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IntroductionIn 2012, the estimated global prevalence of pre-diabetes was 280 million, and the prevalence is expected to rise to 400 million by 2030. Oat-based foods are a good source of beta-glucans, which have been shown to lower postprandial blood glucose. Studies to evaluate the effectiveness of the long-term intake of beta-glucan-enriched bread as part of a habitual diet among individuals with pre-diabetes are needed. Therefore, we designed a multicentre intervention study in adults with pre-diabetes to investigate the effects of consumption of an oat-derived beta-glucan-enriched bread as part of a normal diet on glycated haemoglobin (HbA1c) in comparison to consumption of whole-grain wheat bread.Methods and analysisThe CarbHealth trial is a multicentre double-blind randomised controlled 16-week dietary intervention trial in participants 40–70 years of age with a body mass index of ≥27 kg/m2 and HbA1c of 35–50 mmol/mol. The study is conducted at four universities located in Norway, Sweden and Germany and uses intervention breads specifically designed for the trial by Nofima AS. The aim is to recruit 250 participants. The primary outcome is the difference in HbA1c between the intervention and the control groups. The main analysis will include intervention group, study centre and baseline HbA1c as independent variables in an analysis of covariance model.Ethics and disseminationThe study protocol was approved by respective ethical authorities in participating countries. The results of the study will be communicated through publication in international scientific journals and presentations at (inter)national conferences.Trial registration numberNCT04994327.

Intake of soluble fibers including beta-glucan, is known to improve post-prandial glycemic response. The mechanisms have been attributed to the viscous gel forming in the stomach and small intestine, giving a longer absorption time. However, recent evidence suggests a link between intake of beta-glucan and improved glycemic regulation at subsequent meals through the gut microbiota. We investigated the short-term effect of granola with different amounts of cereal beta-glucan on glycemic response and gut microbiota. After a two-week run-in period (baseline), fourteen healthy, normal weight adults completed a dose-response dietary crossover study. Different amounts of cereal beta-glucan (low: 0.8 g, medium: 3.2 g and high: 6.6 g) were provided in granola and eaten with 200 ml low-fat milk as an evening meal for three consecutive days. Blood glucose and insulin were measured fasted and after an oral glucose tolerance test (OGTT) the following day, in addition to peptide YY (PYY) and glucagon-like peptide (GLP-2), fasting short chain fatty acids (SCFA) in blood, breath H2, and gut microbiota in feces. Only the intervention with medium amounts of beta-glucan decreased blood glucose and insulin during OGTT compared to baseline. Fasting PYY increased with both medium and high beta-glucan meal compared to the low beta-glucan meal. The microbiota and SCFAs changed after all three interventions compared to baseline, where acetate and butyrate increased, while propionate was unchanged. Highest positive effect size after intake of beta-glucan was found with Haemophilus, followed by Veillonella and Sutterella. Furthermore, we found several correlations between different bacterial taxa and markers of glycemic response. In summary, intake of granola containing 3.2 g cereal beta-glucan as an evening meal for three consecutive days reduced the glycemic response after an OGTT 0-180 min and changed gut microbiota composition. Since we cannot rule out that other fiber types have contributed to the effect, more studies are needed to further explore the effect of cereal beta-glucan on glycemic regulation.Clinical Trial Registration[www.clinicaltrials.gov], identifier [NCT03293693].

Cell wall disrupted and dried Microchloropsis gaditana (Mg), Tetraselmis chui (Tc) and Chlorella vulgaris (Cv) microalgae biomasses, with or without ethanol pre-treatment, were added to wheat bread at a wheat flour substitution level of 12%, to enrich bread protein by 30%. Baking performance, protein quality and basic sensory properties were assessed. Compared to wheat, Mg, Tc and Cv contain higher amounts of essential amino acids and their incorporation markedly improved protein quality in the bread (DIAAS 57–66 vs. 46%). The incorporation of microalgae reduced dough strength and bread volume and increased crumb firmness. This was most pronounced for Cv and Tc but could be improved by ethanol treatment. Mg gave adequate dough strength, bread volume and crumb structure without ethanol treatment. To obtain bread of acceptable smell, appearance, and colour, ethanol treatment was necessary also for Mg as it markedly reduced the unpleasant smell and intense colour of all algae breads. Ethanol treatment reduced the relative content of lysine, but no other essential amino acids. However, it also had a negative impact on in vitro protein digestibility. Our results show that Mg had the largest potential for protein fortification of bread, but further work is needed to optimize pre-processing and assess consumer acceptance.

