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Glucansucrase was obtained from Lactobacillus reuteri SK24.003 and its characterizations and in vitro biosynthesis for glucose polymer and oligosaccharides were investigated. The final specific activity of glucansucrase was 1.3IU/mg protein with 8.6-fold purification and 8.7% recovery. The molecular weight of purified enzyme was 166.0kDa. The glucansucrase exhibited optimum activity at 30-35°C, whereas the maximum activity was obtained at pH 5.0-5.5. The double-charged ions including Mg 2+ , Mn 2+ , Ni 2+ , Co 2+ , Ca 2+ , Fe 2+ and Zn 2+ activated the glucansucrase activities and Ca 2+ ion highly stimulated the activity by approximately 4 times. K m , V max and k cat of purified glucansucrase were calculated to be 3.7mM, 0.8IU/mg and 18.21/s, respectively. For in vitro biosynthesis, glucansucrase synthesized 1,6-,1,4-α-d-glucan with a molecular weight of 2.5×10 7 g/mol from sucrose as initial primer. Moreover, maltose acceptor-products synthesized by glucansucrase were composed of panose, maltotriose, maltotetraose, tetrasaccharide and pentasaccharide products analogues with an α-1,4/α-1,6 alternating structure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The structure of ethanol precipitated fraction from 1M KOH extracted flaxseed kernel polysaccharides (KPI-EPF) was studied for better understanding the molecular structures of flaxseed kernel cell wall polysaccharides. Based on methylation/GC-MS, NMR spectroscopy, and MALDI-TOF-MS analysis, the dominate sugar residues of KPI-EPF fraction comprised of (1,4,6)-linked-β-d-glucopyranose (24.1mol%), terminal α-d-xylopyranose (16.2mol%), (1,2)-α-d-linked-xylopyranose (10.7mol%), (1,4)-β-d-linked-glucopyranose (10.7mol%), and terminal β-d-galactopyranose (8.5mol%). KPI-EPF was proposed as xyloglucans: The substitution rate of the backbone is 69.3%; R1 could be T-α-d-Xylp-(1→, or none; R2 could be T-α-d-Xylp-(1→, T-β-d-Galp-(1→2)-α-d-Xylp-(1→, or T-α-l-Araf-(1→2)-α-d-Xylp-(1→; R3 could be T-α-d-Xylp-(1→, T-β-d-Galp-(1→2)-α-d-Xylp-(1→, T-α-l-Fucp-(1→2)-β-d-Galp-(1→2)-α-d-Xylp-(1→, or none. The Mw of KPI-EPF was calculated to be 1506kDa by static light scattering (SLS). The structure-sensitive parameter (ρ) of KPI-EPF was calculated as 1.44, which confirmed the highly branched structure of extracted xyloglucans. This new findings on flaxseed kernel xyloglucans will be helpful for understanding its fermentation properties and potential applications., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

