The solution conformation of hyaluronan (HA), a biopolymer in the glycosaminoglycan (GAG) family, is still not well understood despite its simple primary structure. The main origin of the structural flexibility of HA is its two alternating β(1 → 4) and β(1 → 3) glycosidic linkages and several studies have reported apparently contrasting evidence on the structural and dynamic behavior of these two linkages. Meanwhile, only short HA oligosaccharides have been investigated thoroughly by NMR spectroscopy and few studies have focused on comparisons between short and longer HA chains. We report herein a detailed conformational investigation of HA octasaccharide and 20-mer by NMR spectroscopy based on NOEs and 3J-couplings, focusing on the glycosidic linkages and the N-acetyl group. This is the first NMR study that provides experimental support for the existence of minor conformations of the β(1 → 4) glycosidic linkage and validation of the predominant conformation of the β(1 → 3) glycosidic linkage previously established by MD simulations. This approach provides methodologies for conformational analysis among different chain lengths in other glycans.

Improved efficacy of probiotics can be achieved by using different strategies, including the optimization of production parameters. The impact of fermentation parameters on bacterial physiology is a frequently investigated topic, but what happens during the formulation, i.e., the step where the lyoprotectants are added prior to freeze-drying, is less studied. In addition to this, the focus of process optimization has often been yield and stability, while effects on bioactivity have received less attention. In this work, we investigated different metabolic activities of the probiotic strain Limosilactobacillus reuteri DSM 17938 during formulation with the freeze-drying protectant sucrose. We discovered that the strain consumed large quantities of the added sucrose and produced an exopolysaccharide (EPS). Using NMR, we discovered that the produced EPS was a glucan with α-1,4 and α-1,6 glycosidic bonds, but also that other metabolites were produced. The conversion of the lyoprotectant is hereafter designated lyoconversion. By also analyzing the samples with GCMS, additional potential bioactive compounds could be detected. Among these were tryptamine, a ligand for the aryl hydrocarbon receptor, and glycerol, a precursor for the antimicrobial compound reuterin (3-hydroxypropionaldehyde). To exemplify the bioactivity potential of lyoconversion, lyoconverted samples as well as purified EPS were tested in a model for immunomodulation. Both lyoconverted samples and purified EPS induced higher expression levels of IL-10 (2 times) and IL-6 (4–6 times) in peripheral blood mononuclear cells than non-converted control samples. We further found that the initial cultivation of DSM 17938 with sucrose as a sugar substrate, instead of glucose, improved the ability to convert sucrose in the lyoprotectant into EPS and other metabolites. Lyoconversion did not affect the viability of the bacteria but was detrimental to freeze-drying survival, an issue that needs to be addressed in the future. In conclusion, we show that the metabolic activities of the bacteria during the formulation step can be used as a tool to alter the activity of the bacteria and thereby potentially improve probiotic efficacy.

Increased consumption of plant-based foods and better utilisation of side-streams can reduce the environmental impact of food consumption. A promising crop for production of protein-rich plant-based foods is faba bean, which can serve as a local alternative to soy in cold-climate regions. This study investigated faba bean protein gelation at multiple pH values and the effect of adding a fibre-rich side-stream from protein extraction. Two different sources were used to extract the fibre (cotyledon and hull). The gels were characterised in terms of textural properties, microstructure and water mobility. Gels produced at pH 4 and 5 showed reduced fracture stress, fracture strain and water-holding capacity, but higher Young’s modulus, than gels produced at pH 7. The effect of adding fibre (at fixed solids content) varied with pH. Differences observed were attributed to the gel microstructure, as light and scanning electron micrographs showed coarse, aggregated microstructure at pH 4 and 5 and a fine-stranded protein network at pH 7. Irrespective of fibre source (cotyledon/hull), addition of fibre had comparable effects on textural properties. Low-field NMR revealed differences in water mobility between gels at pH 4-5 versus pH 7, and between gels with/without added fibre, likely related to contrasting microstructures and the water-binding properties of the fibre fractions.

