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Homoexopolysaccharides (HoEPS) such as α-glucans and β-fructans are synthesized by lactic and acetic acid bacteria. Methylation analysis is an important and well-established tool for the structural analysis of these polysaccharides, however, multiple steps are required for polysaccharide derivatization. Because ultrasonication during methylation and the conditions during acid hydrolysis may influence the results, we investigated their role in the analysis of selected bacterial HoEPS. The results reveal that ultrasonication is crucial for water insoluble α-glucan to swell/disperse and deprotonate prior to methylation whereas it is not necessary for water soluble HoEPS (dextran and levan). Complete hydrolysis of permethylated α-glucans requires 2 M trifluoroacetic acid (TFA) for 60/90 min at 121 °C while levan is hydrolyzed in 1 M TFA for 30 min at 70 °C. Nevertheless, levan was also detectable after hydrolysis in 2 M TFA at 121 °C. Thus, these conditions can be used to analyze a levan/dextran mixture. However, size exclusion chromatography of permethylated and hydrolyzed levan showed degradation and condensation reactions at harsher hydrolysis conditions. Application of reductive hydrolysis with 4-methylmorpholine-borane and TFA did not lead to improved results. Overall, our results demonstrate that conditions used for methylation analysis have to be adjusted for the analysis of different bacterial HoEPS.

Dextransucrases are extracellular enzymes, which are exclusively expressed by lactic acid bacteria (LAB) and produce α−1→6 linked glucose polymers from sucrose. In this study, two dextransucrases derived from water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827 were identified and comparatively investigated. Differences between both proteins mainly arise from an additional C-terminal glucan-binding domain and the presence of a signal motif in the L. nagelii TMW 1.1827 dextransucrase. L. hordei TMW 1.1822 released the enzyme only in the presence of its substrate sucrose in contrast to L. nagelii TMW 1.1827, while both strains functionally expressed the dextransucrases independently of sucrose. Both enzymes could be recovered as crude protein extracts in culture supernatants, as they are not covalently bound to the cell surface. This enabled the formation of dextrans at equal reaction conditions as well as their subsequent structural analysis in terms of molecular structure and molecular weight. The volumetric transglycosylation and hydrolysis activities were distinctly different for both enzymes, which produced O3-branched dextrans with a comparable degree of branching. Moreover, identical oligosaccharides were obtained for both dextrans upon endo-dextranase digestion, while some differences in the polysaccharide fine structures could be identified from the varying portions of certain oligosaccharides. Dextrans synthesized by the dextransucrase released by L. nagelii exhibited an averaged molecular weight (M w) of 7.9×107 Da, while those produced by the dextransucrase released by L. hordei exhibited an M w of 6.1×107 Da. Moreover, glycosylation of glucansucrases by LAB was identified for the first time for the released dextransucrase of L. nagelii TMW 1.1827. Our study therefore reveals new molecular insights into how dextransucrases released by water kefir borne L. hordei TMW 1.1822 and L. nagelii TMW 1.1827 contribute to the complex formation of the traditional beverage water kefir.

Chromatographic analysis of endo-dextranase liberated branched oligosaccharides proved to be a valuable approach to differentiate dextrans from fermented food products or isolated lactic acid bacteria. Because these hydrolysis products also yield valuable information on the dextran fine structures, several branched isomalto-oligosaccharides were liberated from different dextrans and chromatographically purified. Mass spectrometry and two-dimensional NMR spectroscopy were used for structural characterization of the oligomers. Isomalto-oligosaccharides from L. reuteri TMW 1.106 dextrans were exclusively O4-branched. Furthermore, they contained only monomeric side chains and at least one unsubstituted backbone unit between ramified residues. In contrast, O3-branched oligosaccharides with monomeric as well as elongated side chains were isolated. The varying abundance of the O3-branched oligosaccharides suggests that side chain length is a major factor for structural differences between dextrans. Overall, we demonstrated that chromatographic analysis of endo-dextranase liberated isomalto-oligosaccharides provides valuable information on the structural properties and supplements conventional methods such as methylation analysis.