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.

Agar-based extracts from Gelidium sesquipedale were obtained by applying a conventional hot water treatment and alternative ultrasound- and microwave-assisted methods, with and without the application of an alkaline pre-treatment. The alkaline pre-treatment produced refined extracts with higher purity; however, extraction yields increased from 2–5% to 7–19% by omitting this step. In particular, the ultrasound-assisted extraction allowed reducing 4-fold the extraction time, while keeping constant or even increasing the yield (up to 19% for the 1 h extraction) with respect to the conventional protocol. Interestingly, the presence of proteins and polyphenols conferred the semi-refined extracts a relatively high antioxidant capacity (19–24 μmol TE/g extract). The refined extract produced by the standard protocol formed the strongest hydrogels (>1000 g/cm2). On the other hand, the semi-refined extracts produced by the alternative protocols formed slightly stronger hydrogels (337–438 g/cm2) than the refined counterparts (224–311 g/cm2), due to their greater molecular weights of the former ones. LCA assessment showed lower global warming potential for the semi-refined extracts, especially the ultrasound-assisted extraction, hence highlighting the potential of this method to produce more sustainable agar-based extracts for food-related applications., This work was financially supported by the “Agencia Estatal de Investigación” (PCI2018-092886 Grant) and co-funded by the European Union's Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2).

While the harmonized INFOGEST model provides a physiologically relevant platform for simulated digestion, it needs to be combined with adequate analytical methods to enable quantification and comparison of protein digestibility in different food matrices. We have shown that size exclusion chromatography (SEC) can be used to estimate the proportion of small peptides potentially available for uptake. Combined with determination of total dissolved protein, the % of small peptides per total protein was calculated as a physiologically relevant estimate of protein digestibility (DSEC). Values for DSEC differed for casein (87.6%), chicken mince (72.6%), heated pea protein concentrate (67.8%), bread (63%), beef entrecote (57.7%) and pea protein concentrate (57.8%). In contrast to existing methods (TCA soluble protein, free NH2-groups), the proposed SEC based method gives separate insight into the two fundamental processes during protein digestion (solubilization and break-down), while maintaining the ability to rank digestibility of very different food proteins.

The sensitivity of microorganisms to low pH can be utilized in food protection by preparing coatings based on macromolecular acids. Due to limited diffusivity of macromolecules low pH occurs primarily at the surface, while the interior parts of the food remain unaffected. This principle is demonstrated using food approved alginic acid in various types of coatings (aqueous, emulsions, dispersions, dry coating) on a wide range of foods including meat, fish, chicken, shrimp and boiled rice. Significant delay or inhibition of the natural flora is generally demonstrated, particularly when exposed to ‘temperature abuse’. Specifically, we show that the coatings reduce or inhibit regrowth of pathogens (Bacillus cereus, B. weihenstephanensis, Listeria monocytogenes serotype 1 and Staphylococcus aureus). In special cases like boiled rice, alginic acid may largely replace acetic acid for acidification and preservation, as demonstrated studying regrowth of added spores of B. cereus. Most formulations allow easy removal prior to further processing (cooking, frying). Temporary side effects such as ‘acid cooking’ obtained for high acid concentrations on sensitive surfaces (e.g. salmon) disappear during processing, recovering the normal taste and texture. The coating is hence suitable for a large variety of foods. © 2018. This is the authors’ accepted and refereed manuscript to the article. Locked until 16.8.2019 due to copyright restrictions. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

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.

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.

Agar-based extracts from Gelidium sesquipedale were generated by heat and combined heat-sonication, with and without the application of alkali pre-treatment. Pre-treatment yielded extracts with greater agar contents; however, it produced partial degradation of the agar, reducing its molecular weight. Sonication produced extracts with lower agar contents and decreased molecular weights. A gelation mechanism is proposed based on the rheological and small angle scattering characterization of the extracts. The formation of strong hydrogels upon cooling was caused by the association of agarose chains into double helices and bundles, the sizes of which depended on the agar purity and molecular weight. These different arrangements at the molecular scale consequently affected the mechanical performance of the obtained hydrogels. Heating of the hydrogels produced a gradual disruption of the bundles; weaker or smaller bundles were formed upon subsequent cooling, suggesting that the process was not completely reversible., Synchrotron experiments were performed at NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff (2018022638 project). This work was financially supported by the “Agencia Estatal de Investigación” (PCI2018-092886 Grant) and co-funded by the European Union’s Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2). Part of this work was supported by the COST Action ES1408 European network for algal-bioproducts (EUALGAE).

