Fine structures of different dextrans assessed by isolation and characterization of endo-dextranase liberated isomalto-oligosaccharides

Carbohydrate Polymers 215 (2019) 296–306

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Fine structures of different dextrans assessed by isolation and characterization of endo-dextranase liberated isomalto-oligosaccharides

T

Franziska Münkel, Daniel Wefers

⁎

Department of Food Chemistry and Phytochemistry, Institute of Applied Biosciences, Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131 Karlsruhe, Germany

ARTICLE INFO

ABSTRACT

Keywords: Glucans Structure NMR spectroscopy Chromatography HPAEC-PAD Enzymatic fingerprinting

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 isomaltooligosaccharides were liberated from different dextrans and chromatographically purified. Mass spectrometry and two-dimensional NMR spectroscopy were used for structural characterization of the oligomers. Isomaltooligosaccharides 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.

1. Introduction Dextrans are α-glucans which are comprised of a backbone of α-1,6linked glucose units. Because the backbone may be ramified at position O2, O3, and/or O4, these polysaccharides have a high structural complexity (Torino, de Valdez, & Mozzi, 2015; Zannini, Waters, Coffey, & Arendt, 2016). Dextrans are enzymatically synthesized by dextransucrases which are secreted by various lactic acid bacteria. These enzymes use the glucose residue from sucrose to form polysaccharides (Leemhuis et al., 2013). Besides multiple applications in the medical and cosmetic sector, dextrans can also be used to influence the properties of various food products (Torino et al., 2015; Zannini et al., 2016). Besides the application of isolated, pre-produced dextrans, it is possible to directly produce dextrans in fermented food products. For example, dextrans or dextran producing lactic acid bacteria were successfully applied to beneficially influence the properties of gluten-free sourdough bread (Chen, Levy, & Gänzle, 2016; Di Cagno et al., 2006; Galle et al., 2012b; Katina et al., 2009; Rühmkorf et al., 2012; Wolter, Hager, Zannini, Czerny, & Arendt, 2014). In addition, it was

demonstrated that type and degree of branching influences the functional properties of dextrans in sourdough (Chen et al., 2016; Galle et al., 2012a; Rühmkorf et al., 2012). In situ produced dextrans are also present in water kefir which is made by fermentation of a sucrose solution (Fels, Jakob, Vogel, & Wefers, 2018). In water kefir, dextrans most likely play a major role for aggregation of lactic acid bacteria and yeast, which was shown to depend on the structural composition, too (Xu et al., 2018). Furthermore, dextran structures are most likely responsible for the physiological properties of these polysaccharides in the human gastrointestinal tract. However, details on dextran fine structures are scarcely reported. In a recent study, Maina, Virkki, Pynnonen, Maaheirno, & Tenkanen (2011) isolated and characterized endo-dextranase/α-glucosidase resistant oligosaccharides from a dextran produced by the potential sourdough starter culture Weissella confusa. By analyzing the oligosaccharide structures, they demonstrated that the investigated dextran contains elongated side chains which are bound to position O3 of the α-1,6-linked dextran backbone. Thus, enzymatically liberated oligosaccharides yield information on substitution type and side chain length. Chromatographic analysis of enzymatically

Abbreviations: HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detection; PMAAs, partially methylated alditol acetates; HSQC, Heteronuclear Single Quantum Coherence; H2BC, Heteronuclear Two-bond Correlation; COSY, H,H-Correlated Spectroscopy; TOCSY, Total Correlated Spectroscopy; HMBC, Heteronuclear Multiple Bond Correlation ⁎ Corresponding author. E-mail address: [email protected] (D. Wefers). https://doi.org/10.1016/j.carbpol.2019.03.027 Received 27 January 2019; Received in revised form 9 March 2019; Accepted 10 March 2019 Available online 24 March 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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liberated oligosaccharides was already used to differentiate between structurally different dextrans (Fels et al., 2018; Katina et al., 2009; Shukla et al., 2014; Xu et al., 2018). These studies clearly demonstrated the potential of this approach, because it can be used to analyze complex matrices and to detect differences in dextrans with a comparable structural composition. For example, dextrans from multiple L. hordei strains and L. nagelii TMW 1.1827 were only distinguishable by the ratios of the oligosaccharides liberated by endo-dextranase (Xu et al., 2018). However, the oligosaccharides characterized to date were exclusively O3-branched and in part only obtained as a mixture. Because endo-dextranase hydrolysis of structurally different dextrans most likely yields different oligosaccharides, the aim of this study was to characterize endo-dextranase liberated isomalto-oligosaccharides from dextrans produced by different food-associated lactic acid bacteria. For oligosaccharide purification, dextrans were produced by fermentation/ recombinant dextransucrases and subsequently hydrolyzed by endodextranase. The enzymatically liberated isomalto-oligosaccharides were purified by different chromatographic approaches and their structure was determined by using mass spectrometry and two-dimensional NMR spectroscopy. Eventually, structures and abundance of the characterized oligosaccharides were used to obtain information on the fine structures of different dextrans.