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

The properties of the glucopolymer dextran are versatile and linked to its molecular size, structure, branching, and secondary structure. However, suited strategies to control and exploit the variable structures of dextrans are scarce. The aim of this study was to delineate structural and functional differences of dextrans, which were produced in buffers at different conditions using the native dextransucrase released by Liquorilactobacillus (L.) hordei TMW 1.1822. Rheological measurements revealed that dextran produced at pH 4.0 (MW = 1.1 * 108 Da) exhibited the properties of a viscoelastic fluid up to concentrations of 10% (w/v). By contrast, dextran produced at pH 5.5 (MW = 1.86 * 108 Da) was gel-forming already at 7.5% (w/v). As both dextrans exhibited comparable molecular structures, the molecular weight primarily influenced their rheological properties. The addition of maltose to the production assays caused the formation of the trisaccharide panose instead of dextran. Moreover, pre-cultures of L. hordei TMW 1.1822 grown without sucrose were substantial for recovery of higher dextran yields, since the cells stored the constitutively expressed dextransucrase intracellularly, until sucrose became available. These findings can be exploited for the controlled recovery of functionally diverse dextrans and oligosaccharides by the use of one dextransucrase type.

Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks. The microbial enzymes mostly target the energy present in cellulose, hemicellulose, and pectin, three major forms of energy storage in plants. In the effort to develop bioenergy as an alternative to fossil fuel, a common strategy is to harness microbial enzymes to hydrolyze cellulose to glucose for fermentation to fuels. However, the conversion of plant biomass to renewable fuels will require both cellulose and hemicellulose, the two largest components of the plant cell wall, as feedstock to improve economic feasibility. Here, we explore the enzymes and strategies evolved by two well-studied bacteria to depolymerize the hemicelluloses xylan/arabinoxylan and mannan. The sets of enzymes, in addition to their applications in biofuels and value-added chemical production, have utility in animal feed enzymes, a rapidly developing industry with potential to minimize adverse impacts of animal agriculture on the environment.

The generation of protein-polysaccharide conjugates via dry-heating can overcome several disadvantageous properties of proteins. It has already been demonstrated that conjugates of whey protein isolate (WPI) and pectin yield clearly improved emulsifying properties. However, less attention has been paid to the connection between the amount/portion of conjugates and the emulsifying properties. In addition, the molecular background of the conjugation reaction is yet unknown. This study was focused on the evaluation of the molecular background of conjugate formation. Before further characterization, WPI-pectin conjugates were produced by using various reaction times and analyzed by various approaches. The results were used to select a reaction time which yields high conjugate portions, high solubility and good droplet stabilization. To obtain information about the influence of residual unconjugated WPI in that mixture, a fractionation procedure based on the solubility in different buffers was developed. This approach allowed for the isolation of fractions with increasing portions of conjugated WPI, which was confirmed by analyzing the protein content. In addition, the amino acid profile of the isolated fractions indicated that increasing portions of conjugates correlate with decreasing portions of lysine. Unambiguous evidence for the conjugation of the e-lysine amino groups with pectin was obtained from the isolation and characterization of a corresponding Maillard reaction product. Therefore, the results of this study demonstrated that conjugate formation between protein-bound lysine and pectin is responsible for the improved emulsifying properties of WPI-pectin conjugates. Furthermore, the emulsifying properties of the conjugate-enriched fractions confirmed that increasing conjugate portions allow for the stabilization of smaller droplets.

In-situ exopolysaccharide (EPS) formation by starter cultures is frequently exploited in the dairy-industry, where they function as natural hydrocolloids and improve both rheological and textural characteristics of these food products. In contrast, applications in fermented meat products remain widely unexplored - neglecting beneficial effects such as fat replacement or enhanced serum binding capacity. Since amount and structure of EPS determine their functional effects in foods, we investigated the impact of cold and salt stress, which are important parameters in the manufacturing of meat products, for their impact on quantity and structural properties of the dextran produced by Lactobacillus sakei TMW 1.411. Upon growth under different (stress) conditions, the amount of dextran was lowest (0.5 g/L) at 30 °C/9.5% NaCl, while about fourfold increased amounts were obtained at 10 °C/0% NaCl (6.7 g/L) compared to 30 °C/0% NaCl (1.6 g/L). The highest molecular weights and particle sizes were determined for the dextran produced at 10 °C/0% NaCl, which displayed a decreased degree of branching. Comparable results regarding amounts and sizes were obtained when dextrans were produced by resting cells in buffers under the same conditions used during growth experiments in mMRS media. Our data suggest that amount, molecular and macromolecular structure of extracellular dextrans produced by Lactobacillus sakei TMW 1.411 are markedly influenced by the applied temperature, while high salt concentrations strongly limit dextran polymerization from sucrose. These findings provide new insights into possible future applications aiming at an in-situ EPS formation in meat-based food products with improved properties.

