Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley

A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.     

Binding capacity of a barley beta-D-glucan to the beta-glucan recognition molecule dectin-1

To clarify whether barley beta-glucans exhibit their biological effects via binding to dectin-1, a pivotal receptor for beta-1,3-glucan, the structure of barley beta-glucan E70-S (BBG-70) was unambiguously investigated by NMR spectroscopy and studied for its binding capacity and specificity to dectin-1 by ELISA. NMR spectroscopy confirmed that BBG-70 contains two different linkage glucans, namely, alpha-glucan and beta-glucan, which are not covalently attached to one another. Beta-glucan within BBG-70 is a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose residues but does not contain the continuous 1,3-linkage. Competitive ELISA revealed that highly purified barley beta-glucan E70-S (pBBG-70) inhibits the binding of soluble dectin-1 to sonifilan (SPG), a beta-1,3-glucan, although at a concentration higher than that of SPG and laminarin. It was found that barley beta-glucan can be recognized by dectin-1, implying that barley beta-glucan might, at least in part, exhibit its biological effects via the recognition by dectin-1 of the ligand sugar structure, which may be formed by 1,3-beta- and 1,4-beta-glucosyl linkage.     

Variation in total and soluble beta-glucan content in hulless barley: effects of thermal, physical, and enzymic treatments

Total and soluble beta-glucan content and effects of various treatments of barley grain on extractability and molecular characteristics of soluble beta-glucan were studied. Four types of hulless barley (normal, high amylose, waxy, and zero amylose waxy) from 29 registered and experimental genotypes were analyzed. For each, moisture, protein, amylose, 100 kernel weight, starch, beta-glucan (total and soluble), beta-glucanase activity, and slurry viscosity were determined. Significant differences in total beta-glucan were observed among the groups, with average values of 7. 49%, 6.86%, 6.30%, and 4.38% for high amylose, waxy, zero amylose waxy, and normal barley, respectively. The extractability of beta-glucan in high amylose barley was relatively low (20.6-29.7%) compared to that in normal (29.8-44.3%), zero amylose waxy (34.0-52. 5%), and waxy (36.7-52.7%) barley genotypes. Viscosity of barley flour slurries was affected by the content of soluble beta-glucans, beta-glucanase activity, and molecular weight of beta-glucans. Hydrothermal treatments (autoclaving and steaming) of barley had no effect on extractability of beta-glucans, but prevented enzymic hydrolysis of beta-glucans, and thereby substantially improved their molecular weight. The addition of enzymes (protease and esterase) during extraction and/or physical treatments (sonication) increased extractability of beta-glucans from barley.     

Improved quantitative analysis of oligosaccharides from lichenase-hydrolyzed water-soluble barley beta-glucans by high-performance anion-exchange chromatography

Cereal beta-glucan is a linear biopolymer linked by beta-(1,3)/(1,4)-glycosidic bonds. More specifically, the beta-(1,4)-linked glucose chain is interrupted with beta-(1,3)-linkages in cereal beta-glucan structure. Elucidation of the exact length and distribution of linear beta-(1,4)-linked portion facilitates the understanding of the fine structure of cereal beta-glucan. A HPAEC assisted by lichenase treatment has been used for the structural and quantitative analysis of cereal beta-glucan. The absence of authentic standard oligosaccharides, putatively 3-O-beta-cellobiosyl-D-glucose (DP3) and 3-O-beta-cellotriosyl-D-glucose (DP4), was a potential problem to the characterization of beta-glucan structure. In this study, two major lichenase-hydrolyzed products were generated from the barley beta-glucan, and putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose were separated and highly purified by recycling preparative HPLC technology. Structural analysis of highly purified putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose was performed by TLC and LC-MS analysis. Two putative DP3 and DP4 displayed the nonreducing end/(1,4)/(1,3) linkage ratios of 1:0.96:0.90 and 1:2.18:1.16, respectively; the molecular masses (m/z) of their sodium adducts were 527.0 and 689.0, respectively. Using these structurally confirmed oligosaccharides, the exact amounts of beta-glucan lichenase hydrolysates from domestic barley cultivars were quantified. The amount of two major DP3 and DP4 accounted for only 71.4-73.3% of water-extractable beta-glucan fraction, and the (1,4)/(1,3) linkage ratios of the extracted beta-glucans were almost identical in the range of 2.24-2.25 among the barley cultivars tested.     

