Lichenase (endo-1,3:1,4-β-D-Glucanase)
(Bacillus subtilis)

We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 3.2.1.73) in crude enzyme extracts. Two assay formats based on a) a direct cleavage or b) an enzyme coupled substrate were initially investigated. The ‘direct cleavage’ substrate, namely 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-3¹-cellotriosyl-β-glucopyranoside (MBG4), was found to be the more generally applicable reagent. This substrate was fully characterised using a crude malt β-glucanase extract, a bacterial lichenase (Bacillus sp.) and a non-specific endo-1,3(4)-β-glucanase from Clostridium thermocellum (EC 3.2.1.6). Standard curves were derived that allow the assay absorbance response to be directly converted to β-glucanase/lichenase activity on barley β-glucan. The specificity of MBG4 was confirmed by analysing the action of competing glycosyl hydrolases that are typically found in malt on the substrate. Manual and automated assay formats were developed for the analysis of a) β-glucanase in malt flour and b) lichenase enzyme extracts and the repeatability of these assays was fully investigated.

A commercially available enzymic method for the quantitative measurement of (1→3),(1→4)-β-glucan has been simplified to allow analysis of up to 10 grain samples in 70 min or of 100–200 samples by a single operator in a day. These improvements have been achieved with no loss in accuracy or precision and with an increase in reliability. The glucose oxidase/peroxidase reagent has been significantly improved to ensure colour stability for periods of up to 1 h after development. Some problems experienced with the original method have been addressed and resolved, and further experiments to demonstrate the quantitative nature of the assay have been designed and performed.

A method developed for the quantification of (1→3)(1→4)-β-D-glucan in barley flour has been modified to allow its use in the measurement of this component in malt, wort, beer and spent grain. For malt samples, free D-glucose was first removed with aqueous ethanol. Quantification of the polymer in wort and beer samples involved precipitation of the β-glucan with ammonium sulphate followed by washing with aqueous ethanol to remove free D-glucose. Spent grain was lyophilised and milled and then analysed by the method developed for malt. In all cases, the β-glucan was depolymerised with lichenase and the resultant β-gluco-oligosaccharides hydrolysed to D-glucose with β-D-glucosidase. The released D-glucose was then specifically determined using glucose oxidase-peroxidase reagent.

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in values are determined for each sample allowing the direct measurement of β-glucan in malt samples. α-Amylase does not interfere with the assay. The method is suitable for the routine analysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0-1 for barley flour samples containing 3-8 and 4-6% (w/w) β-glucan content.

Improving the redispersion and recycling of dried cellulose nanofibrils (CNFs) without compromising their nanoscopic dimensions and inherent mechanical properties are essential for their large-scale applications. Herein, mixed-linkage (1,3;1,4)-β-d-glucan (MLG) was studied as a rehydration medium for the redispersion and recycling of dried CNFs, benefiting from the intrinsic affinity of MLG to both cellulose and water molecules as inspired from plant cell wall. MLG from barley with a lower molar ratio of cellotriosyl to cellotetraosyl units was found homogeneously coated on CNFs, facilitating rehydration of the network of individualized CNFs. The addition of barley MLG did not impair the mechanical properties of the CNF/MLG composites as compared to neat CNFs nanopaper. With the addition of 10 wt% barley MLG, dry CNF/MLG composite film was successfully redispersed in water and recycled with well-maintained mechanical properties, while lichenan from Icelandic moss, cationic starch, and xyloglucan could not help the redispersion of dried CNFs.

Bacterial exopolysaccharides (EPS) are secreted biopolymers with often critical roles in bacterial physiology and ecology. In addition to their biological role, there is increasing interest for EPS in various industrial sectors. β-glucans are among the most important ones including cellulose as the most abundant organic polymer on earth, but also newcomers, such as the bacterial Mixed Linkage β-Glucan (MLG), displaying a unique repeating unit suggestive of biotechnological potential. In this work we describe Rhizobium etli as the first bacterium reported to be able to produce these two linear β-glucans cellulose and MLG. Rhizobium etli is an agronomic relevant rhizobacteria able to perform Biological Nitrogen Fixation (BNF) in a symbiotic association with common bean plants. The production and regulation of cellulose and MLG by Rhizobium etli CFN42 is discussed and their impact on its free-living and symbiotic lifestyles evaluated.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin “mining” conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