Water kefir granules are supposed to mainly consist of dextrans produced by Lactobacillus (L.) hilgardii. Still, other microorganisms such as L. hordei, L. nagelii, Leuconostoc (Lc.) citreum and Saccharomyces (S.) cerevisiae are commonly isolated from water kefir granules, while their contribution to the granule formation remains unknown. We studied putative functions of these microbes in granule formation, upon development of a simplified model system containing hydrophilic object slides, which mimics the hydrophilic surface of a growing kefir granule. We found that all tested lactic acid bacteria produced glucans, while solely those isolated from the four different L. hordei strains induced yeast aggregation on the hydrophilic slides. Therefore, structural differences between these glucans were investigated with respect to their size distributions and their linkage types. Beyond the finding that all glucans were identified as dextrans, those of the four L. hordei strains were highly similar among each other regarding portions of linkage types and size distributions. Thus, our study suggests the specific size and structural organization of the dextran produced by L. hordei as the main cause for inducing S. cerevisiae aggregation and network formation on hydrophilic surfaces and thus as crucial initiation of the stepwise water kefir granule growth.

Water kefir is a beverage which is produced by initiating fermentation of a fruit extract/sucrose solution with insoluble kefir grains. Exopolysaccharides that are formed from sucrose play a major role in the kefir grain formation, but the exopolysaccharides in the kefir beverage and the detailed structural composition of the whole kefir grains have not been studied yet. Therefore, kefir grains and the corresponding kefir beverage were analyzed for exopolysaccharides by multiple chromatographic approaches and two-dimensional NMR spectroscopy. Furthermore, different fractionation techniques were applied to obtain further information about the exopolysaccharides. The exopolysaccharide-fraction of the investigated kefir beverage was predominantly composed of O3- and O2-branched dextrans as well as lower amounts of levans. The insoluble dextrans from the kefir grains were mostly O3-branched and contained an elevated portion of 1,3-linked glucose units compared to the soluble dextrans. The structurally different exopolysaccharides in water kefir suggest the involvement of multiple bacteria.

Pectins extracted from sugar beet pulp are promising additives for the food and beverage industry due to their good emulsifying properties. However, the relationship between molecular structure and emulsifying properties is still not fully understood; thus, systematic optimization of pectin extraction for a better emulsifying activity is not possible. In this study, we assess the underlying relationship between molecular structure and emulsifying properties by linking relevant structural parameters of 43 acid-extracted sugar beet pectins to their emulsifying properties. Sugar beet pectins with distinct structural characteristics were produced by acid extraction using varying conditions. The extracted polysaccharides were used as emulsifying agents in oil-in-water emulsions. Resulting droplet sizes were measured to correlate molecular characteristics with emulsifying properties. Analyses of protein content, degree of methylation, degree of acetylation, mean molecular weights, and trans-ferulic acid contents showed that these parameters affect the emulsifying properties to some degree, but cannot fully explain the differences observed among all pectins. However, a linear correlation between the proportion of neutral sugar side chains and droplet sizes was observed. The influence of galactose (18–30 mol%) was negligible, but increasing amounts of branched arabinans (up to 57 mol% arabinose) positively affected the emulsification result. Based on these results, it can be concluded that particularly branched arabinan side chains play an important role in the sugar beet pectin-based stabilization of emulsions. Thus, the arabinose content appears to be a suitable indicator to estimate the emulsifying properties of acid-extracted sugar beet pectins.