The cell-associated glucansucrase from Leuconostoc citreum SK24.002 was isolated, purified, characterized and used for de novo synthesis of α-glucan and acceptor-products. The final specific glucansucrase activity was 1.4U/mg protein with 13.2-fold purification. The obtained glucansucrase had a molecular weight of 186kDa,Tm of 61.7 °C and △H of 176.7kJ/mol. The enzyme showed maximum activity at pH 5.0-6.0 and 45°C. The enzyme activity was enhanced by Ca(2+), Mn(2+) or Co(2+) ions, whereas the activity decreased as the methanol, ethanol, n-butanol, DMSO or isopropanol concentration increased. The chemical inhibitors including BD, DTNB, EDC or NBS also significantly inhibited enzyme activity. Km, Vmax and kcat of glucansucrase were 10.9mM, 3.6U/mg and 306.6 1/s, respectively. For de novo synthesis from sucrose, α-glucan polymer with molecular weight of 1.5×10(7)g/mol and maltose acceptor products (trisaccharide, tetrasaccharide and pentasaccharide) were obtained by glucansucrase via glucosyltransfer reactions, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sulfated modification was carried out to modify a water-insoluble polysaccharide from Ganoderma atrum (AGAP). The effects of sulfation on structure, physicochemical and functional properties of AGAP were investigated. Three sulfated derivatives were prepared, designated as S-1, S-2 and S-3 with degree of substitution (DS) of 0.35, 0.74 and 1.14, respectively. AGAP was elucidated as an α-(1→3)-glucan with few branches terminated by single mannose or xylose residues. The molecular weight (Mw) and radius of gyration (Rg) were estimated to be 1665 kDa and 65.49 nm, respectively. After sulfated modification, non-selective sulfation occurred preferably at O-6, partially at O-2 and O-4 positions of the glucosyl residues. The water-solubility of the derivatives was significantly improved in a DS-dependent manner. Mw of the derivatives showed a sharp decrease, and the chain conformation was estimated to be expanded stiff in phosphate buffer. In vitro tests showed that sulfated modification improved its antioxidant activities and anti-proliferative ability against S-180 tumor cells. This study suggested that sulfated modification was an effective approach to improve the water-solubility and functional properties of insoluble polysaccharides., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The structural features of a neutral extracellular glucan derived from Lactobacillus reuteri SK24.003 were investigated. Colonies of the strain SK24.003 exhibited a creamy and slimy morphological appearance on MRS solid medium and were identified as L. reuteri via 16S rDNA sequence analysis. The exopolysaccharide produced from sucrose was composed exclusively of glucose, and the weight-average molecular weight was 4.31 × 10(7)g/mol. The polysaccharide exhibited an α-(1→4) backbone with an α-(1→6) branch at every fourth residue, as deduced from both NMR and GC-MS data. The exopolysaccharide acted as a natural steel corrosion inhibitor. The results suggested that a novel α-glucan produced by L. reuteri SK24.00 could be broadly used in food and material field., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

The structure and digestibility of endosperm water-soluble α-glucans from different sugary-1 maize mutants (Zhongtian 8#, Zhongtian 2# and Pintian 8#) were investigated. The yield of pure glucan was in the range of 25.91-34.38%. The α-glucan belonged to a typical native nano-scale particle and the average particle size was in the following order: Zhongtian 8# >Pintian 8# >Zhongtian 2#. The weight-average molar mass of glucans ranged from 1.69 to 2.08×10(7)g/mol. The branch densities and α-1,6 linkages of Zhongtian 8#, Zhongtian 2# and Pintian 8#, were 8.60%, 8.77% and 9.51%, 7.71%, 6.58% and 6.81%, respectively. The resistant starch (10.06%) of Pintian 8# was lower than other two cultivars. The study showed that water-soluble glucan exhibited α-1,4-linked backbone with α-1,6 branch sites and digestibility was influenced by granule size, ratio of α-1,4 to α-1,6 linkages, molecular fine structure in this set of sugary maize mutants., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Abstract: Molecular modelling was used to study the conformational properties of cereal β-glucans and compared with experimental results. Followed the steps of exploring global minima of β 1-4 and β 1-3 linked disaccharides and building up the repeating units, the consecutive cellotriosyl units, which are considered as reaction sites of β-glucan chains, were created and then characterized as a three fold helix with a pitch of 41.35 Å. Mostly importantly, the 3D molecular model of cereal β-glucans was created and the moderately extended sinuous chain conformation was first visualized. The conformational parameters of cereal β-glucans were calculated by RMMC simulation, which are in good agreement with experimental results. The calculated parameters also revealed that the chain stiffness of β-glucans increased with the tri/tetra ratio. [Copyright &y& Elsevier]

Abstract: Cereal β-glucans form aggregates even in dilute aqueous solution. Their aggregation behaviours were studied by static and dynamic light scattering. It was shown that the aggregation of cereal β-glucans in aqueous solution was a very fast dynamic process and the equilibrium of molecular association and dissociation were reached quickly. The concentration dependence of the average apparent diameters of cereal β-glucans suggested that the cluster–cluster aggregation was dominant. The structural features of cereal β-glucans played an important role on their aggregation behaviours. As molecular weight increased, the degree of aggregation decreased due to the lower diffusion rate of large molecules. For the more rigid conformation (lower diffusion rate) of β-glucans with higher tri/tetra ratio, their degrees of aggregation were lower. These results suggested that the aggregation process was diffusion limited. [Copyright &y& Elsevier]