In the present work, we explored Lewis acid catalysis, via FeCl3, for the heterogeneous surface functionalization of cellulose nanofibrils (CNFs). This approach, characterized by its simplicity and efficiency, facilitates the amidation of nonactivated carboxylic acids in carboxymethylated cellulose nanofibrils (c-CNF). Following the optimization of reaction conditions, we successfully introduced amine-containing polymers, such as polyethylenimine and Jeffamine, onto nanofibers. This introduction significantly enhanced the physicochemical properties of the CNF-based materials, resulting in improved characteristics such as adhesiveness and thermal stability. Reaction mechanistic investigations suggested that endocyclic oxygen of cellulose finely stabilizes the transition state required for further functionalization. Notably, a nanocomposite, containing CNF and a branched low molecular weight polyethylenimine (CNF-PEI 800), was synthesized using the catalytic reaction. The composite CNF-PEI 800 was thoroughly characterized having in mind its potential application as coating biomaterial for medical implants. The resulting CNF-PEI 800 hydrogel exhibits adhesive properties, which complement the established antibacterial qualities of polyethylenimine. Furthermore, CNF-PEI 800 demonstrates its ability to support the proliferation and differentiation of primary human osteoblasts over a period of 7 days.

A wide range of articles describe the role of different probiotics in the prevention or treatment of various diseases. However, currently, the focus is shifting from whole microorganisms to their easier-to-define components that can confer similar or stronger benefits on the host. Here, we aimed to describe polysaccharide B.PAT, which is a surface antigen isolated from Bifidobacterium animalis ssp. animalis CCDM 218 and to understand the relationship between its structure and function. For this reason, we determined its glycerol phosphate-substituted structure, which consists of glucose, galactose, and rhamnose residues creating the following repeating unit: To fully understand the role of glycerol phosphate substitution on the B.PAT function, we prepared the dephosphorylated counterpart (B.MAT) and tested their immunomodulatory properties. The results showed that the loss of glycerol phosphate increased the production of IL-6, IL-10, IL-12, and TNF-α in bone marrow dendritic cells alone and after treatment with Lacticaseibacillus rhamnosus GG. Further studies indicated that dephosphorylation can enhance B.PAT properties to suppress IL-1β-induced inflammatory response in Caco-2 and HT-29 cells. Thus, we suggest that further investigation of B.PAT and B.MAT may reveal distinct functionalities that can be exploited in the treatment of various diseases and may constitute an alternative to probiotics.

Nanocellulose–oligoproline hydrogels, able to respond to high levels of ROS and protect cells from oxidative environments, are promising candidates for the treatment of chronic wounds and other clinical conditions associated with high levels of ROS.

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.

Starch and protein are major components in many foods, contributing to nutritional and textural properties. Understanding how the behaviour and interactions of these components contribute to different textures is important. In this study, mixed gel systems were created with different ratios of starch to protein (constant solid content 12%) extracted from faba bean, a promising crop for locally produced plant-based foods in cold climate regions. The mixed starch-protein gels were characterised in terms of pasting, texture and microstructure. Starch-rich mixtures showed higher water binding and water absorption than samples with higher protein content. A tendency for more efficient hydration in starch-rich samples was confirmed by NMR. Iodine affinity appeared to be lower for high-protein samples, particularly at higher temperatures. Mixtures with high starch content also showed higher viscosity during pasting, higher storage modulus throughout gelation, lower tan delta and lower frequency dependence of the final gel. Characterisation by compression tests showed stronger and more elastic gels with increasing starch content. Light microscopy revealed that starch granules were tightly packed, espe-cially at higher starch content, with protein filling the spaces between starch granules. SEM micrographs revealed a network structure with larger pores and thicker strands in samples with higher starch content. Overall, increasing protein content reduced viscosity during pasting and caused softer gels, likely owing to different gelation and hydration properties of starch and protein.