Despite several hurdles, which hinder bacterial growth in beer, certain bacteria are still able to spoil beer. One type of spoilage is characterized by an increased viscosity and slimy texture caused by exopolysaccharide (EPS) formation of lactic acid bacteria (LAB). In this study, we characterize for the first time EPS production in a beer-spoiling strain (TMW 1.2112) of Lactobacillus brevis, a species commonly involved in beer spoilage. The strain's growth dynamics were assessed and we found an increased viscosity or ropiness in liquid or on solid media, respectively. Capsular polysaccharides (CPS) and released EPS from the cells or supernatant, respectively, were analyzed via NMR spectroscopy and methylation analysis. Both are identical β-(1→3)-glucans, which are ramified with β-glucose residues at position O2. Therefore, we assume that this EPS is mainly produced as CPS and partially released into the surrounding medium, causing viscosity of e.g. beer. CPS formation was confirmed via an agglutination test. A plasmid-located glycosyltransferase-2 was found as responsible for excess β-glucan formation, chromosomal glucanases were proposed for its degradation. The glycosyltransferase-2 gene could also be specifically identified in beer-spoiling, slime-producing Lactobacillus rossiae and Lactobacillus parabuchneri strains, suggesting it as promising marker gene for the early detection of β-glucan-producing Lactobacilli in breweries.

Kozakia baliensis NBRC 16680 produces a heteropolysaccharide (HePS), whose biosynthesis is similar to the biosynthesis of the exopolysaccharide acetan. To elucidate structural components and macromolecular properties of this HePS, we carried out methylation analysis, NMR spectroscopy, rheological measurements and size determinations. In accordance with acetan, the HePS are composed of 1,4-substituted Glcp, 1,3,4-substituted Glcp, 1,2-substituted Manp, 1,4-substituted GlcpA, and 1,6-substituted Glcp units. In contrast to acetan, rhamnose and acetylation of side chains were not detected. Furthermore, a putative, unidentified uronic acid and 1,6-substituted Galp units were found to be HePS constituents, both of which could not be correlated with the responsible HePS biosynthesis in contrast to the other present structural elements. Depending on the initial carbon source, K. baliensis HePS were produced in different amounts and exhibited different rheological properties and elution profiles during AF4 and HPSEC separations. In conclusion, we propose an acetan-like HePS with slight molecular weight variations depending on the production conditions.

The objective of this study was to investigate the influence of molecular weight of κ-carrageenan on its ice recrystallization inhibition (IRI) activity. To reduce the molecular weight, acid hydrolysis with 0.1 M trifluoroacetic acid (TFA) at 80 °C or 0.1 M hydrochloric acid (HCl) at 60 °C was performed. In addition, molecular weight was reduced by dialyzing κ-carrageenan against demineralized water. It was demonstrated that IRI activity of κ-carrageenan decreases with decreasing molecular weight, which is contrary to previous studies. It was shown in our study that the different results may be attributed to an aggregation of κ-carrageenan molecules due to high NaCl concentration, originated from HCl and subsequent neutralization with NaOH. This aggregation causes a decrease in IRI activity. Furthermore, it was shown that the addition of a small amount of NaCl can lead to an increase in IRI activity of κ-carrageenan.

The popularity of kiwifruit (Actinidia deliciosa) is steadily rising among consumers since their introduction in the global market 60 years ago. Kiwifruit are ripened postharvest for marketing. A good technique to follow the complex changes throughout postharvest ripening is metabolomics. So far research on the kiwifruit metabolome has been performed on kiwifruit development or on-vine ripening. This study aimed at a comprehensive description of processes during postharvest ripening of kiwifruit. Metabolic changes in kiwifruit during six well-defined stages of postharvest ripening and two non-marketable stages due to water loss were monitored using an untargeted multi-platform approach consisting of GC × GC–MS, GC–MS and NMR. In addition, postharvest cell wall polysaccharide modifications were followed using multiple chromatographic approaches. Using the multi-platform approach, it was possible to follow the process of postharvest ripening kiwifruit. Investigation of soluble metabolites in kiwifruit yielded mainly sugars, sugar-related substances and organic acids as well as other known and unknown metabolites. Sugars predominantly increased during ripening while organic acids predominantly decreased. In addition, unexpected changes in the concentration of some known and unknown metabolites were observed. Changes of the non-starch polysaccharides were dominated by a continuous loss of neutral pectic side chains, whereas minor changes were observed for homogalacturonan and hemicellulosic polysaccharides only. Overall the three applied platforms and the cell wall characterization were complementary and allowed a comprehensive description of the kiwifruit metabolome during postharvest ripening.