The fate of β-glucan (BG) health promoting properties during food production is crucial, but not predictable yet. Therefore, high molar mass BG (hBG) and control BG (cBG) were extracted from two barley varieties, characterized and added to wheat breads at levels of 3% and 6%. Bread quality criteria, carbohydrate contents and BG content and structural properties were determined. Additionally, breads were subjected to an in vitro digestion. The BG content in the chyme, molar mass, molar ratio, viscosity and bile acid retention were determined. The hBG and the cBG decreased loaf volume and increased crumb hardness with increasing BG content. The reduction in BG content during bread making was similar for hBG and cBG, but the molar mass of cBG decreased to a greater extent. As a result, only 10% of cBG entering in vitro digestion were found in the chyme afterwards, while 40% of the ingested hBG were detected. Molar mass reduction was much more severe for cBG compared to hBG. The use of hBG showed higher viscosity and better bile acid retention, indicating cholesterol lowering properties, compared to similar or higher amounts of cBG. These results provide valuable knowledge on the criteria to select BG-rich raw materials for ideal health promoting properties.

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBP_MLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBP_MLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBP_MLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBP_MLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. 

Novel anti-biofilm and dispersal agents are currently being investigated in an attempt to combat biofilm-associated wound infections. Glycoside hydrolases (GHs) are enzymes that hydrolyze the glycosidic bonds between sugars, such as those found within the exopolysaccharides of the biofilm matrix. Previous studies have shown that GHs can weaken the matrix, inducing bacterial dispersal, and improving antibiotic clearance. Yet, the number of GH enzymes that have been examined for potential therapeutic effects is limited. In this study, we screened sixteen GHs for their ability to disperse mono-microbial and polymicrobial biofilms grown in different environments. Six GHs, α-amylase (source: A. oryzae), alginate lyase (source: various algae), pectinase (source: Rhizopus sp.), amyloglucosidase (source: A. niger), inulinase (source: A. niger), and xylanase (source: A. oryzae), exhibited the highest dispersal efficacy in vitro. Two GHs, α-amylase (source: Bacillus sp.) and cellulase (source: A. niger), used in conjunction with meropenem demonstrated infection clearing ability in a mouse wound model. GHs were also effective in improving antibiotic clearance in diabetic mice. To examine their safety, we screened the GHs for toxicity in cell culture. Overall, there was an inverse relationship between enzyme exposure time and cellular toxicity, with twelve out of sixteen GHs demonstrating some level of toxicity in cell culture. However, only one GH exhibited harmful effects in mice. These results further support the ability of GHs to improve antibiotic clearance of biofilm-associated infections and help lay a foundation for establishing GHs as therapeutic agents for chronic wound infections.

White wheat salted noodles containing oats have a slower digestion rate those without oats, with potential health benefits. Oat β-glucan may play an important role in this. Effects of sheeting and shearing during noodle-making and subsequent cooking on β-glucan concentration, solubility, molecular size and starch digestibility were investigated. The levels of β-glucan were reduced by 16% after cooking, due to the loss of β-glucan into the cooking water. Both the noodle-making process and cooking increased the solubility of β-glucan but did not change its average molecular size. Digestion profiles show that β-glucan in wholemeal oat flour did not change starch digestion rates compared with isolated starch, but reduced the starch digestion rate of oat-fortified wheat noodles compared to the control (wheat noodles). Confocal laser scanning microscopy suggests that interaction between β-glucan and protein contributes to the starch-protein matrix and changes noodle microstructure, and thus alters their digestibility.

Water-insoluble β-glucan has been reported to have beneficial effects on human health. However, no studies have thoroughly characterized the structure and function of water-insoluble β-glucan in oat bran. Thus, the structure and effect of water-insoluble β-glucan on weight gain and lipid metabolism in high-fat diet (HFD)-fed mice were analyzed. First, water-insoluble β-glucan was isolated and purified from oat bran. Compared with water-soluble β-glucan, water-insoluble β-glucan had higher DP3:DP4 molar ratio (2.12 and 1.67, respectively) and molecular weight (123,800 and 119,200 g/mol, respectively). Notably, water-insoluble β-glucan exhibited more fibrous sheet-like structure and greater swelling power than water-soluble β-glucan. Animal experiments have shown that oral administration of water-insoluble β-glucan tended to lower the final body weight of obese mice after 10 weeks treatment. In addition, water-insoluble β-glucan administration significantly improved the serum lipid profile (triglyceride, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels) and epididymal adipocytes size. What is more, water-insoluble β-glucan reduced the accumulation and accelerated the decomposition of lipid in liver. In conclusion, water-insoluble β-glucan (oat bran) could alleviate obesity in HFD-fed mice by improving blood lipid level and accelerating the decomposition of lipid.

