (r2=0.9316), whereas the expansion ratio (r2=0.9307) and water absorption index (r2=0.9061) increased. Wheat flour generally caused an increase in L values from 57.81 to 62.94 providing bright samples. Few cells were observed at high wheat–oat flour ratios under a scanning electron microscope, and a smooth surface was noted. Meanwhile, the total (r2=0.9867) and soluble (r2=0.9848) β-glucan contents were inversely proportional to the wheat–oat flour ratio. Extrudates with added wheat flour had a high molecular weight, but wheat flour had no significant (P&lt;0.05) effect on the viscosity of β-glucan extracts. Conclusively, incorporation of wheat flour at a wheat–oat flour ratio of 2.33 provides ready-to-eat food based on whole oat flour, on account of improving the texture and providing sufficient β-glucan contents (0.806 g/100 g) without significantly affecting β-glucan viscosity.

Production of oat bran functional probiotic beverage using Bifidobacterium lactis

A non-dairy probiotic beverage based on oat bran extract was produced through fermentation of beverage using a probiotic strain Bifidobacterium lactis. Physicochemical characteristics, viability of probiotic bacteria, and sensory specification were investigated during three weeks of refrigerated storage with various concentration of oat bran extract and carbon dioxide in grape flavor beverage. It was observed that the acidity of the beverage samples significantly increased by increasing the percentage of oat bran extract and carbon dioxide, while the Brix decreased during storage and reached the lowest value after 21 days. The viability of the bacteria was significantly enhanced by increasing the amount of carbon dioxide and oat bran extract concentration until the 14th day after fermentation. The viability extremely decreased with increasing the storage time to 21 days. Oat bran is a rich source of nutrients including carbohydrates, protein, minerals, vitamins and soluble β-glucan. The use of oat bran as a profitable substrate to produce a non-dairy grape flavor probiotic beverage was examined. Due to high viability of the Bifidobacterium lactis after two weeks of storage at 4 °C, the formulation was characterized with 15% (w/v) oat bran extract and 1% carbon dioxide. The highest sensory score was obtained by 15% (w/v) oat bran extract and 0.5% carbon dioxide sample. Therefore, incorporation of the oat bran in formulated grape flavor probiotic could enhance the nutritional value of the beverage.

Pre-harvest glyphosate application effects on properties of β-glucan from oat groats

Pre-harvest glyphosate is applied to cereal grains to control weed growth. However, it has been claimed that oat (Avena sativa L.) composition is affected by pre-harvest glyphosate application. The research was conducted to evaluate differences in properties of β-glucan in grains of pre-harvest glyphosate treated versus untreated oat plants. Two oat cultivars (Rockford and Souris) were grown at Minot and Prosper, ND, in 2015, and glyphosate was sprayed during the soft dough stage, hard dough stage, or not applied. β-glucan viscosity was not significantly (p > 0.05) affected by treatment at soft dough (1082 cP) or mature (1166 cP) stages compared with untreated (1150 cP) controls. Applying glyphosate at the soft dough stage significantly (p < 0.05) reduced the content and solubility of the β-glucan versus untreated samples. β-glucan content and solubility in oat treated at soft dough were 4.35% and 52.1%, respectively, while in untreated samples were 4.65% and 60.6%, respectively. Treatment at soft dough and hard dough stages significantly (p < 0.05) increased weight average molecular weight (Mw) of the high molecular weight fraction of soluble β-glucan (4.4 × 106 and 3.8 × 106, respectively), compared with untreated controls (3.5 × 106). The Mw of the low molecular weight fraction of soluble β-glucan fraction significantly (p < 0.05) increased at soft and hard dough treatments (5.5 × 105 and 3.3 × 105, respectively), versus untreated samples (3.0 × 105). Therefore, glyphosate can be applied when the grain has reached physiological maturity or thereafter.

Expression and characterization of TbCel12A, a thermophilic endoglucanase with potential in biomass hydrolysis

Abstract To increase economic benefit and reduce pollutants, agricultural biomass can be hydrolyzed to monosaccharides, which serve as a feedstock for biorefinery production of fuels and fine chemicals. In a search for new enzymes to apply to this process, Thermobispora bispora TbCel12A endoglucanase was identified by rice straw compost metagenome analysis and expressed in recombinant Escherichia coli. TbCel12A hydrolyzed both soluble and insoluble β-glucan substrates. Its highest hydrolysis rate was observed for cellopentaose compared to other cellooligosaccharides, barley mixed linkage β-glucan, and carboxymethyl cellulose. Low hydrolysis rates were observed for α-cellulose, and avicel. The primary product detected was cellobiose and a smaller amount of glucose. TbCel12A exhibited highest activity at 65 °C and pH 5.0 and retained 86% relative activity after incubation at 60 °C overnight. TbCel12A conserved >80% activity in 10% alcohol and at high salt concentrations. Hydrolysis of pretreated rice straw using commercial cellulase and TbCel12A released slightly more reducing sugars than hydrolysis using only commercial cellulase. Therefore, TbCel12A is a promising enzyme for further development for use in biomass conversion.