Levan is a fructan-type exopolysaccharide, which is produced by many microbes from sucrose via extracellular levansucrases. The hydrocolloid properties of levan depend on its molecular weight, while it is unknown why and to which extent levan is functionally diverse in dependence of its size. The aim of our study was to get deeper insights into the size-dependent, functional variability of levan. For this purpose, levans of different sizes were produced using the water kefir isolate Gluconobacter albidus TMW 2.1191 and subsequently rheologically characterized. Three levan types could be identified, which are similarly branched, but significantly differ in their molecular size and rheological properties among each other. The smallest levan (< 107 Da) produced without adjustment of the pH exhibited Newton-like flow behavior up to a specific concentration of 25% (w/v). On the contrary, larger levans (> 108 Da) produced at pH ≥ 4.5 were shear-thinning and showed a gel like behavior at ≥ 5% (w/v). A third (intermediate) levan variant was obtained via production in buffers at pH 4.0 and exhibited the properties of a viscoelastic fluid at ≥ 5% (w/v). Our study reveals that the variable size and composition of levan are controllable and more decisive for its functionality than the amount of exerted levan.

Levan is a fructan-type exopolysaccharide which is produced by many microbes from sucrose via extracellular levansucrases. The hydrocolloid properties of levan depend on its molecular weight, while it is unknown why and to what extent levan is functionally diverse depending on its size. The aim of our study was to gain deeper insight into the size-dependent functional variability of levan. For this purpose, levans of different sizes were produced using the water kefir isolate Gluconobacter albidus TMW 2.1191 and subsequently rheologically characterized. Three levan types could be identified, which are similarly branched, but differ significantly in their molecular size and rheological properties. The smallest levan (108 Da) produced at pH ≥ 4.5 were shear-thinning, and the levan produced at pH 5.0 showed a gel-like behavior at 5% (w/v). A third (intermediate) levan variant was obtained through production in buffers at pH 4.0 and exhibited the properties of a viscoelastic fluid up to concentrations of 15% (w/v). Our study reveals that the rheological properties of levan are determined by its size and polydispersity, rather than by the amount of levan used or the structural composition.

Glucansucrases can be used to glucosylate various plant-derived phenolic compounds by using sucrose as donor substrate. We applied Lactobacillus reuteri TMW 1.106 dextransucrase to glucosylate the acceptor substrates caffeic acid and gallic acid. Subsequently, monoglucosylated and in particular oligo- and polyglucosylated conjugates were characterized by using different chromatographic techniques and two-dimensional NMR spectroscopy. Both acceptors were substituted at positions O3 and O4. Under the conditions used, two monoglucosylated products were formed for caffeic acid, whereas only one O3-monosubstituted conjugate was detected for gallic acid. However, both acceptors resulted in O4-substituted oligo- and polyglucosylated conjugates, the amount of which was higher from gallic acid than from caffeic acid. Profile analysis tensiometry suggested that, in contrast to unmodified dextrans, oligo- and polymeric glucoconjugates of gallic acid are highly interfacially active. Overall, we provide the first detailed characterization of enzymatically conjugated oligo- and polymeric dextrans, which may have further potential as functional ingredients.

Proteins are plant cell wall components but they are not included in the definition of dietary fiber. Therefore, dietary fiber preparations have to be corrected for their residual protein contents. This is commonly done by calculating the residual protein concentrations from the nitrogen contents after Kjeldahl digestion. Here, three different methods to determine nitrogen in Kjeldahl digests were compared: conventional titration with hydrochloric acid after steam distillation, a colorimetric assay (24-well microplates and cuvettes), and the determination by using an ammonia electrode. All assays gave similar results but detection using the ammonia electrode was found to be the most time-efficient approach. Also, an amino-acid profiling method, which is not based on commercial kits and which is suitable for routine analysis of dietary fiber preparations, was established. For this purpose, an HPLC-FLD method following amino acid derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was optimized for fiber samples. Although all commonly used dietary fiber preparation methods involve the application of proteases the amino acid profiles of fiber samples from different sources were shown to be quite diverse. Considering the amino acid composition of the residual protein in various dietary fiber preparations, residual protein is probably not only based on structural proteins.