Introduction The Automated Quantification Algorithm (AQuA) is a rapid and efficient method for targeted NMR-based metabolomics, currently optimised for blood plasma. AQuA quantifies metabolites from 1D- 1 H NMR spectra based on the height of only one signal per metabolite, which minimises the computational time and workload of the method without compromising the quantification accuracy. Objectives To develop a fast and computationally efficient extension of AQuA for quantification of selected metabolites in highly complex samples, with minimal prior sample preparation. In particular, the method should be capable of handling interferences caused by broad background signals. Methods An automatic baseline correction function was combined with AQuA into an automated workflow, the extended AQuA, for quantification of metabolites in plant root exudate NMR spectra that contained broad background signals and baseline distortions. The approach was evaluated using simulations as well as a spike-in experiment in which known metabolite amounts were added to a complex sample matrix. Results The extended AQuA enables accurate quantification of metabolites in 1D- 1 H NMR spectra with varying complexity. The method is very fast (< 1 s per spectrum) and can be fully automated. Conclusions The extended AQuA is an automated quantification method intended for 1D- 1 H NMR spectra containing broad background signals and baseline distortions. Although the method was developed for plant root exudates, it should be readily applicable to any NMR spectra displaying similar issues as it is purely computational and applied to NMR spectra post-acquisition.

Eutrophication is an important threat to aquatic ecosystems world-wide, and reliable identification of areas vulnerable to phosphorus (P) losses from diffuse sources is essential for high efficiency of mitigation measures. In this three-step study we investigated (i) relationships between the agronomic (Olsen-P and P-AL) and environmental soil P tests (P-CaCl 2 ) with molecular techniques ( 31 P NMR and XANES) followed by (ii) rainfall simulation experiment on topsoil lysimeters and (iii) comparison to long-term field measurements of water quality. Soil samples were collected from seven sites indicated to be vulnerable to nutrient losses due to underlying geology. High P release correlated to standard agronomic P tests (Olsen P, r = 0.67; and P-AL, r = 0.74) and low P sorption capacity (r = − 0.5). High content of iron-bound P compounds indicated more labile P and higher release of dissolved P (r = 0.67). The leaching experiment showed that three out of four soils with high initial soil P status had both higher P leaching concentrations before fertilization (0.83–7.7 mg P l −1 ) compared to soil with low initial soil P status (0.007–0.23 mg P l −1 ), and higher increase in P concentrations after fertilization. Higher soil P sorption capacity reduced P leaching losses. Finally, long-term monitoring data show no significant trends in P losses in a field with low initial P content and moderate P fertilization rates whereas high and over time increasing P losses were recorded in a field with high initial soil P content and repetitively high P fertilization rates.

Bifidobacteria are among the most common bacteria used for their probiotic properties and their impact on the maturation and function of the immune system has been well-described. Recently, scientific interest is shifting from live bacteria to defined bacteria-derived biologically active molecules. Their greatest advantage over probiotics is the defined structure and the effect independent of the viability status of the bacteria. Here, we aim to characterize Bifidobacterium adolescentis CCDM 368 surface antigens that include polysaccharides (PSs), lip-oteichoic acids (LTAs), and peptidoglycan (PG). Among them, Bad368.1 PS was observed to modulate OVA-induced cytokine production in cells isolated from OVA-sensitized mice by increasing the production of Th1-related IFN-gamma and inhibition of Th2-related IL-5 and IL-13 cytokines (in vitro). Moreover, Bad368.1 PS (BAP1) is efficiently engulfed and transferred between epithelial and dendritic cells. Therefore, we propose that the Bad368.1 PS (BAP1) can be used for the modulation of allergic diseases in humans. Structural studies revealed that Bad368.1 PS has an average molecular mass of approximately 9,99 x 106 Da and it consists of glucose, galactose, and rhamnose residues that are creating the following repeating unit: [-> 2)-beta-D-Glcp-(1 -> 3)-beta-L-Rhap-(1 -> 4)-beta-D-Glcp-(1 -> 3)-alpha-L-Rhap-(1 -> 4)-beta-D-Glcp-(1 -> 3)-alpha-D-Galp-(1 -> ]n