Abstract: A high purity wheat β-d-glucan (91.58%) was obtained from white wheat bran after alkali extraction and multi-precipitation with ammonium sulphate compared to previous reported purity of only 70%. The molecular weight (M w) of the purified wheat β-d-glucan was 487,000g/mol with a broad M w distribution (polydispersity is M w/M n =1.65). By applying an ammonium sulphate gradient precipitation method, the purified wheat β-d-glucan was fractionated into six fractions with M w ranged from 43,000 to 758,000g/mol and much narrower M w distribution (polydispersity is M w/M n=1.03 to 1.26). Structural analysis revealed that there were no significant differences between the six fractions and between the fractions and the original sample. This set of sample was used to investigate the effect of M w on physical properties of wheat β-d-glucan. Dynamic rheometry and scanning calorimetric studies revealed that the gelation rate and the melting enthalpy (ΔH) of wheat β-d-glucan increased with the decrease of molecular weight indicating smaller wheat β-d-glucan molecules (must be above a minimum M w) is easier to form junction zones and establish stronger three-dimensional network due to their high mobility and structural regularity (high ratio of tri/tetra). In contrast, the melting temperature of wheat β-d-glucan gels increased with the increase of molecular weight, suggesting a more extended structural network was formed for high M w wheat β-d-glucans. [Copyright &y& Elsevier]

A water-soluble polysaccharide, named NCSP-50, was obtained from natural Cordyceps sinensis by hot water extraction and ethanol fractionation precipitation. It was eluted as a single symmetrical peak and had an average molecular weight of 9.76 × 10 5 Da. The structure was determined by monosaccharide composition, methylation analysis, 1D/2D NMR spectroscopy, and enzymatic hydrolysis and characterization of the oligosaccharides by MALDI-TOF mass spectrometry. The repeating unit of this polysaccharide was proposed as follows: Image 2 This glucan showed potent immunostimulatory activity on the basis of its significant abilities to promote macrophage proliferation, enhance NO production, as well as and cytokines (IL-1β and TNF-α) secretion. [ABSTRACT FROM AUTHOR]

Abstract: β-d-glucans, which are widely distributed in the cell walls of microorganisms, mushrooms and plants, have received much attention with respect to their biological functions. Traditional method for preparing β-d-glucans from Saccharomyces cerevisiae cells was based on repeated extractions with acid or alkali solutions, which resulted in degradation of the β-d-glucan chains. A new mild method to extract β-d-glucans from S. cerevisiae cells was proposed in this paper, which was composed of induced autolysis, water and organic solvent treatment, homogenization and protease hydrolysis. Compared with traditional isolation methods, this new method had distinct advantages that it gave high yields and purity of β-d-glucans and kept β-d-glucans native conformation. Furthermore, this isolation procedure did not pollute the environment and could be easily scaled-up to an industrial process. The experimental data indicated that each extraction step had its significant effect on β-d-glucans preparation, especially, the induced autolysis and homogenization. After 24h of induced autolysis, 48% (w/w) cellular substances were released, whereas the cell wall nearly remained intact. Treated by hot water, the mechanical strength of the yeast cell wall decreased with the removal of the mannoproteins, which caused the yeast cell walls easy to be disrupted. As a result, the ratio of broken yeast cell slightly exceeded 95% after three passes in homogenization treatment in 70MPa. Finally, β-d-glucans were obtained at a yield of 91% of the original ratio in the yeast cell walls and with a purity of up to 93% (w/w). [Copyright &y& Elsevier]