Dextransucrases are extracellular enzymes, which are exclusively expressed by lactic acid bacteria (LAB) and produce α-1→6 linked glucose polymers from sucrose. In this study, two dextransucrases derived from water kefir borne

The water kefir organism Leuconostoc citreum TMW 2.1194 forms highly branched dextrans with O3- and O4-bound side chains. To obtain detailed information on the enzymatic synthesis of these polymers, the four glucansucrases encoded by Leuconostoc citreum TMW 2.1194 were cloned, heterologously expressed, and used for polysaccharide production. Molecular and macromolecular structure of the synthesized glucans were analyzed by methylation analysis, two-dimensional NMR spectroscopy, oligosaccharide analysis after partial hydrolysis, and asymmetric flow field-flow fractionation. It was demonstrated that two glucansucrases form insoluble glucans with variously branched dextran sections and varying portions of consecutive, 1,3-linked glucose units. In contrast, the other two glucansucrases synthesized O3- (Lc6255) and O4-branched (Lc1785) soluble dextrans. Analysis, isolation, and characterization of enzymatically liberated oligosaccharides showed that monomeric and elongated side chains are abundant in both polysaccharides. From the structures and size distributions it was concluded that Lc1785 is mainly responsible for synthesis of fermentatively produced soluble dextrans.

Microbial transglutaminase (mTG) catalyzes the crosslinking of bulk and interfacial proteins, thereby influencing the physical properties of food dispersions. However, little is known about the impact of the spatial distribution of proteins in a gelled matrix on the enzymatic crosslinking. In this study, free and incorporated emulsions were subjected to mTG to induce crosslinking of emulsion interfaces. First, simple caseinate-stabilized emulsions were fabricated by high shear blending (20,000 rpm, 6 min). Second, emulsion samples were embedded into hydrogel beads using an injection technique. Different alginate (0.5–1.5%) and CaCl2 levels (50–500 mM) were used to modulate the hydrogel pore size and number of junction zones. Third, free and incorporated emulsions were mixed with mTG to investigate the diffusion behavior of the enzyme. Results showed that mTG diffused into the beads to an extent of more than 50%. A delay in ammonia release was observed when emulsions were incorporated into hydrogel beads, whereas protein in free emulsions was instantly crosslinked after mTG was added. These results illustrate that the spatial make-up of biopolymers in gelled matrices play a key role on the enzyme accessibility.

Arabinans and galactans are complex pectic polysaccharides, which greatly influence the physicochemical and physiological properties of plants and plant-based foods. Conventional methods to characterize these challenging polymers are based on derivatization and/or unselective chemical cleavage of the glycosidic bonds of the polysaccharides, resulting in partial loss of essential information such as anomeric configuration. Here, endo-arabinanase and endo-galactanase were used to selectively cleave pectic arabinans and galactans. The liberated oligosaccharides were purified and characterized by LC-MS and one- and two-dimensional NMR spectroscopy resulting in known but also several previously unknown pectic structural elements. For the routine analysis of pectin hydrolysates by HPAEC-PAD, incubation conditions, chromatographic parameters, and relative response factors of the isolated pectic oligosaccharides against an internal standard were determined. The applicability of the method was demonstrated by analyzing different well-characterized plant cell wall materials. It was demonstrated that the developed method yields additional information about pectic arabinan and galactan structures that is not obtained from conventional methods such as methylation analysis.

In this study, the structural composition of commercial kappa (κ)-, iota (ι)-, lambda (λ)-carrageenans was examined by using 1H NMR spectroscopy, and the IRI activity of these different carrageenan samples was examined in sucrose solution. The 1H NMR spectra showed that both λ-carrageenans consist of κ- and ι-units which are either present as a mixture of the two carrageenan types or as a κ/ι-copolymer. According to the IRI activity results, κ-carrageenan samples showed the highest IRI activity among the different carrageenans. In the presence of κ-carrageenan, the round ice crystals transformed into a more angular and elongated shape, while this effect was less visible in the presence of the ι- and λ-carrageenans. Viscosity measurements indicated that there is no correlation between the viscosity and the IRI activity of carrageenans. The IRI activities of the investigated carrageenans are different for the suppliers, and the results show that they are based on the structural differences and possibly also other compositional differences.