EDITORIAL article Front. Microbiol., 29 November 2022Sec. Food Microbiology Volume 13 - 2022 | https://doi.org/10.3389/fmicb.2022.1089461

The structure and physicochemical properties of starch isolated from the cotyledon and hull of faba beans and from wheat (as reference) were examined using 16 different methods. The amylose content in faba bean cotyledon and hull starch was 32% and 36%, respectively, and that in wheat starch was 21%. The faba bean cotyledon and hull starch were structurally alike both displaying C-polymorphic pattern, a similar degree of branching and similar branch chain length distributions. Wheat starch had a significantly greater prevalence of short amylopectin chains (DP < 12) and a higher degree of branching. Granules in both faba bean starches exhibited surface cracks and were more homogenous in size than the smoother wheat starch granules. Gelatinisation temperature was higher for the faba bean starches, likely as an effect of high amylose content and longer starch chains delaying granular swelling. Cotyledon starch produced pastes with the highest viscosities in all rheological measurements, probably owing to larger granules. Higher prevalence of lipids and resistant starch reduced the viscosity values for hull starch. For all starches, viscosity increased at faster heating rates. During the rheological analyses, the samples were exposed to different instruments, heating rates and temperatures ranges, differing from standard rheological procedures, which could help predict how different processing techniques effect the final starch textures. Graphical abstract

We have recently presented an Automated Quantification Algorithm (AQuA) and demonstrated its utility for rapid and accurate absolute metabolite quantification in 1H NMR spectra in which positions and line widths of signals were predicted from a constant metabolite spectral library. The AQuA quantifies based on one preselected signal per metabolite and employs library spectra to model interferences from other metabolite signals. However, for some types of spectra, the interspectral deviations of signal positions and line widths can be pronounced; hence, interferences cannot be modeled using a constant spectral library. We here address this issue and present an improved AQuA that handles interspectral deviations. The improved AQuA monitors and characterizes the appearance of specific signals in each spectrum and automatically adjusts the spectral library to model interferences accordingly. The performance of the improved AQuA was tested on a large data set from plasma samples collected using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant (n = 772). These spectra provided a suitable test system for the improved AQuA since EDTA signals (i) vary in intensity, position, and line width between spectra and (ii) interfere with many signals from plasma metabolites targeted for quantification (n = 54). Without the improvement, ca. 20 out of the 54 metabolites would have been overestimated. This included acetylcarnitine and ornithine, which are considered particularly difficult to quantify with 1H NMR in EDTA-containing plasma. Furthermore, the improved AQuA performed rapidly (<10 s for all spectra). We believe that the improved AQuA provides a basis for automated quantification in other data sets where specific signals show interspectral deviations.

There is currently a huge need for new, improved therapeutic approaches for the treatment of chronic wounds. One promising strategy is to develop wound dressings capable of modulating the chronic wound environment (e.g., by controlling the high levels of reactive oxygen species (ROS) and proteases). Here, we selected the thiol-containing amino acid cysteine to endow wood-derived cellulose nanofibrils (CNF) with bioactivity toward the modulation of ROS levels and protease activity. Cysteine was covalently incorporated into CNF and the functionalized material, herein referred as cys-CNF, was characterized in terms of chemical structure, degree of substitution, radical scavenging capacity, and inhibition of protease activity. The stability of the thiol groups was evaluated over time, and an in vitro cytotoxicity study with human dermal fibroblasts was performed to evaluate the safety profile of cys-CNF. Results showed that cys-CNF was able to efficiently control the activity of the metalloprotease collagenase and to inhibit the free radical DPPH (1,1-Diphenyl-2-picrylhydrazyl radical), activities that were correlated with the presence of free thiol groups on the nanofibers. The stability study showed that the reactivity of the thiol groups challenged the bioactivity over time. Nevertheless, preparing the material as an aerogel and storing it in an inert atmosphere were shown to be valid approaches to increase the stability of the thiol groups in cys-CNF. No signs of toxicity were observed on the dermal fibroblasts when exposed to cys-CNF (concentration range 0.1–0.5 mg/mL). The present work highlights cys-CNF as a promising novel material for the development of bioactive wound dressings for the treatment of chronic wounds.

Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four Cα-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether-containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by Cα H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,β-dehydro-amino acid intermediate during Cα-thioether bridge LC-MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.

Band-selective NMR experiments are presented that allow selective suppression of unwanted signals (SUN) from the spectra of complex metabolite mixtures. As a result, spectral overlap and dynamic range problems are substantially reduced and low-intensity signals normally covered by dominant signals can be observed. The usefulness of the experiments is exemplified with selective suppression of sugar signals from the NMR spectra of fruit juice and a plant sample. Other possible applications include blood, milk, and wine samples.

Cross-linked hyaluronic acid (HA) hydrogels are used in many biomedical applications but their characterization in order to distinguish between physicochemical properties is challenging. Longitudinal (T1) and transverse (T2) relaxation times and diffusion coefficient (D) of water protons in diepoxide 1,4-butanediol diglycidyl ether (BDDE)-cross-linked HA hydrogels were analyzed by high-field NMR spectroscopy to distinguish between different physicochemical properties. Hydrogels of different degrees of modification and cross-linking, representing a range of gel content, swelling ability, elastic and viscous behavior were studied, as well as solutions of native HA of different molecular weights. T1, T2 and D were measured for several concentrations of HA and as a function of temperature. D and T1 showed a weak concentration dependence, but did not differ between the hydrogels. T2, dominated by chemical exchange between water protons and exchangeable protons of HA, varied significantly between the different hydrogels and the temperature profiles changed dramatically between different concentrations.

The global prevalence of type 2 diabetes is increasing rapidly; consequently there is great need for new and novel therapeutic options. Gynostemma pentaphyllum (GP) is a traditional medicinal plant, mainly present in Southeast Asian countries, that has been reported to exert antidiabetic effects, by stimulating insulin secretion. The specific compound responsible for this effect is however as yet unidentified. Screening for discovery and identification of bioactive compounds of an herbal GP extract, was performed in isolated pancreatic islets from spontaneously diabetic Goto-Kakizaki (GK) rats, a model of type 2 diabetes, and from non-diabetic control Wistar rats. From this herbal extract 27 dammarane-type saponins, including two novel compounds, were isolated and their structure was elucidated by mass spectrometry and NMR spectroscopy. One of the dammarane-type triterpenoid showed a glucose-dependent insulin secretion activity. This compound, gylongiposide I, displays unique abilities to stimulate insulin release at high glucose levels (16.7 mM), but limited effects at a low glucose concentration (3.3 mM). Further studies on this compound, also in vivo, are warranted with the aim of developing a novel anti-diabetic therapeutic with glucose-dependent insulinogenic effect.

Hyaluronic acid (HA) cross-linked with 1,4-butanediol diglycidyl ether (BDDE) are hydrogels with many biomedical applications. Degree of substitution, cross-linking and substitution position of the cross-linker might influence the properties of the hydrogels. We showed earlier that the most common substitution position of the cross-linker on the hyaluronan chain was the 4-hydroxyl of N-acetylglucosamine. This result has led us to investigate unsulfated chondroitin (CN) which only differ from HA in the primary structure by the configuration at C4 of the aminoglycan. In the present study, we have investigated (i) the substitution positions of the cross-linker in CN using NMR and LC-MS and compared the results to the data obtained for HA (ii) the effect of alkali on the 13C and 1H chemical shifts in CN and HA (iii) the temperature coefficients and chemical shifts of hydroxyl protons in CN and HA. In CN, the 2-hydroxyl of glucuronic acid and 6-hydroxyl of N-acetylgalactosamine were found to be the major sites of substitution by BDDE. Moreover, while chondroitinase was not able to cleave HA tetrasaccharide substituted at the 4-hydroxyl GlcNAc reducing end by BDDE, it is able to degrade CN-BDDE down to disaccharide units.