Rheological and microstructural investigation of oat β-glucan isolates varying in molecular weight

The rheological properties and microstructure of aqueous oat β-glucan solutions varying in molecular weight were investigated. The structural features and molecular weights (MW) were characterized by ¹³C NMR spectroscopy and high performance size-exclusion chromatography (HPSEC), respectively. The microstructure of the β-glucans dispersions was also examined by atomic force microscopy (AFM). The samples with β-glucan content between 78 and 86% on a dry weight basis had MW, intrinsic viscosity ([η]) and critical concentration (c*) in the range of 142-2800 × 10³ g/mol, 1.7-7.2 dl/g and 0.25-1.10 g/dl, respectively. The flow and viscoelastic behaviour was highly dependent on MW and on the concentration of the β-glucans dispersions. Pseudoplastic behaviour was exhibited at high concentrations and Newtonian behaviour was evident at low concentrations. At the same concentration, the viscosity was higher for higher MW samples. The Cox-Merz rule was applicable for the lower molecular weight samples at higher concentrations whereas the high molecular weight sample deviated at concentrations greater than 1.0%, w/v. The mechanical spectra with variation of both MW and concentration were typical of entangled biopolymer solutions. AFM images revealed the formation of clusters or aggregates linked via individual polymer chains scattered heterogeneously throughout the system. The aggregate size increased with the molecular weight of the samples investigated and has been linked to the rheological behaviour of the samples.     

Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes

Background  

Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved β-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda.     

Post-translational processing of β-d-xylanases and changes in extractability of arabinoxylans during wheat germination

Endo-1,4-β-d-xylanase (EC 3.2.1.8, β-d-xylanase) activity, and arabinoxylan (AX) level and extractability were monitored for the first time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to 24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3–4 DPI). This increase was not accompanied by the discernible presence of homologues of the barley X-I β-d-xylanase as established by immunodetection. This suggests that other, yet unidentified β-d-xylanases operate in this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of unprocessed 67 kDa X-I like β-d-xylanase, which was much more abundant in the presence of GA. The occurrence of higher levels of the unprocessed enzyme was related with higher β-d-xylanase activities and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa β-d-xylanase. During the late stage (up to 24 DPI) gradual processing of the 67 kDa β-d-xylanase occurred and was associated with a drastic increase in β-d-xylanase activity. Up to 120-fold higher activity was recorded at 24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during the late stage, suggesting that the β-d-xylanase is processed into more active forms to achieve extensive AX breakdown.     

γ-Irradiation of malic acid in aqueous solutions

The -irradiation of malic acid in aqueous solutions was studied under initially oxygenated and oxygen-free conditions in an attempt to determine the possible interconversion of malic acid into other carboxylic acids, specifically those associated with Krebs cycle. The effect of dose on product formation of the system was investigated. Gas-liquid chromatography combined with mass spectrometry was used as the principal means of identification of the non-volatile products. Thin layer chromotography and direct probe mass spectroscopy were also employed.The findings show that a variety of carboxylic acids are formed, with malonic and succinic acids in greatest abundance. These products have all been identified as being formed in the -irradiation of acetic acid, suggesting a common intermediary. Since these molecules fit into a metabolic cycle, it is strongly suggestive that prebiotic pathways provided the basis for biological systems.     

Deprotonation of β-cyclodextrin in alkaline solutions

Variable pH ¹³C NMR and ¹H NMR spectroscopic studies of the β-cyclodextrin (β-CD) in alkaline aqueous solutions revealed that β-CD does not deprotonate at pH < 12.0. Further increase in solution pH results in the deprotonation of OH-groups adjacent to C-2 and C-3 carbon atoms of β-CD glucopyranose units, whereas the deprotonation of OH-groups adjacent to C-6 carbon atoms is expressed less markedly. The pK_a values for β-CD OH-groups adjacent to C-2 and C-3 carbon atoms are rather close, pK_a1,2 being 13.5 ± 0.2 (22.5 °C).     