Abstract: Cereal β-glucans can form aggregates in aqueous solution. The presence of aggregates in cereal β-glucan solutions led to inaccurate determination of molecular weights and it was believed that intermolecular hydrogen bonding caused the aggregation. To eliminate aggregates, a carbanilation method for molecular weight determination of cereal β-glucans was developed. Wheat β-glucan samples were selected for investigation. The carbanilation method can prevent intermolecular hydrogen bonding by blocking hydroxyl groups with phenyl carbamate groups. The carbanilates of cereal β-glucans were prepared by the reaction of cereal β-glucans with phenylisocyanate catalyzed by DMSO and pyridine. To avoid degradation during the carbanilation reaction, relatively mild conditions were used, which led to incomplete substitution (DS: ∼2). However, after the carbanilation reaction, the carbanilates dissolved completely in 1,4-dioxane solution without any detectable aggregates, which allowed accurate molecular weight determination. The degree of substitution (DS) of carbanilates was determined by both a nitrogen content method and an FT-IR method. The FT-IR method proved to be the more effective for DS estimation. Using this method, the converted molecular weights of cereal β-glucans were in good agreement with the results measured in 0.5M NaOH solution, which previously was shown to be a good solvent for cereal β-glucans. After the carbanilation reaction, conformational changes of carbanilates were studied by static and dynamic light scattering techniques. The fractal dimension (d f =2.27) and the structure sensitive parameters (ρ >2) suggested a porous globular structure for partially carbanilated β-glucans. [Copyright &y& Elsevier]

β- d -glucan (GLP20) isolated from Ganoderma lucidum fruiting bodies was successfully fractionated into five fractions with different weight-average molecular weights ( M w ) through ultrasonic irradiation. The M w , radius of gyration ( R g ), hydrodynamic radius ( R h ) and intrinsic viscosity ([ η ]) of these fractions in phosphate buffered saline (PBS) aqueous solution were determined using HPSEC-MALLS-RI-VS system. The results indicated that β- d -glucan displayed rigid chain conformations with ρ values lager than 2.06 for the five fractions. By applying the polymer solution theory, the exponent ( ν and α ) values of R g  =  kM w ν and [ η ] =  kM w α were calculated as 0.72 and 1.05, respectively, which confirmed the rigid chain conformation of glucans with M w from 2.9 × 10 5 to 2.42 × 10 6  g·mol −1 . According to the known theory for chains, the molar mass per unit contour length M L , persistence length q and contour length h per main-chain glucose residue were estimated to be 2150 nm −1 , 128 nm and 0.30 nm, respectively, which indicated that the glucan existed as triple-helical chains in PBS aqueous solution. Bioactivity study of glucan fractions on lymphocytes and THP-1 macrophages demonstrated that all these fractions could stimulate lymphocyte proliferation, promote macrophages to form pseudopodia, and enhance the levels of inflammatory cytokines IL-6 and TNF-α. Meanwhile, the glucan fractions with M w higher than 1.82 × 10 6  g·mol −1 exhibited better activity on enhancing the release of inflammatory cytokines. [ABSTRACT FROM AUTHOR]

Oat β-glucan-amino acid/peptide conjugates were prepared through Maillard reaction between oat β-glucan and amino acid/peptide. FT-IR, high performance size exclusion chromatography (HPSEC), and rheometry were used to characterize the structural and rheological properties of the amino acid/peptide conjugates. The results showed that the amino acid and peptide were successfully bonded with oat β-glucan to give β-glucan-amino acid conjugates and β-glucan peptide conjugates, respectively. The 2% (w/v) solution of β-glucan-amino acid conjugates showed shear thinning behavior as non-Newtonian fluid over the entire measured shear rates at 25 °C. The apparent viscosity of β-glucan-amino acid conjugates was increased compared with that of oat β-glucan. On the contrary, the viscosity of β-glucan peptide conjugates decreased sharply and showed a Newtonian fluid when shear rate was higher than 10 s −1 . The storage modulus (G′) of β-glucan-amino acid conjugates was much higher than that of β-glucan and β-glucan peptide conjugates at the frequency of 1 Hz. The G′ was higher than the loss modulus (G″) at all frequencies for all the oat β-glucan conjugates solutions at 7% (w/v) concentration, indicating that both of β-glucan-amino acid conjugates and β-glucan peptide conjugates presented as a solid-like behavior. [ABSTRACT FROM AUTHOR]