Two exopolysaccharide (EPS) producing Lactococcus lactis strains that differed largely in their technofunctional properties (gel stiffness, ropiness) were used to relate their functionality in concentrated acid milk gel suspensions to the structural and macromolecular properties of the EPS. LL-1+ produced ropy and cell-bound EPS that contributed to acid milk gels with a higher gel stiffness, improved serum retention, and resulted in concentrated acid milk gel suspensions with smaller particles and a more ‘creamy’ texture compared with the non-ropy EPS from LL-2. From the basic structural and macromolecular analysis of the EPS it is concluded that mainly the higher hydrodynamic radius of the ropy EPS from LL-1+ is responsible for these features. The use of ropy and cell-bound EPS producing strains has, therefore, potential for the manufacture of low-fat fresh cheese with tailored functionality.

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood. Here, we identify a PUL that encodes multiple esterases for degradation of complex arabinoxylans in Bacteroides species. The PUL is specifically upregulated in the presence of complex arabinoxylans. We characterize some of the esterases biochemically and structurally, and show that they release ferulic acid from complex arabinoxylans. Growth of four different colonic Bacteroidetes members, including Bacteroides intestinalis, on complex arabinoxylans results in accumulation of ferulic acid, a compound known to have antioxidative and immunomodulatory properties., Human gut bacteria can degrade arabinoxylans, polysaccharides found in dietary fiber. Here, Pereira et al. identify a bacterial gene cluster encoding esterases for degradation of complex arabinoxylans. The action of these enzymes results in accumulation of ferulic acid, a phenolic compound with antioxidative and immunomodulatory properties.

Background: Low wall shear stress (LSS) is an established cause of pro-atherogenic endothelial pathophysiology, yet it has never been demonstrated in the cerebral circulation affected by intracranial atherosclerotic disease (ICAD). Noninvasive CT angiography (CTA) computational fluid dynamics (CFD) enables high-resolution investigation of detailed post-stenotic phenomena. We used all available CTA data in the SAMMPRIS trial of ICAD to detect and quantify post-stenotic LSS. Methods: CTA source images from SAMMPRIS were reconstructed in 3D followed by geometry refinements to generate a mesh of the diseased arterial lesion and adjacent segments. CFD was performed with Ansys (ICEM, Fluent), applying reference boundary conditions with k-omega turbulence and non-Newtonian modeling of the traversing blood viscosity. 3D CFD parameter maps illustrated velocity, velocity swirling and corresponding wall shear stress. Results: 144 subjects enrolled in SAMMPRIS had CTA at baseline, including 140 with CTA source images enabling CFD. Post-stenotic velocity profiles revealed vortices in all cases, quantified by swirling and turbulent kinetic energy. These luminal flow changes were adjacent to focal regions of LSS in the post-stenotic region (Figure). Conclusions: Low wall shear stress is associated with vortices of fluid flow in CTA CFD modeling of ICAD from SAMMPRIS. CTA source images may be used to noninvasively quantify LSS and model this pro-atherogenic factor in ICAD across a wide variety of lesions. Future studies should examine the related endothelial biology and potential link with plaque evolution.

Microbial exopolysaccharides (EPS) like xanthan are widely exploited as natural biopolymers in diverse industrial sectors. In foods, in-situ EPS formation by starter cultures allows the manufacturing of “clean labeled” products with improved textural and nutritional properties. We performed structural analyses of the cell surface-associated EPS produced by Lactobacillus plantarum TMW 1.1478, which is a promising starter culture for fermented foods. Chromatographic analyses and NMR experiments suggested an acetylated heptameric repeating unit comprised of glucose, rhamnose and galactose as major components, whereas analysis of the macromolecular HePS structure suggested an apparent molecular mass of Mr ∼2 × 106 and a root mean square (RMS) radius of ca. 60 nm. Genetic analyses enabled the identification of the respective EPS biosynthesis cluster, and its modular organization supports the chemically identified, novel EPS structure. The obtained results broaden the understanding of complex EPS formation from activated sugar nucleotides by Lactobacillus plantarum.