α-Chymotrypsin superactivity in aqueous solutions of cationic surfactants

α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl-l-phenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. k_cat significantly increased while K_m was little changed after correction to free substrate concentration. The ratio of k_cat to K_m was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10⁻² M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants.     

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.     

High-temperature solvolysis of 2′-deoxyribonucleosides in aqueous amine solutions

Abstract

Degradation of 2′-deoxyribonucleosides in 0.5?M aqueous pyrrolidine at 110?°C proceeds at different rates, ordered as deoxyuridine?>?deoxyadenosine?>?deoxycytidine?>?deoxyguanosine ??deoxythymidine. Deoxyadenosine degradation produces the free base, adenine, while deoxycytidine by deamination produces deoxyuridine, and then uracil. The solvolysis of deoxyadenosine has an activation energy of 23.3?kcal/mol. Ammonolysis is slower than pyrrolidinolysis for deoxyadenosine, but faster for deoxyguanosine. In pyrrolidinolysis of the trinucleotides, d-TGT and d-TAT, the guanine moiety reacts faster than the adenine moiety. These trends are interpreted in terms of the ionization of the guanine moieties under basic conditions, rendering them less susceptible to nucleophilic attack.     

A theoretical framework for β-glucan degradation during barley malting

During malting, barley germinates and produces hydrolytic enzymes that de-structure the endosperm, making the grains soft and friable. This process starts close to the embryo and spreads throughout the whole grain. It is leaded by the degradation of cell walls, which are mainly constituted of β-glucans. Fast and extended breakdown of β-glucans occurs by means of an expanding reaction front driven by β-glucanase, and appears to follow pseudo-first-order kinetics. Endosperm permeabilization to macromolecules is closely linked to the dismantling of cell walls, thus that access to β-glucans by β-glucanase itself is limited. It is shown that the kinetics of β-glucan degradation during malting are consequent to this condition, and can be explained according to an anomalous evolution of the reverse quasi-steady-state approximation (rQSSA) for enzymatic reactions. In fact, kinetics based on the rQSSA include a transient phase wherein fast substrate depletion is indeed of pseudo-first-order. In the germinating barley, the conditions in which the physical modification of the endosperm occurs are shown to be suitable for the fast transient to persist in dynamic equilibrium while it progressively expands throughout the grain, depleting most β-glucans and, then, establishing the overall kinetics of β-glucan breakdown.     

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Solubilization of poorly water-soluble bioactive molecules in neutral aqueous media by complexation with renatured β-1,3-1,6-glucan nanoparticles

The design of scaffolds for solubilizing/dispersing poorly water-soluble bioactive molecules in neutral aqueous media is a major challenge of functional food, pharmaceuticals, and cosmetics development, as highlighted by the plethora of corresponding solubilization/dispersion strategies. Herein, renatured β-1,3-1,6-glucan (r-glucan) nanoparticles prepared by neutralization of alkali-denatured β-1,3-1,6-glucan and subsequent centrifugation are used as a host to disperse water-insoluble bioactive molecules (curcumin, all-trans-retinoic acid, and rebamipide) by simple mixing of host and guest solutions. Curcumin in the r-glucan cavity is found to be stacked in the form of J-aggregates and twisted along the helix, and is demonstrated to be retained for significantly longer than curcumin in the corresponding γ-cyclodextrin (γ-CD) complex. Specifically, curcumin incorporated in γ-CD is released within 5.5 hours, whereas that in the r-glucan complex is released very slowly, with 12% of curcumin in the latter complex retained after 31-day incubation at 37°C. Thus, inclusion protocol simplicity and slow release ability make r-glucan nanoparticles a potential carrier scaffold for various applications.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

Dietary calcium phosphate content and oat β-glucan influence gastrointestinal microbiota, butyrate-producing bacteria and butyrate fermentation in weaned pigs