β-Glucan benefits are related with its molar mass and it would be of interest to better understand how this parameter can be changed by processing and variety for design of food with specific health effects. For this purpose, extracts from barley malts and brewers' spent grain, processed at different conditions, were analysed regarding β-glucan content, molar mass, and protein content. Molar mass distribution was assessed using asymmetric flow field-flow fractionation (AF4) with multiangle light scattering (MALS), differential refractive index (dRI) and fluorescence (FL) detection. β-Glucan was detected in a wide molar mass range, <2000 to approximately 6.7 × 10 6 g/mol. Differences in molar masses were more noticeable between barley varieties and steeping malting conditions than by mashing of malt. Barley products processed to preserve β-glucan contained more β-glucan of high molar mass with potential to shift the fermentation site to the distal colon. Enzymatic degradation of proteins indicated presence of aggregates containing β-glucan and protein. [ABSTRACT FROM AUTHOR]

Effects of non-chemical modification on structure and fermentability of α-D-glucan from Leuconostoc citreum SK24.002 were investigated. It was found that the apparent viscosity, molecular weight and percentage of α-1,6 linkage of α-D-glucan decreased after endodextranase or ultrasonic treatment. During in vitro fermentation, a strong increase in bacterial population was observed and the largest changes took place during the first 12 h of incubation. A decreased pH was also observed and gas production after 48 h fermentation increased at the following sequence: enzymatic treated α-D-glucan < ultrasonic treated α-D-glucan < α-D-glucan. The fermentation of modified sample resulted in a consistent generation of SCFA, particularly in the 12–48 h part of incubation. The concentrations of propionic acid and butyric acid at 48 h of fermentation were 60.4 and 19.1 mM for enzymatic treated α-D-glucan, respectively, higher than ultrasonic treated α-D-glucan or α-D-glucan. This showed that enzymatic hydrolysis could be a promising degradation method for the production of potential prebiotics. [ABSTRACT FROM AUTHOR]

The objective of this work was to produce the bioengineered α-D-glucan from Leuconostoc citreum SK24.002 and study its molecular structure and prebiotics properties. The connectivities and substitutions of α-D-glucan were measured using 2D NMR and the chain structure of the repeating unit was deduced. During in vitro fecal fermentation, a strong increase in bacterial population was observed and the largest changes took place during the first 24 h of incubation. A decrease pH (approximately 2.0) was also observed for the fermentation from 0 h to 48 h compared to the blank (pH 6.8–6.6). Total short-chain fatty acid (SCFA) increased during batch fecal fermentation in the presence of α-D-glucan and it reached the maximum level of 30.01 mM after 48 h of fermentation. The lowest gas production and highest butyric acid production at 48 h of incubation was recorded with α-D-glucan as compared with dextran or pullulan. Moreover, human fecal microbiota on α-D-glucan produced more butyric acid between 24 and 48 h than other test carbohydrates. These results suggest that α-D-glucan would be a good candidate slowly fermentable fiber and may have a positive effect for the gut microbiota homeostasis. [ABSTRACT FROM AUTHOR]

A high molecular weight bioengineered α-D-glucan was obtained from Leuconostoc citreum SK24.002 and certain physicochemical properties were investigated. Scanning electron microscopy of α-D-glucan demonstrated a cementitious material with agglutinated granules. Atomic force microscopy showed the irregularly dendritic shaped structure, revealing some side chains. The glucan solution (3%, v/w) has opalescent, bluish-white colour and the concentration affected the depth of colour. The degradation process of 265–345 °C and melting point of 133.9 °C were determined from the thermal analysis. The glucan viscosity increased exponentially with concentration in the range of 5%–10% (w/v). The glucan solution (9%, w/v) showed a non-Newtonian pseudoplastic behaviour at shear rates of 0.01–100 1/s. The changes of elastic modulus and viscous modulus indicated the glucan could form a weak gel. The glucan solution also had good resistance to acidic or thermal hydrolysis. Three-dimensional model revealed α-D-glucan molecule has a helical conformation with some inter-molecular hydrogen bonds, suggesting a high viscosity in solution and this could make it suitable for usage as a biothickener or stabilizer agent in food systems. [ABSTRACT FROM AUTHOR]