Marine green microalgae are a sustainable source of high-quality protein. However, due to their green pigmentation and composition of volatiles, their incorporation into food products is a challenge. Incorporation of cell-wall disrupted Tetraselmis chuii (Tc) into bread (0, 4, 8, 12 and 16% wheat flour substitution) affected dough rheology and bread quality negatively. These effects were more pronounced at addition levels necessary for a EFSA nutrition claim on protein enrichment (12 and 16%). Treatment of Tc with ethanol not only removed much of the green pigmentation and volatiles, but also reduced the negative impact on dough rheology and bread quality. Doughs prepared with ethanol treated Tc (TcEt) showed a clear improvement in dough rheology evident as increased dough-stability-time (DST), resistance to extension (Rmax) and elastic-recovery-compliance (Je) particularly at substitution levels >4%. This was accompanied by an increase in bread quality e.g. at 12% substitution level specific volume increased from 2.1 to 2.69 mL/g, crumb firmness decreased from 1358 to 297 g and slice brightness increased from 25.2 to 49.0. Ethanol treatment of algae may be a feasible strategy to address the sensory and structural challenges that hinder incorporation of algae into foods at levels that can potentially confer nutritional benefits.

To examine the role of realistic serving sizes of broccoli, broccoli fibre and cellulose co-consumed with mash potato, or mashed potato eaten alone, on glycaemic and insulinaemic responses (GR and IR) in healthy adults. A non-blind randomized crossover trial was conducted with thirteen healthy subjects consuming four different meals. Capillary blood samples between 0 and 180 min were analysed for glucose and insulin. The incremental area under the fasting blood glucose and insulin curves (iAUC) was calculated for different time increments. Differences in GR and IR between meals were assessed by repeated measures analysis of variance. The immediate GR and IR to one serving of mashed potato eaten with two servings of broccoli were significantly lower than mashed potato eaten alone. The peak, incremental peak and iAUC0–30min for GR and iAUC0–30min for IR were all significantly lower for the broccoli–potato meal. This meal also takes longer to return to fasting baseline with a time-delayed lag in IR and GR compared to the potato only meal. The iAUC60–120min for IR was significantly greater for the broccoli–potato meal compared to the other meals. Yet there was no corresponding significant difference between the broccoli–potato meal and the other meals for peak, incremental peak IR or any other iAUCs for GR and IR. For the potato meals containing added broccoli fibre or cellulose, no significant differences in GR or IR were observed when compared with the potato eaten alone. Co-consumption of cooked broccoli with mashed potato has a significant effect on glycaemic and insulinaemic responses compared to potato eaten alone. Our study suggests broccoli eaten with potato improves glucose homeostasis and therefore indicates a general beneficial nutritional role for broccoli when eaten with a carbohydrate staple.

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.

In this study, the use of two modern portable blood glucose monitors (PBGMs) and a fruit juice-specific system (RQFlex) from Merck was evaluated for determination of glucose concentrations in potato juice, as a representation of the whole potato. The two PBGMs (LifeScan OneTouch Ultra Easy and Bayer Contour) both provided squared correlation coefficients (R 2, p

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.

Summary We have studied mixing, rheology and stability of potato-cereal flour doughs. These were prepared using samples of two varieties of cooked and cold-stored potatoes. Dependent on variety a minimum duration of 24–48 h cold-storage was required. Dough mixing torque plateau was inversely related to potato water content. This should be 2 Nm at a specific total mechanical energy input of 18–22 kJ kg−1. The resting temperature of the dough was also inversely related to its strength (plateau elastic modulus at time zero). Target dough strength should be about 25 kPa in a bob-cup. Above 15 °C it decreased as a function of time and within 1 h it became too sticky for commercial processing. At 4 °C the dough remained stable. Sufficient potato starch retrogradation is a pre-requisite to make optimal potato-cereal flour dough for commercial processing.

