Keith W. Waldron

Arabinoxylan and mono- and dimeric ferulic acid release from brewer’s grain and wheat bran by feruloyl esterases and glycosyl hydrolases from Humicola insolens

An enzyme preparation from the thermophilic fungus Humicola insolens, Ultraflo L, was able to solubilise more than half of the biomass of brewer’s grain and wheat bran, two agro-industrial co-products. While almost all of the ferulic acid was released in the free form, the majority of diferulates were released still attached to soluble feruloylated oligosaccharides, except for the 8,5′ benzofuran form, which remained mostly in the residue. H. insolens also produced an esterase capable of releasing over 50% of p-coumaric acid present in wheat bran, but only 9% from the brewer’s grain. The polysaccharide content in the residues after enzyme treatment comprised mostly cellulose and arabinoxylan, which suggests that part of the arabinoxylan in these residues is inaccessible to the xylanases of H. insolens. Differences in the solubilised arabinose-to-xylose ratio coupled to high free ferulate release suggest that the structure of feruloylated arabinoxylan in barley and wheat may differ.

Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acids from cereal arabinoxylan.

The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1→4)-β-d-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5′ form of diferulic acid from arabinoxylan (AX) derived from brewers’ spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.

Signaling from an Altered Cell Wall to the Nucleus Mediates Sugar-Responsive Growth and Development in Arabidopsis thaliana

Sugars such as glucose function as signal molecules that regulate gene expression, growth, and development in plants, animals, and yeast. To understand the molecular mechanisms of sugar responses, we isolated and characterized an Arabidopsis thaliana mutant, high sugar response8 (hsr8), which enhances sugar-responsive growth and gene expression. Light-grown hsr8 plants exhibited increased starch and anthocyanin and reduced chlorophyll content in response to glucose treatment. Dark-grown hsr8 seedlings showed glucose-hypersensitive hypocotyl elongation and development. The HSR8 gene, isolated using map-based cloning, was allelic to the MURUS4 (MUR4) gene involved in arabinose synthesis. Dark-grown mur1 and mur3 seedlings also exhibited similar sugar responses to hsr8/mur4. The sugar-hypersensitive phenotypes of hsr8/mur4, mur1, and mur3 were rescued by boric acid, suggesting that alterations in the cell wall cause hypersensitive sugar-responsive phenotypes. Genetic analysis showed that sugar-hypersensitive responses in hsr8 mutants were suppressed by pleiotropic regulatory locus1 (prl1), indicating that nucleus-localized PRL1 is required for enhanced sugar responses in hsr8 mutant plants. Microarray analysis revealed that the expression of many cell wall–related and sugar-responsive genes was altered in mur4-1, and the expression of a significant proportion of these genes was restored to wild-type levels in the mur4-1 prl1 double mutant. These findings reveal a pathway that signals changes in the cell wall through PRL1 to altered gene expression and sugar-responsive metabolic, growth, and developmental changes.

Spontaneous Mutation Reveals Influence of Exopolysaccharide on Lactobacillus johnsonii Surface Characteristics

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

In vitro evaluation of the prebiotic properties of almond skins (Amygdalus communis L.).

In this study we investigated the potential prebiotic effect of natural (NS) and blanched (BS) almond skins, the latter being a byproduct of the almond-processing industry. A full model of the gastrointestinal tract, including in vitro gastric and duodenal digestion, followed by colonic fermentation using mixed faecal bacterial cultures, was used. Both NS and BS significantly increased the population of bifidobacteria and Clostridium coccoides/Eubacterium rectale group, resulting in a prebiotic index (3.2 for BS and 3.3 for NS) that compared well with the commercial prebiotic fructo-oligosaccharides (4.2) at a 24-h incubation. No significant differences in the proportion of gut bacteria groups and in short-chain fatty acid production were detected between NS and BS, showing that polyphenols present in almond skins did not affect bacterial fermentation. In conclusion, we have shown that dietary fibre from almond skins altered the composition of gut bacteria and almond skins resulting from industrial blanching could be used as potential prebiotics.

Mechanical properties of lettuce

The fracture properties of Spanish Iceberg and English Round lettuce tissues were investigated using a tensile test on notched specimens. The level of notch sensitivity was investigated for samples of differing colour and vein orientation. Vein orientation perpendicular to the test direction proved to be the most notch sensitive and samples with vein orientation parallel to the test direction proved to be very notch insensitive, samples with a diagonal (45°) orientation showed an intermediate response. This response was interpreted in terms of the interaction of veins with the crack path. The strengths of English Round tissues were broadly comparable with those of Spanish Iceberg although the upper limits depended on vein orientation and were in the order: parallel > diagonal ≈ perpendicular. A similar ranking of vein orientation was found in estimates of stiffness.