In vivo modeling of beta-glucan degradation in contrasting barley (Hordeum vulgare L.) genotypes

An important determinative of malt quality is the malt beta-glucan content, which in turn depends on the initial barley beta-glucan content as well as the beta-glucan depolymerization by beta-glucanase (EC 3.2.1.73) during malting. Another enzyme, named beta-glucan solubilase, has been suggested to act prior to beta-glucanase; its existence, however, has not been unequivocally proven. We monitored changes in beta-glucan levels and in the development of beta-glucan-degrading enzymes during malting of five lots of contrasting barley genotypes. Two models of in vivo kinetics for beta-glucan degradation were then compared as follows: (i) a biphasic model based on the sequential action of beta-glucan solubilase and beta-glucanase and (ii) a monophasic model assuming that all beta-glucans are depolymerized by beta-glucanase without the previous intervention of another enzyme. Confirmatory regression analysis was used to test the fit of the models to the observed data. Our results show that beta-glucan degradation is mostly monophasic, although some enzyme other than beta-glucanase seems to be required for the early solubilization of a small fraction of insoluble beta-glucans (on average, 7% of total beta-glucans). Furthermore, the genotype-dependent kinetic rate constant (indicating beta-glucan degradability), in addition to beta-glucanase activity, is suggested to play a major role in malting quality.     

Improvement of malt modification by use of Rhizopus VII as starter culture

The development of a selected starter culture on malting barley and its effects on malt quality aspects were studied. Application of Rhizopus sporangiospores in a malting process resulted in increased beta-glucanase and xylanase contents of the malting barley and improved starchy endosperm cell-wall degradation. Activation of the sporangiospores and optimization of the inoculation procedure led to a further increase in enzyme levels and to larger and more consistent impacts on cell-wall modification. Whereas the main effect of the starter culture on beta-glucan degradation was observed during malting, a further decrease in beta-glucan during mashing suggests that the microbial enzymes that survived the kilning step were active during mashing. Other quality aspects that were influenced by the starter culture activity were protein modification, wort color, and wort pH. The level of microbial enzymes produced was related to the amount of barley kernels infected with the starter culture.     

Pearling of hull-less barley: product composition and gel color of pearled barley flours as affected by the degree of pearling

Barley grains from two hull-less varieties, Phoenix and Candle, were pearled to various degrees (10-80%). The composition (starch, protein, beta-glucan, lipid, and ash) of pearled grain (PG) and pearling flour (PF) was determined. Effect of pearling on Hunter L, a, and b color parameters of uncooked and cooked (gel) barley flour (milled from PG) was investigated over a 3 day storage at 4 degrees C.     

Correlation of malt quality parameters and beer flavor stability: multivariate analysis

Malt is known to have an impact on beer flavor stability mainly due to the presence of antioxidants. In this study, five barley varieties were malted at industrial and micro scale, and quality parameters of the resulting malts were measured (diastatic power, friability, beta-glucan content, antiradical power, reducing power, lipoxygenase activity, and nonenal potential) and correlated with the sensory data obtained for the corresponding fresh and forced aged beers. A statistical strategy using multiple linear regressions was applied to explore relationships between the malt chemical parameters and beer sensory data, showing antiradical power as the major contribution of malt to beer flavor stability. Additionally, the measured antiradical power, which is well correlated with the polyphenolic content, was found to be very similar for malt and barley, emphasizing the key role of barley endogenous polyphenols.     

Xylanase,beta-glucanase,and other side enzymatic activities have greater effects on the viscosity of several feedstuffs than xylanase and beta-glucanase used alone or in combination