The diet provides carbohydrates that are non-digestible in the upper gut and are major carbon and energy sources for the microbial community in the lower intestine, supporting a complex metabolic network. Fermentation produces the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, which have health-promoting effects for the human host. Here we investigated microbial community changes and SCFA production during in vitro batch incubations of 15 different non-digestible carbohydrates, at two initial pH values with faecal microbiota from three different human donors. To investigate temporal stability and reproducibility, a further experiment was performed 1 year later with four of the carbohydrates. The lower pH (5.5) led to higher butyrate and the higher pH (6.5) to more propionate production. The strongest propionigenic effect was found with rhamnose, followed by galactomannans, whereas fructans and several α- and β-glucans led to higher butyrate production. 16S ribosomal RNA gene-based quantitative PCR analysis of 22 different microbial groups together with 454 sequencing revealed significant stimulation of specific bacteria in response to particular carbohydrates. Some changes were ascribed to metabolite cross-feeding, for example, utilisation by Eubacterium hallii of 1,2-propanediol produced from fermentation of rhamnose by Blautia spp. Despite marked inter-individual differences in microbiota composition, SCFA production was surprisingly reproducible for different carbohydrates, indicating a level of functional redundancy. Interestingly, butyrate formation was influenced not only by the overall % butyrate-producing bacteria in the community but also by the initial pH, consistent with a pH-dependent shift in the stoichiometry of butyrate production.

A modified digestion method was used to hydrolyze large amounts of quinoa meal to study polysaccharide-bound phenolic acids. Besides the well-characterized oligosaccharide O-(2-O-trans-feruloyl-α-l-arabinofuranosyl)-(1→5)-l-arabinofuranose, O-(2-O-trans-coumaroyl-α-l-arabinofuranosyl)-(1→5)-l-arabinofuranose was isolated from the hydrolysate and identified by LC-MS, GC-MS, and NMR experiments. This study demonstrates for the first time that p-coumaric acid can be linked to pectic side chains of dicotyledonous plants.

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α- l -arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β- l -arabinopyranosidase (CpAbp27A), and two GH127 β- l -arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para -nitrophenyl ( p NP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α- l -arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l -arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus . IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus . The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β- l -arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s-1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98A resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.

Exopolysaccharide (EPS) producing lactic acid bacteria are known for their positive effect on the texture of fermented dairy products, with their high water binding capacity causing changes in rheology and syneresis reduction. During manufacture, product texture is affected by shearing with an unknown impact on the properties of the EPS. By subjecting aqueous solutions of viscosity enhancing EPS from Streptococcus thermophilus DGCC7710 or dextran to defined shear treatments with a Microfluidizer, a cell disruption system or a toothed ring dispersion unit to apply different energy input, we investigated macromolecular properties and the behaviour in solution. Despite the breakage of covalent bonds, spectroscopic NMR analyses revealed that repeating unit structure remained unchanged, whereas changes in macromolecular properties such as intrinsic viscosity, hydrodynamic volume, and molecular mass strongly depended on energy input. The decrease of the dynamic viscosity of the polysaccharide solutions was significantly related to the observed decrease in molecular mass, and the impact of the EPS on technofunctional properties such as contribution to gel stiffness during acid gel formation was also affected. The results therefore indicate that mechanical energy input through shearing should be considered because product texture may be altered as a consequence of a mechanical breakdown of EPS molecules.

Alginate lyases can be used for alginate oligosaccharide production and for structural characterization or modification of alginates. For these applications it is important to obtain detailed information on mode of action and substrate specificities of alginate lyases. In this study, five alginate lyase genes were cloned from Cellulophaga algicola DSM 14237 genomic DNA, heterologously expressed, and characterized by using HPSEC-RI and HPAEC-PAD/MS. It was demonstrated that these analytical approaches can provide detailed information on preferred substrates, extent of hydrolysis, and the liberated products. The recombinant enzymes cleaved alginates endolytically (CaAly1, CaAly2, CaAly3) or exolytically (CaAly4, CaAly5). The three endolytic alginate lyases predominantly hydrolyzed guluronic acid-rich alginates, only CaAly1 also showed activity on mannuronic acid-rich alginates. The oligosaccharide profiles further demonstrated that the endolytic enzymes have rather narrow but slightly different substrate specificities and that the two exolytic alginate lyases mainly cleaved unsaturated guluronic acid oligosaccharides to monomers.