This study aimed to evaluate the effects of oat β-glucan in combination with low- and high-dietary calcium phosphate (CaP) content on gastrointestinal bacterial microbiota, prevalence of butyrate-production pathway genes and fermentation end-products in 32 weaned pigs allocated to four diets: a cornstarch-casein-based diet with low [65% of the calcium (Ca) and phosphorous (P) requirement] and high CaP content (125% and 115% of the Ca and P requirement, respectively); and low and high CaP diets supplemented with 8.95% of oat β-glucan concentrate. Pigs were slaughtered after 14 days, and digesta were collected for quantitative PCR analysis, and quantification of short-chain fatty acids and lactate. The high CaP content reduced gastric lactate and streptococci and propionate in the large intestine. Oat β-glucan distinctly raised gastric bacterial numbers, and colonic lactobacilli and bifidobacteria. Although not reflected by gene copies of butyrate-production pathway genes, oat β-glucan also increased gastric, caecal and colonic butyrate concentrations, which may be favourable for intestinal development in weaned pigs. Thus, a high CaP content negatively affected the intestinal abundance of certain fermentation end-products, whereas oat β-glucan generally enhanced bacterial numbers and activity. The results emphasize the importance of the stomach for bacterial metabolism of oat β-glucan in weaned pigs.     

Stabilization of monovalent nickel in aqueous solutions by complexation with the β-isomer of C-5,12-racemic-1,4,5,7,7,8,11,12,14,14-decamethyl-1,4,8,11-tetraazacyclotetradecane

The monovalent nickel complex formed by the reduction of the β-isomer of the complex of C-5,12- racemic-1,4,5,7,7,8,11,12,14,14-decamethyl-1,4,8,11- tetraazacyclotetradecane nickel(II), NiL₁²⁺ in 0.1 M HCO₂Na, pH 7.6, has a half-life longer than 90 h. The redox potential of the couple NiL₁⁺/NiL₁²⁺ is −0.94 V vs. Ag/AgCl. The absorption spectrum of NiL₁⁺ consists of a band with λ_max = 335 nm and ϵ_max = 2200 M⁻¹ cm⁻¹. For the analogous complex with C-5,12-racemic-5,7,7,12,14,14-hexamethyl-1,4, 8,11-tetraazacyclotetradecane, L₂, the half-life time of NIL₂⁺ is less than 1 min and the redox potential is −1.44 V vs. Ag/AgCl. These results are similar to those reported earlier for the analogous nickel complexes with the meso-isomers of the ligands. The results thus indicate that both the kinetic and thermodynamic stabilization of monovalent nickel by N-methylation of tetraazamacrocyclic ligands is not significantly affected by the configuration of the ligand.     

Activation of members of a MAPK module in β-glucan elicitor-mediated non-host resistance of soybean

Plants recognize microbial pathogens by discriminating pathogen-associated molecular patterns from self-structures. We study the non-host disease resistance of soybean (Glycine max L.) to the oomycete, Phytophthora sojae. Soybean senses a specific molecular pattern consisting of a branched heptaglucoside that is present in the oomycetal cell walls. Recognition of this elicitor may be achieved through a β-glucan-binding protein, which forms part of a proposed receptor complex. Subsequently, soybean mounts a complex defense response, which includes the increase of the cytosolic calcium concentration, the production of reactive oxygen species, and the activation of genes responsible for the synthesis of phytoalexins. We now report the identification of two mitogen-activated protein kinases (MAPKs) and one MAPK kinase (MAPKK) that may function as signaling elements in triggering the resistance response. The use of specific antisera enabled the identification of GmMPKs 3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. Elicitor specificity of MAPK activation as well as the sensitivity against inhibitors suggested these kinases as part of the β-glucan signal transduction pathway. An upstream GmMKK1 was identified based on sequence similarity to other plant MAPKKs and its interaction with the MAPKs was analyzed. Recombinant GmMKK1 interacted predominantly with GmMPK6, with concomitant phosphorylation of the MAPK protein. Moreover, a preferential physical interaction between GmMKK1 and GmMPK6 was demonstrated in yeast. These results suggest a role of a MAPK cascade in mediating β-glucan signal transduction in soybean, similar to other triggers that activate MAPKs during innate immune responses in plants. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. The nucleotide sequences encoding the MAPKs and MAPKK1 from soybean can be accessed through the GenBank database under GenBank accession numbers AF104247, AF329506, and AY070230.     