This study was carried out to evaluate the effects of a pure xylanase, a pure beta-glucanase, a mix of the two pure enzymes, and a commercial enzyme preparation (Quatrazyme HP, Nutri-Tomen Les Ulis, France) on the viscosity exhibited by water-soluble nonstarch polysaccharides of several feedstuffs (Rialto wheat, Sidéral wheat, Isengrain wheat, triticale, rye, barley, oats, corn, wheat bran, rice bran, wheat screenings, soybean meal, rapeseed meal, sunflower meal, and peas). The viscosity depended on the feedstuffs and varieties of the same feedstuff. There was a correlation (R (2) = 0.86) between viscosity of cereals and their arabinoxylan and beta-glucan contents. The correlation was greater (R (2) = 0.99) when the type of cereal was taken into account. The addition of pure xylanase significantly decreased the viscosity of all feedstuffs except sunflower meal (P < or = 0.05). However, pure beta-glucanase was unable significantly to decrease the viscosity of Isengrain wheat, corn, rice bran, wheat screenings, soybean meal, and sunflower meal. There was a greater decrease in viscosity with the combination of xylanase and beta-glucanase than with addition of xylanase or beta-glucanase alone. This synergistic action of xylanase and beta-glucanase was observed only in Rialto wheat, Sidéral wheat, triticale, rye, barley, oats, and peas. Finally, the commercial enzyme preparation produced a greater reduction (P < or = 0.05) in viscosity for all feedstuffs compared to xylanase or beta-glucanase used alone or in combination. The greater effectiveness of the commercial enzyme preparation was due to the presence of side enzymatic activities (arabinofuranosidase, xylosidase, glucosidase, galactosidase, cellulase, and polygalacturonase).     

Phytochemical and dietary fiber components in barley varieties in the HEALTHGRAIN Diversity Screen

Ten different barley varieties grown in one location were studied for their content of tocols, folate, plant sterols, alkylresorcinols, and phenolic acids, as well as dietary fiber components (arabinoxylan and beta-glucan). The samples included hulled and hull-less barley types and types with normal, high-amylose, and waxy starch. The aim was to study the composition of raw materials, and therefore the hulls were not removed from the hulled barleys. A large variation was observed in the contents of all phytochemicals and dietary fibers. Two varieties from the INRA Clermont Ferrand barley program in France (CFL93-149 and CFL98-398) had high content of tocopherols and alkylresorcinols, whereas the variety Dicktoo was highest in dietary fiber content and phenolics. Positive correlations were found between 1000 kernel weight, alkylresorcinols, and tocols, as well as between dietary fiber content and phenolic compounds. The results demonstrate that the levels of phytochemicals in barley can likely be affected by breeding and that the contents of single phytochemicals may easily be adjusted by a right selection of a genotype.     

Growth of Trichoderma viride on crude cell wall preparations from barley

Trichoderma viride can utilize crude cell wall preparations from barley starchy endosperm as sole source of carbon and energy. In the process beta-(1-->3)(1-->4)-glucan and arabinoxylan are released. The onset of release of the latter preceded that of glucan, consistent with arabinoxylan being encountered and utilized first. The release of several enzymes was observed during growth of Trichoderma on this substrate: endo-beta-(1-->3)(1-->4)-glucanase, endo-beta-(1-->4)-glucanase, endo-xylanase, arabinofuranosidase, esterase, carboxypeptidase, and "beta-glucan solubilase". It is inferred that each of these activities is necessary for the digestion of this substrate.     

Isolation of an in vitro and ex vivo antiradical melanoidin from roasted barley

The antiradical properties of water-soluble components of both natural and roasted barley were determined in vitro, by means of DPPH* assay and the linoleic acid-beta-carotene system, and ex vivo, in rat liver hepatocyte microsomes against lipid peroxidation induced by CCl4. The results show the occurrence in natural barley of weak antioxidant components. These are able to react against low reactive peroxyl radicals, but offer little protection against stable DPPH radicals deriving from peroxidation in microsomal lipids. Conversely, roasted barley yielded strong antioxidant components that are able to efficiently scavenge free radicals in any system used. The results show that the barley grain roasting process induces the formation of soluble Maillard reaction products with powerful antiradical activity. From roasted barley solution (barley coffee) was isolated a brown high molecular mass melanoidinic component, resistant to acidic hydrolysis, that is responsible for most of the barley coffee antioxidant activity in the biosystem.     

