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Recently, we have observed a relationship between poor breadmaking quality and protease activities related to fungal infection. This study aims to identify potential gluten-degrading proteases secreted by fungi and to analyze effects of these proteases on rheological properties of dough and gluten. Fusarium graminearum-infected grain was used as a model system. Zymography showed that serine-type proteases secreted by F. graminearum degrade gluten proteins. Zymography followed by liquid chromatography-mass spectrometry (MS)/MS analysis predicted one serine carboxypeptidase and seven serine endo-peptidases to be candidate fungal proteases involved in gluten degradation. Effects of fungal proteases on the time-dependent rheological properties of dough and gluten were analyzed by small amplitude oscillatory shear rheology and large deformation extensional rheology. Our results indicate that fungal proteases degrade gluten proteins not only in the grain itself, but also during dough preparation and resting. Our study gives new insights into fungal proteases and their potential role in weakening of gluten.

Cereal beta-glucans may reduce fasting LDL-cholesterol and post-prandial blood glucose rise in humans. Several studies have shown that physicochemical properties of beta-glucan such as molecular weight (M) and solubility/extractability as well as the viscosity of food products after in vitro digestion are important for this physiological effect. In this study, we directly compare beta-glucan physicochemical properties and flow behavior of different cereal products after analytical (thermostable alpha-amylase) and physiological (in vitro digestion) extractions. The beta-glucan weight average molecular weights (Mw) were similar for both types of extractions, while differences in beta-glucan solubility between the food products were only evident after in vitro digestion. The extracts after in vitro digestion behaved essentially as pure beta-glucan solutions. Viscosity dropped to 0.8–1.2 mPa after incubation with a beta-glucanase for all extracts, but was not influenced by xylanase addition. The relationship of beta-glucan Mw (or intrinsic viscosity calculated from Mw), concentration and zero shear viscosity measured in the extracts closely resembled the general relationship reported for “random coil” polysaccharides with two distinct regions, below and above the occurrence of coil overlap. A standardized presentation of previously published in vivo data on blood glucose response showed a distinct grouping of samples with significant effect in the range of coil overlap. This points towards an important role of coil overlap for the reduction of post-prandial glycemic response by beta-glucan rich foods and demonstrates the potential of such a general comparison of different studies to increase the understanding of beta-glucan rich food products in vivo effects.

A comprehensive characterization of polysaccharides from the chanterelle was performed. Experiments included both linkage analysis by methylation and GC–MS, monosaccharide composition analysis by methanolysis, SEC-MALLS and several NMR experiments including COSY, HSQC, TOCSY, HSQC-TOCSY, NOESY, and HMBC. A 671 kDa (1 → 6)-linked α- d -mannan with single and short (1 → 2)-linked side chains (WCcF1b) was isolated from the hot water extract, after protease treatment and fractionation by size exclusion chromatography. The hot 1 M NaOH extract contained two types of β-glucan; a water soluble (1 → 6)-linked β- d -glucan with single and short (1 → 3)-linked β-glucan side chains (ACcSw) and a less branched (1 → 3)-linked β- d -glucan (ACcIw/ACcId), proposed to contain short side chains in O -4. Chemical shifts assignments of the α-mannan and β-glucan are presented as well as chemical shift values obtained from a (1 → 6)/(1 → 3)-β- d -glucan derived from Saccharomyces cerevisiae which was used as reference.