Ribonucleic acid–deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide

Hybridization in 6xSSC (SSC, 0.15m-sodium chloride-0.015m-sodium citrate) at 66 degrees C was compared with hybridization in formamide-6xSSC (1:1, v/v) at 35 degrees C. As expected, the RNA hybridization potential was labile in the former system and stable in the latter. DNA retention by filters was poor in the formamide system, but could be improved. Several other properties of the hybridization reaction were explored and it was concluded that the formamide system is generally superior.     

Properties of β-glucan synthetase from Saccharomyces cerevisiae

Properties of -glucan synthetase from S. cerevisiae were studied. The enzyme exhibited optimal activity at pH 6.7 and 24 C. Km for UDP-glucose was 0.12mm. Addition of Mg⁺⁺ or Mn⁺⁺ stimulated its activity by 60% and 21% respectively. High concentrations of EDTA and hydroxyquinoline were inhibitory. Glucan synthetase was fully active in cell-free extracts. Small concentrations of trypsin or subtilopeptidase A from Bacillus subtilis, caused only a slight increase in glucosyl transferase activity, but larger concentrations destroyed -glucan synthetase. Acid proteases were neither stimulatory nordestructive. Thus it seemsunlikelythat -glucan synthetase exists in a zymogen form. Glucan synthetase was unstable. It was inactivated more rapidly at 28 C than at 0 C. The presence of substrate, -glucan or the protease inhibitors PMSF, Antipain or Pepstatin A did not protect -glucan synthetase from inactivation. Glucan synthetase was not stimulated by addition of cellobiose or -glucans. The synthesis of -glucans was competitively inhibited by UDP (K_i=0.45mm). Glucono--lactone, a known inhibitor of -glucosidases was a strong non-competitive inhibitor of -glucan synthetase.This work was supported by grants PNCB 00071 and 847 of the Consejo Nacional de Ciencia y Tecnología, México.     

Quantitative trait loci influencing β-glucan content in oat (Avena sativa, 2n=6x=42)

The β-glucan content of oat grain is of inter-est due to its positive human health role as a dietary component influencing serum cholesterol levels and its relation to the energy intake of livestock feed. Two recombinant inbred populations sharing a common parent (Kanota × Ogle and Kanota × Marion), and containing 137 individual lines each, were used to identify genomic regions that influence the β-glucan content in cultivated oat. Single-factor ANOVA, a backward elimination process, simple interval mapping (SIM) and simplified composite interval mapping (sCIM) were used to identify quantitative trait loci (QTLs). Regions on linkage groups 11 and 14 of the hexaploid oat RFLP map influenced β-glucan levels in both populations and over environments. Other genomic regions were identified whose effects varied depending on the genetic background, but were significant over measurements for a given population. Kanota and Ogle exhibit similar β-glucan levels and each parent contributed about the same number of positive β-glucan alleles in the Kanota × Ogle cross. Marion is higher in β-glucan content than Kanota and contributed all of the positive alleles in the Kanota × Marion cross. Three of the β-glucan QTL regions identified have been previously implicated as having a significant influence on the groat oil content in oat. These correlated QTL regions were either in coupling phase, with a region from one parent having the same effect on both traits, or were in repulsion phase. Identification of coupling- and repulsion-phase QTL regions for β-glucan and oil content facilitates the use of markers in manipulating these traits in oat breeding. Received: 8 September 1999 / Accepted: 25 March 2000