Lactobacillus plantarum and Pediococcus pentosaceus starter cultures as a tool for microflora management in malting and for enhancement of malt processability

Lactobacillus plantarum VTT E-78076 (E76) and Pediococcus pentosaceus VTT E-90390 (E390) starter cultures were added to the steeping water of normal malting barley in order to balance the microbial community and to enhance malt processability. In this study, we also investigated the effects of lactic acid-acidified MRS-spent medium (MRS-LA) on malting performance. Malting trials with five different two-row barley varieties were carried out in 25 kg pilot scale. The starter cultures promoted yeast growth during malting and restricted the growth of harmful bacteria and Fusarium fungi. Furthermore, they had positive effects on malt characteristics. Reduction in wort viscosity and beta-glucan content and enhanced xylanase and microbial beta-glucanase activities were observed. Starter cultures notably improved lautering performance. Some of the beneficial effects were due to the lactic acid and low pH, as similar effects were obtained with MRS-LA. Starter cultures offer a tool for tailoring of malt properties.     

Structural and biological characterization of aminated-derivatized oat beta-glucan

beta-Glucan extracted from oats was subjected to reductive amination, producing a cationic beta-glucan derivative of which physiological properties were characterized. The degree of substitution was obtained from elemental analysis, which was 0.48. In addition, the distribution of amino groups in the beta-glucan derivative was investigated by FT-IR analysis. In vitro bile acid binding capacity of the aminated beta-glucan was examined, showing significantly higher bile acid binding activity than native beta-glucan. Moreover, the beta-glucan derivative showed pronounced antimicrobial effects against Escherichia coli and Bacillus subtilis, and ACE (angiotensin-converting enzyme) inhibition activities which were dependent on its concentration. Furthermore, bronchoalveolar lavage (BAL) experiments demonstrated that the beta-glucan derivative stimulated the synthesis of nitric oxide. The improved functionalities of the derivative could be explained by its polycationic characteristics.     

In vitro bile acid binding of flours from oat lines varying in percentage and molecular weight distribution of beta-glucan

Two experimental high beta-glucan oat (Avena sativa) lines (7.64 and 8.05%) and two traditional lines (4.77 and 5.26% beta-glucan) were used to evaluate the effect of beta-glucan quantity and molecular weight on bile acid (BA) binding. The oat flour samples were digested by an in vitro system that simulated human digestion. No significant differences among oat type were found in the overall beta-glucan, starch, and pentosan digestibilities. Considering the standard, cholestyramine, as 100% bound, the relative BA binding for the oat flour samples on a dry matter basis was in the range of 7.5-14.8%, which is higher than the values determined for some other grains and plant materials in the literature. Although the high beta-glucan flours bound a high amount of BA, no significant correlations were found between beta-glucan content in the flours and BA binding. Significant correlations were found between BA binding and insoluble dietary fiber content. Partial hydrolysis with lichenase of the beta-glucan molecules did not affect the BA binding. A summary of all data suggested that BA binding is a multicomponent-dependent process.     

Effect of steam explosion treatment on barley bran phenolic compounds and antioxidant capacity

A steam explosion pretreatment process followed by methanol extraction has been applied for releasing and extracting phenolic compounds, as well as other effective components, from barley bran. The steam explosion treatment was performed at different temperatures ranging from 210 to 250 °C, with a residence time of 30 s. The effect of residence time was also studied in the range 10 s to 120 s at 220 °C. The extracts were evaluated for their total soluble phenolic content (TSPC) including total free phenolic acids (TFPC) and total soluble conjugates (TSC), identified phenolic acids, total antioxidant capacity (TAC), water-soluble carbohydrates (WSC) and total methanol extracts (TME). High-performance liquid chromatography (HPLC) coupled with a photodiode array detector (PDA) was used in this study for the analysis of p-coumaric acid and ferulic acid in barley bran before and after steam explosion. Our results indicate that TSPC and TAC increased with residence time. They also increased dramatically with temperature up to 220 °C. After steam explosion at 220 °C for 120 s, the TSPC reached 1686.4 gallic acid equivalents mg/100 g dry weight, which was about 9-fold higher than that of the untreated sample. The TSPC and TAC obtained were highly positively correlated (r = 0.918-0.993), which meant that the increase of TAC for the steam explosion pretreated barley bran extracts was due, at least in part, to the increase of TSPC in the methanol soluble fraction. Also, under optimum conditions, the WSC in aqueous solution was 5 times as much as that of the untreated sample, which demonstrated that steam explosion also hydrolyzes carbohydrates into water-soluble sugars. It can be concluded that a proper and reasonable steam explosion pretreatment could be applied to release the bound phenolic compounds and enhance the antioxidant capacity of barley bran extracts.     