The correct identification of immune-modulatory activity of polysaccharides is often hampered by immune-stimulatory contaminants, with pyrogens such as lipopolysaccharide (LPS) as a very potent example. In order to avoid false positive immuno-stimulatory properties to be attributed to polysaccharides, accurate quantification and inactivation of LPS in test samples is crucial. To quantify LPS in polysaccharide preparations of different origin and structure we used two different limulus amoebocyte lysate test kits in two different laboratories. We observed larger variation in detection of LPS contamination between kits than between labs. LPS quantification proved unreliable for some polysaccharide preparations as spike controls resulted in spike recoveries outside the acceptable range. We designed a cellular in vitro assay as alternative method to detect the presence of functional LPS. This HEK-Blue hTLR4 cell culture provides a reliable assay, when combined with a cell viability test, for determining functional LPS in polysaccharide preparations. Finally, to inactivate LPS in polysaccharide preparations, we setup an alkaline-ethanol-based treatment. With this assay we observed that our treatment (5h incubation in 0.1M NaOH) at 56°C efficiently inactivated LPS in all polysaccharide preparations below immune-stimulatory levels. At this elevated temperature, however, we also observed minimal to severe degradation of polysaccharide preparations as determined with SEC-RI. Taken together, we describe methods and precautions to reliably detect and inactivate LPS in polysaccharide preparations to allow reliable in vitro investigations towards immune-modulatory potential of polysaccharide preparations.

During the production of composite wheat/barley breads β-glucans are rapidly degraded by endogenous wheat and barley flour β-glucanases. Since the physiological effect of β-glucans is related to their molecular weight a protection from degradation is essential in order to retain their full effect on the mitigation of serum cholesterol and blood glucose levels. In this study we have shortened the contact time between barley flour and water while focusing on the production of good quality barley breads. Incorporation of barley flour into already fermented wheat dough or omitting fermentation and shortening proofing times by more yeast and sucrose addition enabled the production of barley bread with increased β-glucan calcofluor weight average molecular weight (Mwcalc) and good bread quality (crumb fineness, bread slice area). The use of barley components with bigger particle size like coarse barley flour or intact barley flakes further increased β-glucan molecular weight in bread. Inactivation (with scalding) or reduction (dry heat treatment) of β-glucanase activity in barley flour prior to use, on the other hand, had little effect on β-glucan molecular weight reduction during one of the optimized short bread making processes.

Scleroglucan, a neutral β(1–3) glucan with β(1–6) glucan branches every third residue, is being considered as an alternative rod-like, shear thinning high molecular weight β-glucan based polysaccharide to xanthan gum for the management of patients with oropharyngeal dysphagia. It is therefore important to understand more fully its hydrodynamic properties in solution, in particular heterogeneity, molecular weight distribution and its behaviour in the presence of mucin glycoproteins. A commercially purified scleroglucan preparation produced by fermentation of the filamentous fungus Sclerotium rolfsii was analysed in deionised distilled water with 0.02% added azide. Sedimentation velocity in the analytical ultracentrifuge showed the scleroglucan preparation to be unimodal at concentrations >0.75 mg/ml which resolved into two components at lower concentration and with partial reversibility between the components. Sedimentation coefficient versus concentration plots showed significant hydrodynamic non-ideality. Self-association behaviour was confirmed by sedimentation equilibrium experiments with molecular weights between ~3 × 106 g/mol to ~5 × 106 g/mol after correcting for thermodynamic non-ideality. SEC-MALS-viscosity experiments showed a transition between a rod-shape at lower molar masses to a more flexible structure at higher masses consistent with previous observations. Sedimentation velocity experiments also showed no evidence for potentially problematic interactions with submaxillary mucin., Graphical abstract Image 1, Highlights • Scleroglucan considered as alternative to xanthan. • Sedimentation velocity shows partial reversible self-association. • Associative behaviour confirmed by sedimentation equilibrium. • SEC-MALS and viscosity show transition between a rod to more flexible structure. • No problematic interactions with submaxillary mucin.