Rheological characterization of wheat flour as modified by adding barley glucagel (a β-glucan isolate) under thermo-mechanical stress using Mixolab

The study was aimed at determining the effect of incorporating glucagel from 9 different hulless barley cultivars to wheat flour on the rheological properties of dough using Mixolab. The mixing properties were significantly affected as glucagel incorporation led up to 11.4% increase in dough consistency at a constant hydration of 56%. Addition of glucagel lowered both the dough stability and protein weakening by up to 18.85% and 10.81%, respectively. The peak viscosity on heating was up to 4.3% higher in samples containing glucagel. Moreover, the incorporation of glucagel to wheat flour caused lowering of shear thinning/breakdown by up to 76.8% which could be evident from the negative correlation (r = − 0.68, p < 0.05) of percent breakdown with the soluble β-glucan content. Retrogradation was up to 13.14% lower in the presence of glucagel. Higher levels of soluble β-glucan in the glucagel lowered retrogradation in the dough and a negative correlation of r = − 0.65 (p < 0.05) existed between the two parameters, demonstrating the pivotal role of glucagel in lowering of staling, therefore glucagel could be utilized in several bakery products.

Delineating functional properties of a cello-oligosaccharide and β-glucan specific cellobiohydrolase (GH5_38): Its synergism with Cel6A and Cel7A for β-(1,3)-(1,4)-glucan degradation

Cellulase cocktails formulated to degrade crystalline cellulose generally contain cellobiohydrolases (CBHs), referred to as CBHI (Cel7A) and CBHII (Cel6A), as the major constituents. The combined hydrolytic activities of CBHI and CBHII improve the release of fermentable sugars (β-1,4-cellobiose as the main product) from crystalline cellulose. In this study, a novel cellobiohydrolase (Exg-D) sourced from a metagenome of hindgut bacterial symbionts of a termite was heterologouly expressed, purified, and functionally characterised. Exg-D specific activity was higher on insoluble barley β-glucan (38.94 U/mg protein), soluble wheat flour β-glucan (12.71 U/mg protein) and oat β-glucan (8.89 U/mg protein) compared to cellulosic substrates; Avicel and CMC. We further explored Exg-D activity on the unpretreated or NaOH-pretreated (mercerised) Avicel and compared its activity to commercially available CBHI and CBHII on these celluloses. CBHI displayed the highest activity of 4.74 U/mg protein on mercerised cellulose followed by CBHII (2.14 U/mg protein), while Exg-D activity on untreated and mercerised cellulose was 1.66 and 1.67 U/mg protein, respectively. The high activity of CBHI was supported by binding assays, which revealed that CBHI has a higher binding capacity towards crystalline cellulose compared to Exg-D and CBHII. Only CBHI and CBHII showed synergism during the hydrolysis of mercerised Avicel, showing a degree of synergy (DS) of about 1.299 and yielded about 1.43 μmol/ml of reducing sugars higher than control. In contrast, Exg-D and CBHII displayed synergism during β-glucan degradation, displaying a DS of about 1.22. Thus, we propose that Exg-D should only be used synergistically with other CBHs to degrade mixed linked-β-(1,3)-(1,4)-glucan.

Differences in anti-inflammatory properties of water soluble and insoluble bioactive polysaccharides in lipopolysaccharide-stimulated RAW264.7 macrophages.

β-Linked polysaccharides including β-glucans are well known to be important functional ingredients, and are known to possess immunomodulatory and anti-tumor activities. This study aimed to investigate the anti-inflammatory properties and participating receptor of water soluble and insoluble bioactive polysaccharides from Grifola frondosa (GFP, non-digestible water soluble polysaccharides), Laminaria digitata (laminarin, a water soluble β-glucan) and Saccharomyces cerevisiae (zymosan, a water insoluble β-glucan) in lipopolysaccharide (LPS)-stimulated parental and Dectin-1 highly expressing RAW264.7 macrophages. Results showed that GFP and laminarin significantly inhibited nitric oxide and prostaglandin E2 production, but only the GFP with high molecular weight exhibited strong inhibition on pro-inflammatory cytokine (TNF-α and IL-6) secretion in a concentration-dependent manner. The activation of NF-κB was also significantly down-regulated by GFP treatment as compared with cells treated with LPS alone. Although GFP and laminarin were able to bind to β-glucan receptor Dectin-1, there was no relationship between the inhibitory potency and the content of β-glucans in GFP, and these inhibitory effects were not affected by the expression level of Dectin-1 in macrophage cells. In contrast, zymosan significantly intensified LPS-induced inflammatory responses through Dectin-1. In conclusion, these results suggest that the inhibitory effects of water soluble polysaccharides on LPS-induced pro-inflammatory mediator production in murine macrophages may not involve β-glucan receptor Dectin-1.