The prevalence of gastritis in humans is constantly growing and a prediction of an increase in this health problem is observed in many countries. For this reason, effective dietary therapies are sought that can alleviate the course of this disease. The objective of this study was to determine the effect of chemically pure oat beta-glucan preparations with different molar masses, low or high, used for 30 days in patients with histologically diagnosed chronic gastritis. The study enrolled 48 people of both genders of different ages recruited from 129 patients with a gastritis diagnosis. Before and after the therapy, hematological, biochemical, immunological and redox balance parameters were determined in the blood and the number of lactic acid bacteria and SCFA concentrations in the feces. Our results demonstrated a beneficial effect of oat beta-glucans with high molar mass in chronic gastritis in humans, resulting in reduced mucosal damage and healthy changes in SCFA fecal concentration and peripheral blood serum glutathione metabolism and antioxidant defense parameters. This fraction of a highly purified oat beta-glucan is safe for humans. Its action is effective after 30 days of use, which sheds new light on the nutritional treatment of chronic gastritis.

A modified TEMPO-catalyzed oxidation of the solvent-exposed glucosyl units of cellulose to uronic acids, followed by carboxyl reduction with NaBD₄ to 6-deutero- and 6,6-dideuteroglucosyl units, provided a robust method for determining relative proportions of disordered amorphous, ordered surface chains, and anhydrous core-crystalline residues of cellulose microfibrils inaccessible to TEMPO. Both glucosyl residues of cellobiose units, digested from amorphous chains of cellulose with a combination of cellulase and cellobiohydrolase, were deuterated, whereas those from anhydrous chains were undeuterated. By contrast, solvent-exposed and anhydrous residues alternate in surface chains, so only one of the two residues of cellobiosyl units was labeled. Although current estimates indicate that each cellulose microfibril comprises only 18 to 24 (1 → 4)-β-D-glucan chains, we show here that microfibrils of walls of Arabidopsis leaves and maize coleoptiles, and those of secondary wall cellulose of cotton fibers and poplar wood, bundle into much larger macrofibrils, with 67 to 86% of the glucan chains in the anhydrous domain. These results indicate extensive bundling of microfibrils into macrofibrils occurs during both primary and secondary wall formation. We discuss how, beyond lignin, the degree of bundling into macrofibrils contributes an additional recalcitrance factor to lignocellulosic biomass for enzymatic or chemical catalytic conversion to biofuel substrates.

Brown seaweed polysaccharides (BSPs) are one of the primary active components from brown seaweed that has a range of pharmaceutical and biomedical applications. However, the quality control of BSPs is a challenge due to their complicated structure and macromolecule. In this study, saccharide mapping based on high-performance liquid chromatography (HPLC), multi-angle laser light scattering, viscometer, and refractive index detector (HPSEC-MALLS-Vis-RID), and Fourier transform infrared (FT-IR) were used to discriminate the polysaccharides from nine different species of brown algae (BA1-9). The results showed that BSPs were composed of β-D-glucans and β-1,3−1,4-glucan linkages. The molecular weight, radius of gyration, and intrinsic viscosity of BSPs were ranging from 1.718 × 10⁵ Da to 6.630 × 10⁵ Da, 30.2 nm to 51.5 nm, and 360.99 mL/g to 865.52 mL/g, respectively. Moreover, α values of BSPs were in the range of 0.635 to 0.971, which indicated a rigid rod chain conformation. The antioxidant activities of BSPs exhibited substantial radical scavenging activities against DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radicals, which indicated that the use of BSPs might be a potential approach for antioxidant supplements. Thus, this study gives insights about the structure-function relationship of BSPs, which will be beneficial to improve the quality of polysaccharides derived from marine algae.

Feruloylated arabinoxylan (AX) is one of the most predominant dietary fiber in cereal grains. In recent decades, soluble AX has gained interest, as a result of its well-established health benefits. Apart from enzymatic degradation during cereal storage, food processing causes AX degradation. These reactions lead to structural modifications and influence both the AX functionalities and its health promoting effects. The aim of this study was to investigate the structural modifications and related property changes of health promoting water-extractable (WE) wheat bran AX through grain milling and extrusion. Multi-detector HPSEC revealed a correlation between M_w, conformational changes and the related viscosity behaviour depending on the processing type. Processing caused molecular degradation of insoluble high M_w AX, which increased the solubility significantly. Moreover, extrusion leaded to a more heterogenic AX fine structure. The detailed characterization of processed dietary fiber may help to facilitate the optimized incorporation of AX in health-promoting foods.