Depletion flocculation is a well-known instability mechanism that can occur in oil-in-water emulsions when the concentration of non-adsorbed polysaccharide exceeds a certain level. This critical flocculation concentration depends on the molecular characteristics of the polysaccharide molecules, such as their molecular weight and hydrodynamic radius. In this study, a range of analytical methods (dynamic shear rheology, optical microscopy, and static light-scattering) were used to investigate the interaction between lipid droplets and polysaccharides (guar gum and β-glucans) of varying weight-average molecular weight and hydrodynamic radius, and concentration. The aim of this work was to see if the health benefits of soluble fibers like β-glucans could be explained by their influence on the structure and digestibility of lipid emulsions. The apparent viscosity of the emulsions increased with increasing polysaccharide concentration, molecular weight, and hydrodynamic radius. Droplet flocculation was observed in the emulsions only at certain polysaccharide concentrations, which was attributed to a depletion effect. In addition, the water-soluble components in oat flakes, flour, and bran were extracted using aqueous solutions, to examine their impact on emulsion stability and properties. Then, the rate and extent of lipolysis of a sunflower oil-in-water emulsion in the presence of these oat extracts were monitored using the pH-stat method. However, the inhibition of lipolysis was not linearly related to the viscosity of the oat solutions. The water-soluble extracts of β-glucan collected from oat flakes had a significant inhibitory effect on lipolysis. The results of this study increase our understanding of the possible mechanisms influencing the impact of oat constituents on lipid digestion. This work also highlights the importance of considering the molecular properties of polysaccharides, and not just their impact on solution viscosity., Graphical abstract The effect of oat and β-glucan on lipid digestion relies on complex mechanisms.Image 1, Highlights • Smaller β-glucans induce depletion flocculation over wider concentration range. • β-glucan molecules occupied the bulk solution around the droplets and drove them together. • Viscosity is not the only factor affecting depletion flocculation. • Compounds released from oat flakes caused the greatest depletion flocculation. • Lipolysis was reduced in the presence of oat flake extract solutions.

International audience; Oat b-glucan has been shown to play a positive role in influencing lipid and cholesterol metabolism. However, the mechanisms behind these beneficial effects are not fully understood. The purpose of the current work was to investigate some of the possible mechanisms behind the cholesterol lowering effect of oat b-glucan, and how processing of oat modulates lipolysis. b-Glucan release, and the rate and extent of lipolysis measured in the presence of different sources of oat b-glucan, were investigated during gastrointestinal digestion. Only a fraction of the original b-glucan content was released during digestion. Oat flakes and flour appeared to have a more significant effect on lipolysis than purified b-glucan. These findings show that the positive action of b-glucan is likely to involve complex processes and interactions with the food matrix. This work also highlights the importance of considering the structure and physicochemical properties of foods, and not just the nutrient content.

The localisation of pectin in Sphagnum moss leaves and its role in preservation has been investigated. Light microscopy using ruthenium red to detect pectin in whole and sectioned Sphagnum papillosum leaves revealed it is abundant in hyaline cell walls, fibrils, papillae, chlorophyllous cell walls and thickenings around hyaline cell pores. Transmission electron microscopy of ultrathin cell walls labelled with poly- l -lysine colloidal gold revealed pectin was distributed throughout the cell wall. The preservative/microbiocidal properties of these pectins are explained by the acid-dissociation properties of galacturonic acid carboxyls and their incorporation in the unique cell arrangement of the Sphagnum leaf. Liquid from a salmon fillet absorbed into S. papillosum leaves and incubated at room temperature for 22 h had a pH around 4.85, was dominated by Lactobacillus sp. and smelled fresh compared to experimental controls. Chlorite-treated Sphagnum leaves could have a potential as a food tray pad that absorbs liquid and prevents the growth of spoilage bacteria inside it.