RHOA-mediated mechanical force generation through Dectin-1.

Dendritic cell-associated C-type lectin 1 (Dectin-1, also known as CLEC7A) is an innate immune pattern recognition receptor that recognizes β-glucan on the Candida albicans cell wall. Recognition of β-glucan by immune cells leads to phagocytosis, oxidative burst, cytokine and chemokine production. We looked for specific mechanisms that coordinate phagocytosis downstream of Dectin-1 leading to actin reorganization and internalization of fungus. We found that stimulation of Dectin-1 by soluble β-glucan leads to mechanical force generation and areal contraction in Dectin-1-transfected HEK-293 cells and M1 macrophages. With inhibitor studies, we found this force generation is a spleen tyrosine kinase (SYK)-independent, but SRC family kinase (SFK)-dependent process mediated through the RHOA-ROCK-myosin light chain (MLC) pathway. We confirmed activation of RHOA downstream of Dectin-1 using activity assays and stress fiber formation. Through phagocytosis assays, we found direct evidence for the importance of RHOA-ROCK-MLC signaling in the process of phagocytosis of C. albicans.

A β-glucan from Grifola frondosa effectively delivers therapeutic oligonucleotide into cells via dectin-1 receptor and attenuates TNFα gene expression

Grifola frondosa is an edible and medicinal mushroom with great nutritional values and bioactivities. In the present study, a soluble homogeneous β-glucan, GFPS, with high molecular mass of 5.42 × 106 Da was purified from the fruit bodies of Grifola frondosa using 5% cold NaOH. The structure of GFPS was determined with FT-IR, NMR, and monosaccharide composition analysis, and was identified to be a β-D-(1-3)-linked glucan backbone with a single β-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. Our results indicated that GFPS had a triple helical structure and could form complex with polydeoxyadenylic acid (poly[A]). Further studies demonstrated that GFPS could interact with poly[A] moiety of a designed antisense oligonucleotide (ASO) targeting the primary transcript of proinflammatory cytokine TNFα (TNFα-A60). This GFPS-based complex could incorporate TNFα-A60 into the macrophage cells via dectin-1 receptor and attenuate lipopolysaccharide-induced secretion of TNFα. Our results suggested that GFPS could be applied to deliver therapeutic oligonucleotides for the treatment of diseases such as inflammation and cancers.

A gravimetric method for the quantitative determination of soluble β-glucan content in oat grain

Background. Oat (Avena sativa L.) is one of the most important sources of protein, oil, starch and dietary fibers, in particular β-glucans. Dietary fiber serves as a source of nutrition for the intestinal microflora and significantly affects not only its composition, but also the normal functioning of the intestine as a whole. In connection with the increased interest in β-glucans as a food component and dietary supplement, there is a need in a convenient and inexpensive method for determination of β-glucans content in grain. The article provides an overview of the existing methods for the isolation and determination of soluble β-glucans in cereals: enzymatic, alkaline, alkaline-enzymatic, colorimetric; their advantages and disadvantages are noted. The main disadvantages of some methods are the complexity and duration of execution, significant cost of the required reagents, and the inability to determine the exact content of β-glucans due to their insufficient purification from various impurities.Results. This study used the example of naked and covered oat cultivars to demonstrate applicability of the gravimetric method that we developed by modifying the alkaline method. Whole grain oat flour was pretreated with a 50% ethanol solution to inactivate β-glucanase and remove free sugars, some lipids, proteins, and other substances. β-glucans were released from the aleurone layer and the endosperm with a 5% sodium hydroxide solution, and finally extracted with a 70% ethanol solution. Β-glucans floated to the surface in the form of a bunch of fibers, which was then dried at a temperature about 100-102°C to constant weight and weighed. The content of β-glucans was calculated from the dry weight (%). The isolated β-glucans were checked for the presence of accompanying substances: the content of nitrogenous substances was determined by the Kjeldahl method, and the presence of starch was determined by a qualitative reaction with Lugolʼs reagent.Conclusions. The content of β-glucans in the grain of the studied samples ranged from 3.12±0.18 up to 4.65±0.17% of the dry weight. As a result of the study, the optimal conditions of β-glucans isolation were selected: the extraction mixtures ratios, as well as the modes of centrifugation, sedimentation and drying were established. It has been shown that this method makes it possible to isolate β-glucans with a minimum impurity of nitrogenous substances (0.07-0.12%) and no traces of starch. One of the advantages of the described method is its availability for mass analysis when studying a collection of grain crops.