Visualization of single and aggregated hulless oat (Avena nuda L.) (1-->3),(1-->4)-beta-D-glucan molecules by atomic force microscopy and confocal scanning laser microscopy

Surfactants were used to disperse oat beta-glucan. Atomic force microscopy (AFM) images of the resulting samples revealed a distribution of extended chainlike molecules and allowed, for the first time, direct visualization of single oat beta-glucan molecules with cross-sectional heights of about 0.44 nm. The number-average contour length (L(n)) and root-mean-square end-to-end distance ((R(ee)2)(1/2)) measured from the AFM images were 938 and 912 nm, respectively. The calculated persistence length (L(p)) was 526 nm. The weight-average molecular weight (M(w)) calculated from single beta-glucan molecules was 4.43 x 10(5). Samples without surfactant showed a strong tendency to form aggregates. The sample concentration, reserving time, and calcofluor as well as freezing could affect the formation of aggregates. These aggregates were visualized by both AFM and confocal scanning laser microscopy. The shape of the aggregates changed from small dots with diameters of approximately 20-50 nm to microfibrils over 3 microm long with the increasing of the concentration of oat beta-glucan from 10 to 100 microg/mL. The particle size distribution obtained by a laser particle size analyzer was 926 nm, which confirmed the size of oat beta-glucan molecules obtained from AFM images.     

In vitro bile acid binding activity within flour fractions from oat lines with typical and high beta-glucan amounts

Whole flours from four oat lines with different amounts of beta-glucan (4.8-8.1%) were examined for their antioxidant activity and total phenolic and lignin concentrations. These data, along with the beta-glucan percentages, were compared with bile acid (BA) binding. Only the lignin concentrations of the flours significantly (P < 0.01) correlated with the BA binding values. The oat flours also were fractionated into bran, protein concentrate, starch, layer above starch, and soluble beta-glucan (SBG)-free flour, and their BA binding capacities were evaluated. The bran fractions were the only fractions that bound greater BA than did the whole oat flours on dry matter basis. Extraction of the soluble beta-glucan to create the SBG-free flour significantly (P < 0.01) decreased the BA binding of the remaining flour. These data suggest that BA binding of the oat flours involves the synergistic interactions of the oat components, with beta-glucan and lignin (insoluble fiber) having a great impact.     

Content and molecular weight of extractable beta-glucan in American and Swedish oat samples

beta-(1-->3),(1-->4)-D-Glucan (beta-glucan) was extracted from 93 Swedish and 41 American oat samples using hot water containing CaCl(2) and thermostable alpha-amylase. The samples showed a large variation in both the content of extractable beta-glucan (0.76-3.68%) and the average molecular weight ((1.25-1.78) x 10(6) g mol(-1)). An analysis of the variance of beta-glucan content and the molecular weight of Swedish oat samples grown in 2000 and 2001 was done with cultivar and harvest year as factors. It showed that the extractable beta-glucan content was a heritable trait whereas molecular weight depended more on environmental factors. The American oat samples had a higher average content of extractable beta-glucan (2.24%) and a somewhat higher average molecular weight (1.58 x 10(6) g mol(-1)) than the Swedish oat samples (1.43% and 1.49 x 10(6) g mol(-1)).     

Antioxidant properties of pearled barley fractions

Two barley varieties (Falcon and AC Metcalfe) were separated by pearling into seven fractions and subsequently extracted with 80% methanol. The extracts, after solvent removal, were evaluated for their radical scavenging efficacy using Trolox equivalent antioxidant capacity (TEAC). The radical scavenging capacity of the extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC(FL)), and superoxide radical assays and a photoinduced chemiluminescence technique. In both barley varieties the outermost fraction (F1) yielded the highest phenolic content. In general, Falcon had a significantly higher total phenolic content than AC Metcalfe. A similar trend was observed for TEAC, DPPH, and superoxide radical scavenging capacities of the extracts. The contents of water-soluble antioxidants of Falcon and AC Metcalfe were 1.15-12.98 and 2.20-12.25 micromol of Trolox equiv/(g of defatted material), while the corresponding lipid-soluble counterparts varied from 1.44 to 4.70 micromol of alpha-tocopherol equiv/(g of defatted material). Phenolic acids, namely, vanillic, caffeic, p-coumaric, ferulic, and sinapic acids, were identified by HPLC in barley fractions.