The carbonyl content of a pectic polysaccharide from Sphagnum papillosum (sphagnan) and periodate oxidised alginates was investigated using three different carbonyl labelling strategies combined with size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and on-line fluorescence or off-line tritium detection. The labelling strategies were tritium incorporation via NaB3H4 reduction, and fluorescent labelling with carbazole carbonyl oxyamine (CCOA), or 2-aminobenzamide (2-AB), respectively. Carbonyl quantification was based on labelled pullulan, dextran and alginate standards possessing only the reducing end carbonyl group. As a result the carbonyl distribution in the polysaccharides could be determined. In sphagnan it was found that the carbonyl content increased with increasing molecular weight, whereas in periodate oxidised alginate the carbonyl content was as expected independent of the molecular weight. The methods proved useful for carbonyl detection in water soluble polysaccharides in general. The tritium incorporation method was preferred for alkali stable polysaccharides, while the CCOA method was most suitable for acid stable polysaccharides with low carbonyl content. The 2-AB method is applicable for all polysaccharides tested with varying carbonyl content; however, it lacks the ability to detect ketone functionalities.

Mild acid hydrolysis was used to release polysaccharides from the chlorite-treated leaves of Sphagnum papillosum . These polysaccharides, collectively known as sphagnan, were physically and chemically characterised. No evidence was found of the presence of a previously described 5-keto- d -mannuronic acid (5-KMA) monomer in sphagnan, or in the chlorite-treated leaves from which sphagnan was extracted. After fractionation by anion-exchange chromatography the majority of polysaccharides were similar to rhamnogalacturonan I-type pectins. They were highly branched and had a weight average molecular weight of 3.9 × 10 g/mol.

Even though size exclusion chromatography (SEC) with post column addition of calcofluor (SEC-calcofluor) has been used for the determination of cereal β-glucan molar mass in foods for many years, there is a lack of systematic evaluation of the method. To address this issue a set of suitable β-glucan standards were generated by preparative SEC and their molar mass characteristics were determined by analytical multi-detection SEC (refractive index (RI), light scattering). Each standard was then analysed by SEC-calcofluor at three different labs. As a direct comparison, the analyses were repeated with a RI detector. For SEC-calcofluor accurate measurements of weight average molar mass (Mw) can be made for β-glucan populations within 10-500×10(3)g/mol. Above this molar mass threshold there is an increasing tendency for underestimation of Mw. Precipitation of some β-glucan-calcofluor complexes may have delayed their transport into the detector.Copyright © 2015 Elsevier Ltd. All rights reserved. Inter-laboratory evaluation of SEC-post-column calcofluor for determination of the weight-average molar mass of cereal β-glucan | Request PDF. Available from: https://www.researchgate.net/publication/273507433_Inter-laboratory_evaluation_of_SEC-post-column_calcofluor_for_determination_of_the_weight-average_molar_mass_of_cereal_b-glucan [accessed Sep 18 2018].

High molecular weight (MW) is a key parameter for cereal β-glucans physiological benefits like decreased serum cholesterol and attenuated post prandial blood glucose. However, the activity of endogenous flour enzymes during bread production results in a decrease of β-glucan MW. The depolymerization of a standard β-glucan solution by different flour extracts (wheat, barley and rye) was followed by measuring the viscosity decrease with a Rheometer. The slope of the inverse viscosity (1/η) against degradation time was used to quantify β-glucanase activity by comparison with slopes obtained with known concentrations of the β-glucanase Lichenase. Results correlated well with depolymerization rates estimated by HPSEC. The viscosity based method is rapid (20 min per sample), accurate (≤6% variation), and a powerful screening tool for identifying flour fractions with low β-glucanase activity, treatments that can inactivate β-glucanases in flour, or the development of β-glucanase inhibitors for the use in e.g. bread making.

A chromatographic method using HPAEC-PAD was developed to accurately quantify the major oligosaccharides derived from lichenase degradation of barley β-glucan. This method was further used to follow β-glucosidase degradation and product formation as progress curves. This approach allowed us to compare the kinetic characteristics of β-glucosidase on each exclusive oligosaccharide substrate and their mixtures. Our results show that when determining the kinetic parameters of exohydrolases on oligosaccharides following the progress curve for the substrates is necessary since calculations based only on released monosaccharide products may lead to an error. The catalytic activity of almond β-glucosidase on laminaribiose (G3G) was approximately half that measured against cellobiose (3.15 S(-1)). The enzyme had 12 times and 560 times less catalytic activity on 3-O-β-cellobiosyl-D-glucose (G4G3G) and on 3-O-β-cellotriosyl-d-glucose (G4G4G3G) respectively than on G3G. Our approach offers a useful tool for the determination of the kinetics of enzymatic or chemical modification of various carbohydrate substrates.