Dough gas cell stability is a prerequisite for obtaining breads with high specific volume and homogeneous crumb. The contribution of cereal endogenous non-starch polysaccharides (NSPs) to gas cell stability during wheat, rye, and oat bread making is still unclear. In this work, the aqueous phases from their fermented doughs were isolated as dough liquor (DL) by ultracentrifugation. The foaming, bulk shear rheology, and air-water (A-W) interfacial properties of wheat and rye DLs (treated with and without endoxylanase) and oat DL (treated with and without both lichenase and β-d-glucosidase) were studied. Enzymatic hydrolysis drastically reduced the apparent bulk shear viscosity of the different DLs and resulted in increased and decreased moduli (or magnitude) of the complex A-W interfacial shear viscosities of wheat and rye DLs, respectively. The latter implies that (non-hydrolyzed) rye DL arabinoxylan strengthens the A-W interfacial film consisting of adsorbed proteins and lipids. No measurable A-W interfacial shear viscosities were obtained for oat DL irrespective of whether its β-D-glucans were hydrolyzed or not. This is probably because lipids dominate the oat DL A-W interfaces. The knowledge generated provides a fundamental basis for specifically modifying the composition of the aqueous phase in wheat, rye, and oat doughs to improve the quality of mixed cereal breads.

Cereal beta-glucan can reduce post-prandial glycaemic responses, which makes it an interesting ingredient to improve the health impact of bread, a staple food with a high glycaemic index (GI). Here we compare the ability of different wheat-based breads prepared with oat bran concentrate and barley flour and a Norwegian type of soft wrap (lompe) for their ability to reduce glycaemic responses in healthy adults. Both breads with the highest beta-glucan content (3.8 g per serving) significantly reduced peak blood glucose rise (PBGR), incremental area under the blood glucose curve (iAUC) and GI compared to wheat control regardless of beta-glucan M_w and solubility. At a medium dose of 1.7 g per serving breads with beta-glucan of high MW and solubility significantly lowered iAUC, but not GI or PBGR compared to white bread. In contrast to previous studies, no significant correlation between viscosity after in vitro digestion and any of the glycaemia variables was found. However, the amount of soluble beta-glucan per serving was inversely correlated with GI. Lompe had a similar medium GI (63) than the high dose beta-glucan breads (56 and 64). However, while “lompe” had significantly lower amounts of rapidly digestible starch, no differences in in vitro starch digestion were found between the different breads. Instead, increased local viscosity at the intestinal border (e.g. soluble beta-glucan interacting with the mucus layer), dilution of nutrients (higher water content and serving size) and/or reduced gastric emptying are proposed as potential explanations for the lower glycaemic responses to high dose beta-glucan breads.

Enhanced bread quality by water-extractable arabinoxylan (WEAX) depends on its inherent structural features. To clarify the underlying mechanism, the current study prepared WEAX with varied structures via graded ethanol precipitation from wheat bran, and their effects on the steamed bread quality in relation to the heat-induced physicochemical changes of dough components were evaluated. The results showed that WEAX with a lower molecular weight (Mw), higher branched degree and ferulic acid content possessed a superior improved effect on the steamed bread quality. The gelatinization of starch was partially inhibited by WEAX, with a more distinct effect by the lower Mw and higher branched WEAX. However, WEAX suppressed the short-term retrogradation of starch, especially for the higher Mw and lower branched WEAX. Both of the heat-induced polymerization degree and rate of gluten were reduced by WEAX, and these reductions were more evident by the lower Mw and higher branched WEAX. The partial inhibition of gluten polymerization by WEAX contributed substantially to the enlarged loaf volume and softer textural property of steamed bread. The current study can provide a theoretical basis for the exploitation of WEAX as a nutritious and technofunctional dough improver.

Background and Aims: Aerenchyma develops in different plant organs and leads to the formation of intercellular spaces that can be used by the plant to transport volatile substances. Little is known about the role of cell walls in this process, although the mechanism of aerenchyma formation is known to involve programmed cell death and some cell wall modifications. We assessed the role that cell wall-related mechanisms might play in the formation of aerenchyma in sugarcane roots. Methods: Sections of roots (5 cm) were subjected to microtomography analysis. These roots were divided into 1-cm segments and subjected to cell wall fractionation. We performed analyses of monosaccharides, oligosaccharides and lignin and glycome profiling. Sections were visualized by immunofluorescence and immunogold labelling using selected monoclonal antibodies against polysaccharide epitopes according to the glycome profiles. Key Results: During aerenchyma formation, gas spaces occupied up to 40 % of the cortex cross-section within the first 5 cm of the root. As some of the cortex cells underwent dissolution of the middle lamellae, leading to cell separation, cell expansion took place along with cell death. Mixed-linkage β-glucan was degraded along with some homogalacturonan and galactan, culminating in the formation of cell wall composites made of xyloglucan, arabinoxylans, cellulose and possibly lignin. Conclusion: The composites formed seem to play a role in the physical–chemical properties of the gas chambers, providing mechanical resistance to forces acting upon the root and at the same time decreasing permeability to gases.