Effect of ripening on the mechanical properties of Portuguese and Spanish varieties of olive (Olea europaea L)

The mechanical properties of the table olive, Hojiblanca and Douro varieties from Spain and Portugal respectively, were determined. The results showed that in a tensile test the skin was stronger and stiffer than the flesh. This was also reflected by the strain at failure of the two tissues, the skin being less deformable than the flesh. During ripening, the strength of both skin and flesh of Hojiblanca decreased, whereas the skin and flesh of Douro increased and decreased in strength respectively. In general, Hojiblanca tissues were stronger than Douro tissues. A softening phenomenon was detected during ripening in the flesh of both varieties. The stiffness of the skin of Hojiblanca decreased significantly during ripening, whereas that of the skin of Douro increased but to a lesser extent. However, in general, the skin and flesh of Hojiblanca were stiffer than those of Douro. A cutting test enabled the determination of skin and flesh toughness. However, it is suggested that the flesh contributed to the measurement of skin toughness in the early stages of ripening, as reported for other commodities. The flesh of Hojiblanca was tougher than that of Douro, with both decreasing in toughness during ripening.

Thermal transitions in freeze-dried carrot and its cell wall components

Abstract Differential scanning calorimetry (DSC) was used to measure the glass transition temperature, T g and a sub- T g event in freeze-dried carrot material and different carrot cell wall components, at moistures ranging from 0% to 20% (w.w.b.). Two glass transition temperatures were detected in the freeze-dried carrot material associated with two phases: a sugar-rich phase and a cell wall-rich phase. The water distribution in the different phases is also important and sorption isotherms of freeze-dried carrot and different cell wall components were determined and compared with published results produced on sugar, pectins and cellulose. For the insoluble carrot cell wall materials, a single T g was detected which increased in the order: a Ca 2+ bound pectin-free residue, an esterified pectin-free residue and a cellulose-rich residue. In all cases T g increased with decreasing moisture. Additionally, a sub- T g endothermic event was observed in all the materials which disappeared on rescanning, consistent with results published on ageing of other biopolymers. The T g of the different cell wall residues was modelled, using the Gordon–Taylor and Kwei equations. The latter showed that water–biopolymers interactions are less prominent in the cellulose-rich cell wall residue, shown by a negative value of q .

Modelling of carrot tissue as a fluid-filled foam

Experimental values of the stiffness of carrot tissue were compared with the predictions from models of fluid-filled closed cell foams. Compared to an experimental value of 7 MPa for fresh carrot, predictions gave moduli in the range 2 to 33 MPa based on a compressible fluid, an isotonic state turgor pressure of 0.8 MPa, a cell wall modulus of 100 MPa and cell lengths from 54 to 3 times the wall thickness. The modulus was predicted to increase linearly with turgor pressure in agreement with experiment for turgor pressures up to about 1 MPa, whereafter the experimental modulus increased more sharply, reaching a value of 14 MPa at a turgor pressure of 2.1 MPa, closer to the predicted rate of increase in an earlier shell model. Predictions based on an incompressible fluid gave initial and equilibrium moduli of 14 and 2 MPa, respectively, in agreement with the experimental values at high and low levels of turgor, again assuming a cell wall modulus of 100 MPa but requiring small cells of length ratio less than three times their wall thickness.

Comparison of saccharification and fermentation of steam exploded rice straw and rice husk.

BackgroundRice cultivation produces two waste streams, straw and husk, which could be exploited more effectively. Chemical pretreatment studies using rice residues have largely focussed on straw exploitation alone, and often at low substrate concentrations. Moreover, it is currently not known how rice husk, the more recalcitrant residue, responds to steam explosion without the addition of chemicals.ResultsThe aim of this study has been to systematically compare the effects of steam explosion severity on the enzymatic saccharification and simultaneous saccharification and fermentation of rice straw and husk produced from a variety widely grown in Vietnam (Oryza sativa, cv. KhangDan18). Rice straw and husk were steam exploded (180–230xa0°C for 10xa0min) into hot water and washed to remove fermentation inhibitors. In both cases, pretreatment at 210xa0°C and above removed most of the noncellulosic sugars. Prolonged saccharification at high cellulase doses showed that rice straw could be saccharified most effectively after steam explosion at 210xa0°C for 10xa0min. In contrast, rice husk required more severe pretreatment conditions (220xa0°C for 10xa0min), and achieved a much lower yield (75xa0%), even at optimal conditions. Rice husk also required a higher cellulase dose for optimal saccharification (10 instead of 6xa0FPU/g DM). Hemicellulase addition failed to improve saccharification. Small pilot scale saccharification at 20xa0% (w/v) substrate loading in a 10xa0L high torque bioreactor resulted in similarly high glucose yields for straw (reaching 9xa0% w/v), but much less for husk. Simultaneous saccharification and fermentation under optimal pretreatment and saccharification conditions showed similar trends, but the ethanol yield from the rice husk was less than 40xa0% of the theoretical yield.ConclusionsDespite having similar carbohydrate compositions, pretreated rice husk is much less amenable to saccharification than pretreated rice straw. This is likely to attenuate its use as a biorefinery feedstock unless improvements can be made either in the feedstock through breeding and/or modern biotechnology, or in the pretreatment through the employment of improved or alternative technologies. Physiological differences in the overall chemistry or structure may provide clues to the nature of lignocellulosic recalcitrance.