Ultrasonication was applied to modify the structural and physicochemical properties of the (1 → 3)(1 → 6)-α- d -glucans from Leuconostoc citreum SK24.002. Upon ultrasound treatment, both the UV absorption at 220 nm and number-average molecular weight decreased substantially in the first 20 min, and then incrementally decreased to a plateau value after 160 min. The molecular weight distribution curves gradually shifted to the low molecular weight region with a narrower distribution and the chain degradation reaction followed the random scission model. NMR results showed that the partial consecutive α-1,6 linkages in the limited regions were ruptured by acoustic cavitations. The viscosity of modified glucan solution dropped remarkably and the glucan after ultrasonication for more than 20 min was dissolved in water to give solutions of 30 g/100 ml or greater. The Newtonian plateaus for sonicated glucan solutions were observed at the high shear rates (1–100 1/s). [ABSTRACT FROM AUTHOR]

Abstract: Physicochemical properties of different β-glucan purified from oat, barley and wheat, were investigated by measuring the composition, molecular weight, intrinsic viscosity ([η]), and rheological properties. The main objective was to investigate the relationship between effects of regulation of cholesterol metabolism and antioxidant property on db/db diabetic mice of β-glucans and their molecular weights and viscosities. Metformin was used as the positive control. The levels of blood glucose, serum lipid, liver lipid, superoxide dismutase (SOD) and malondialdehyde (MDA) in liver were determined. Results showed that average values of molecular weight (MW) of β-glucan from oat, wheat and barley were 172 kDa, 635 kDa and 743 kDa; the viscosities of β-glucans were positively correlated with their corresponded molecular weights, and wheat and barley β-glucan showed significant shear thinning ability compared to oat β-glucan; Barley β-glucans had higher G′ compared to wheat and oat, and the latter had higher G″. Blood glucose differences were not significant due to high variability, however serum TC, TG, liver TC, TG and LDL-C were lower in β-glucan fed groups compared to control. Moreover, the HDL-C was higher in β-glucan fed groups compared to control group. The addition of β-glucan fed to the db/db diabetic mice significantly (P < 0.05) increased their liver SOD activities and reduced their MDA levels (P > 0.05). A correlation between the measured biological parameters and the molecular weight or viscosity of β-glucan was observed. Lack of β-glucanase degeneration was the main cause of the low MW β-glucan and its diminished physiological effect. [Copyright &y& Elsevier]

Highlights: [•] Structure of high Mw β-d-glucan. [•] Increased NO of macrophages. [•] Unique conformation transitions. [ABSTRACT FROM AUTHOR]

Abstract: Oat β-glucan was purified from oat bran and its effects on running performance and related biochemical parameters were investigated. Four-week-old male Sparsgue–Dawley rats, fed with/without oat β-glucan (312.5mgkg−1 d−1) for 7 weeks, were subjected to run on a treadmill system to make them exhausted. All rats were immediately sacrificed after prolonged exercise, and the major metabolic substrates were measured in serum and liver. The results showed feeding dietary oat β-glucan to rats could significantly reduce the body weight and increase the maximum running time compared with normal control (P <0.05). Furthermore, dietary oat β-glucan decreased the levels of blood urea nitrogen, lactate acid, and creatine kinase activity in serum, and increased the levels of non-esterified fatty acids, lactic dehydrogenase activity in serum, and the content of liver glycogen. Therefore, the present study demonstrated that dietary oat β-glucan can enhance the endurance capacity of rats while facilitating their recovery from fatigue. [Copyright &y& Elsevier]