Yeast-derived β-glucans (Y-BG) are potential immune-modulating compounds able to activate macrophages. We investigated whether Y-BG affects the activity of a proteolytic enzyme, legumain, which is expressed in macrophages and involved in inflammatory conditions as well as in cancer development. RAW 264.7 macrophages were stimulated with either particulate (BG-P), water soluble (BG-S) or partly water soluble (BG-PS) Y-BG for 48 h, and cellular legumain activity was determined using a fluorogenic peptide substrate. BG-P inhibited legumain activity in a dose-dependent manner, while BG-S and BG-PS did not. Immunoblotting indicated that the observed inhibition of legumain activity was caused by reduced prolegumain synthesis within the cells, rather than inhibition of legumain proform activation. Blocking of the C-type lectin dectin-1 receptor with antibodies abolished the effect, revealing that this receptor was involved, and it was further demonstrated by confocal microscopy that fluorescently labeled BG-P was internalized into macrophages. RAW 264.7 macrophages stimulated with Y-BG for 6 h were shown to secrete TNF-α, and BG-P was more potent than BG-PS and BG-S in this respect. Y-BG is shown to inhibit legumain activity after internalization into macrophages, providing new insight into the mode of action of β-glucans.

Graphical abstract The effect of oat β-glucan on lipid digestion relies on the delivery form of the polymer., Highlights • The structure of oat tissue is an important factor for determining its influence on (in vitro) lipid digestion. • β-glucan release from oat cell walls during digestion was not complete. • Processing of oats affects the rate and extent of lipolysis. • Viscosity is not the only factor affecting lipolysis., Oat β-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 β-glucan, and how processing of oat modulates lipolysis. β-Glucan release, and the rate and extent of lipolysis measured in the presence of different sources of oat β-glucan, were investigated during gastrointestinal digestion. Only a fraction of the original β-glucan content was released during digestion. Oat flakes and flour appeared to have a more significant effect on lipolysis than purified β-glucan. These findings show that the positive action of β-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.

Sphagnum-dominated peatlands head the list of ecosystems with the largest known reservoirs of organic carbon (C). The bulk of this C is stored in decomposition-resistant litter of one bryophyte genus: Sphagnum. Understanding how Sphagnum litter chemistry controls C mineralization is essential for understanding potential interactions between environmental changes and C mineralization in peatlands. We aimed to separate the effects of phenolics from structural polysaccharides on decay of Sphagnum. We measured aerobic microbial respiration of different moss litter types in a lab. We used chemical treatments to step-wise remove the chemical compounds thought to be important in decay-resistance in three taxonomically distant moss genera. We also focused on the effect of Sphagnum-specific cell-wall pectin-like polysaccharides (sphagnan) on C and N mineralization. Removing polymeric lignin-like phenolics had only negligible effects on C mineralization of Sphagnum litter, but increased mineralization of two other bryophyte genera, suggesting a minor role of these phenolics in decay resistance of Sphagnum but a major role of cell-wall polysaccharides. Carboxyl groups of pectin-like polysaccharides represented a C-source in non-Sphagnum litters but resisted decay in Sphagnum. Finally, isolated sphagnan did not serve as C-source but inhibited C and N mineralization instead, reminiscent of the effects reported for phenolics in other ecosystems. Our results emphasize the role of polysaccharides in resistance to, and active inhibition of, microbial mineralization in Sphagnum-dominated litter. As the polysaccharides displayed decay-inhibiting properties hitherto associated with phenolics (lignin, polyphenols), it raises the question if polysaccharide-dominated litter also shares similar environmental controls on decomposition, such as temperature or nutrient and water availability.

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.

The purpose of this research was to evaluate if pectin-like polysaccharides, collectively known as sphagnan, extracted by acid hydrolysis from the leaves of Sphagnum moss have a unique ability to react with phenylhydrazine, o-phenylenediamine or collagen. A previous assay for determination of carbonyl groups in sphagnan by reaction with phenylhydrazine was disproved due to spectrophotometric interference from furans. The actual carbonyl content of sphagnan is estimated to be much less than previously thought. NMR spectroscopy showed that small amounts of o-phenylenediamine and/or its oxidation products bind to sphagnan probably via imine formation, but evidence of quinoxaline formation was inconclusive. Sphagnan-gelatin mixtures formed complex coacervates at pH 2.0-4.8 at low ionic strength, which is typical of electrostatic polyelectrolyte interactions, rather than covalent carbonyl-amine reactions. Measurements of hydrothermal stability and collagenase-degradation of sphagnan-treated hide powder collagen suggest that sphagnan is a poor tanning agent. The results indicate the suggested preservative properties of sphagnan are not related to tanning.