The specific binding of the fluorescent dye calcofluor to cereal β-glucan results in increased fluorescence intensity of the formed complex and is in use for the quantification of β-glucan above a critical molecular weight (MW) by flow injection analysis. In this study, this method was applied in a fast and easy batch mode. In order to emphasize the spectral information of the emission spectra of the calcofluor/β-glucan complexes, derivative signals were calculated. A linear relationship was found between the amplitude of the second derivative signals and the β-glucan concentration between 0.1 and 0.4 μg/mL. The low detection limit of this new method (0.045 μg/mL) enabled its use to study the transport of cereal β-glucans over differentiated Caco-2 cell monolayers. Additionally, the method was applied to quantify β-glucan in arabinoxylan samples, which correlated well with data by an enzyme based method.

Background Biofilm has been shown to be one way for Salmonella to persist in the feed factory environment. Matrix components, such as fimbriae and cellulose, have been suggested to play an important role in the survival of Salmonella in the environment. Multicellular behaviour by Salmonella is often categorized according to colony morphology into rdar (red, dry and rough) expressing curli fimbriae and cellulose, bdar (brown, dry and rough) expressing curli fimbriae and pdar (pink, dry and rough) expressing cellulose. The aim of the study was to look into the distribution of morphotypes among feed and fish meal factory strains of Salmonella, with emphasis on potential differences between morphotypes with regards to survival in the feed factory environment. Results When screening a total of 148 Salmonella ser. Agona, Salmonella ser. Montevideo, Salmonella ser. Senftenberg and Salmonella ser. Typhimurium strains of feed factory, human clinical and reference collection origin, as many as 99% were able to express rough morphology (rdar or bdar). The dominant morphotype was rdar (74%), however as many as 55% of Salmonella ser. Agona and 19% of Salmonella ser. Senftenberg displayed the bdar morphology. Inconsistency in Calcofluor binding, indicating expression of cellulose, was found among 25% of all the strains tested, however Salmonella ser. Agona showed to be highly consistent in Calcofluor binding (98%). In biofilm, Salmonella ser. Agona strains with bdar mophology was found to be equally tolerant to disinfection treatment as strains with rdar morphotype. However, rdar morphology appeared to be favourable in long term survival in biofilm in a very dry environment. Chemical analysis showed no major differences in polysaccharide content between bdar and rdar strains. Our results indicate that cellulose is not a major component of the Salmonella biofilm matrix. Conclusion The bdar morphotype is common among Salmonella ser. Agona strains isolated from the factory environment. The rdar and the bdar strains were found to be equally tolerant to disinfectants, while the rdar strain was found to be more tolerant to long-term desiccation and nutrient depletion in biofilm than the bdar strain. Cellulose does not appear to be a major component of the Salmonella biofilm matrix.

Alginates comprised of essentially alternating units of mannuronic (M) acid-guluronic (G) acid (MG-alginate), and G-blocks isolated from a seaweed where subjected to partial acid hydrolysis at pH 3.5 The chain-length distribution of oligosaccharides in the hydrolysate were investigated by statistical analysis after their separation with high-performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). Simulated depolymerisation of the MG-alginate provided an estimate of the ratio between two acid hydrolysis rate constants (p=8.3+/-1) and the average distribution of the MM linkages in the original sample of polysaccharide chains. In conclusion, we found HPAEC-PAD together with statistical analysis was a useful method to investigate the fine structure and some properties of binary polysaccharides.