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.

Prediction of dry matter content in whole potatoes is a desired capability in the processing industry. Accurate prediction of dry matter content may greatly reduce waste quantities and improve utilization of the raw material through sorting, hence also reducing the processing cost. The following study demonstrates the use of a low resolution, high speed NIR interactance instrument combined with partial least square regression for prediction of dry matter content in whole unpeeled potatoes. Three different measuring configurations were investigated: (1) off-line measurements with contact between the potato and the light collection tube; (2) off-line measurements without contact between the potato and the light collection tube; and (3) on-line measurements of the potatoes. The offline contact measurements gave a prediction performance of R(2)=0.89 and RMSECV=1.19. Similar prediction performance were obtained from the off-line non-contact measurements (R(2)=0.89, RMSECV=1.23). Significantly better (p=0.038) prediction performance (R(2)=0.92, RMSECV=1.06) was obtained with the on-line measuring configuration, thus showing the possibilities of using the instrument for on-line measurements. In addition it was shown that the dry matter distribution across the individual tuber could be predicted by the model obtained.

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.

Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial profiling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the different cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate profiles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during different phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

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.

Aims: Investigate if the antibacterial effect of sphagnan, a pectin-like carbohydrate polymer extracted from Sphagnum moss, can be accounted for by its ability to lower the pH. Methods and Results: Antibacterial activity of sphagnan was assessed and compared to that of three other acids. Sphagnan in its acid form was able to inhibit growth of various food poisoning and spoilage bacteria on low-buffering solid growth medium, whereas sphagnan in its sodium form at neutral pH had no antibacterial activity. At similar acidic pH, sphagnan had comparable antibacterial activity to that of hydrochloric acid and a control rhamnogalacturonan pectin in its acid form. Conclusions: Sphagnan in its acid form is a weak macromolecular acid that can inhibit bacterial growth by lowering the pH of environments with a low buffering capacity. Significance and Impact of the Study: It has previously been suggested that sphagnan is an antimicrobial polysaccharide in the leaves of Sphagnum moss with a broad range of potential practical applications. Our results now show that sphagnan in its acid form can indeed inhibit bacterial growth, but only of acid-sensitive species. These findings represent increased knowledge towards our understanding on how sphagnan or Sphagnum moss might be used in practical applications.

Oligosaccharides of ([4)-β- d -Man p A-(→] n ) with a degree of polymerisation (DP) of 5, 10 and 15 were generated by partial acid hydrolysis of alginate mannuronan. These were subsequently purified by a combination of size-exclusion chromatography and semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection. The purity of the isolated oligosaccharides was greater than 96%. With automated operation of the chromatography system, milligram quantities can be generated over a period of a few days. Thus, our methodology now offers some significant advantages over earlier, including our own, protocols focused on uronic acid oligomers, where the final products are either not as pure or more starting material is needed to generate an equivalent yield of product. Removal of ammonium ions in collected fractions after size-exclusion chromatography and prior to freeze-drying was found to be essential to prevent the formation of imines and subsequent Maillard browning products.

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.

The dry matter is one of the main quality parameters of raw and processed potatoes. In the present study, the potential of utilizing high throughput commercially available NIR interactance systems for dry matter determination in whole unpeeled potato tubers is investigated. The performance of a 2D NIR interactance instrument was compared with that of a 1D NIR interactance instrument and a traditional underwater weight apparatus. A total of 114 tubers were assessed individually with both of the NIR instruments (760-1040 nm), the underwater weight and an external reference method (freeze drying). The 1D interactance instrument obtained better prediction results than what the 2D instrument could achieve (R2=0.95, RMSECV=0.91, and R2=0.83, RMSECV=1.65, respectively). The underwater weight obtained the highest explained variance (R2=0.97), but the estimation was biased by approximately 1.5% (by weight). The poorer prediction performance of the 2D NIR interactance system can be partly explained by the lower penetration depths of the light compared to the 1D NIR interactance systems.