acetic acid and acetylation was performed by using 1-methylimidazole and acetic anhydride. The partially methylated alditol acetates (PMAAs) were extracted into dichloromethane, washed, and residual water was removed by freezing overnight at −18 °C. PMAAs were identified by GC-MS (GC-2010 Plus and GCMS-QP2010 SE, Shimadzu) and quantitated by GC-FID (GC-2010 Plus, Shimadzu) on a DB5-MS column (30 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA) with the previously described conditions (Fels et al., 2018). Molar response factors according to Sweet, Shapiro, & Albersheim (1975) were used for semiquantitative analyses. All analyses were performed in duplicate. 2.4. Enzymatic hydrolysis For chromatographic analysis of the oligosaccharide profiles as well as for large scale hydrolysis, dextrans were dissolved or suspended in bidistilled water (1 mg/mL). After the addition of at least 0.1 KDU endo-dextranase (from Chaetomium erraticum)/mg dextran, samples were incubated at 30 °C for 24 h. These conditions were previously evaluated to be sufficient for an end point incubation with this enzyme preparation (apparent from the complete hydrolysis of isomaltotriose) (Fels et al., 2018; Maina et al., 2011). For isolation of the oligosaccharide D3-Vb from dextrans produced by L. curvatus TMW 1.624 dextransucrase, polysaccharides were incubated with at least 5 U endo-dextranase from Penicillium sp./mg dextran and 0.5 U oligo-α-1,6-glucosidase/mg dextran. This enzyme combination was used because it provided a much higher yield of oligosaccharide D3-Vb. After incubation, enzymes were inactivated at 95 °C for 10 min and residual solids were, if necessary, removed by centrifugation. Samples for high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis were diluted and analyzed, while large scale hydrolysates were freeze-dried and redissolved in a reduced volume.

2. Experimental 2.1. General If not stated otherwise, all chemicals used were of “p.a.” grade or better and were purchased from Sigma Aldrich (Schnelldorf, Germany), VWR (Radnor, PA), or Carl Roth (Karlsruhe, Germany). Bio-Gel P-2 was from Bio-Rad Laboratories (Hercules, CA). endo-Dextranase (EC 3.2.1.11) preparations from Chaetomium erraticum (≥100 KDU/g) and from Penicillium sp. (100–250 U/mg) were purchased from SigmaAldrich. Oligo-α-1,6-glucosidase (171 U/mg) was purchased from Megazyme (Bray, Ireland).

2.5. High-performance anion exchange chromatography The enzymatic hydrolysates and the purified oligosaccharides were analyzed by HPAEC-PAD on an ICS-5000 system (Thermo Scientific Dionex, Sunnyvale, CA) equipped with a CarboPac PA200 column (250 mm × 3 mm i.d., 5.5 μm particle size, Thermo Scientific Dionex). A flow rate of 0.4 mL/min and a gradient composed of the following eluents was used at 25 °C: (A) bidistilled water, (B) 0.1 M sodium hydroxide, (C) 0.1 M sodium hydroxide + 0.5 M sodium acetate. Before every run, the column was washed with 100% C for 10 min and equilibrated with 90% A and 10% B for 20 min. After injection, the following gradient was applied: 0–10 min, isocratic 90% A and 10% B; 10–20 min, linear from 90% A and 10% B linear to 100% B; 20–90 min, linear from 100% B to 100% C.

2.2. Dextran production L. hordei TMW 1.1822 and L. hilgardii TMW 1.1828 dextrans were produced by growing the respective organisms in sucrose-MRS medium as described previously (Xu et al., 2018). In addition, two of the recombinant dextransucrases described by Rühmkorf et al. (2013) were used to produce dextrans at pH 5.5 (L. curvatus TMW 1.624 dextransucrase) and pH 6.0 (L. reuteri TMW 1.106 dextransucrase) at 30 °C. Polysaccharides were obtained from the centrifuged fermentation broth or reaction mixture by ethanol precipitation and residual low molecular weight compounds were removed by dialysis (Molecular Weight CutOff 3,500 Da). High performance size exclusion chromatography as well as one dimensional NMR spectroscopy demonstrated that high molecular weight dextrans were isolated and that only trace amounts of low molecular weight compounds and buffer salts were present.

2.6. Oligosaccharide purification Redissolved large scale dextran hydrolysates were initially fractionated by size on a Bio-Gel P-2 column (gel bed: 85 × 2.6/1.6 cm). Bidistilled water was used as eluent (0.5–1 mL/min) and the column was heated to 45 °C. Oligosaccharides were detected with a Smartline RI detector 2300 (Knauer, Berlin, Germany) and fractions were automatically collected at fixed time intervals. The resulting fractions were freeze-dried and further fractionated on a semipreparative HPLC-ELSD system (AZURA P 2.1 L pumps, Knauer; Sedex 85 ELSD detector, Sedere, Alfortville, France) equipped with a Hypercarb column (100 mm × 4.6 mm i.d., 5 μm particle size, Thermo Fisher Scientific, Waltham, MA) and an adjustable flow splitter (Analytical Scientific Instruments, Richmond, CA). A flow rate of 2.5 mL/min at 70 °C and the following gradient composed of bidistilled water (A) and acetonitrile (B) was used: 0–1 min, isocratic 100% A; 1–20 min, linear to 80% A and 20% B; 20–24 min, isocratic 20% A and 80% B; 24–28 min, isocratic

2.3. Methylation analysis Methylation analysis was performed as described previously (Fels et al., 2018). Briefly, the samples (2–5 mg) were dissolved in dimethyl sulfoxide and incubated in an ultrasonic bath (90 min) and at room temperature. After addition of methyl iodide, the samples were incubated for 1 h and the methylated polysaccharides were extracted by using dichloromethane. The organic phase was washed with sodium thiosulfate and water. After drying, methylation was repeated once to ensure complete methylation. Subsequently, the residue was hydrolyzed with 2 M TFA at 121 °C for 90 min. After evaporation of the acid, the partially methylated monosaccharides were reduced by using sodium borodeuteride. The reaction was terminated by using glacial 297

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80% A and 20% B. The resulting fractions were freeze-dried and characterized by mass spectrometry and NMR spectroscopy.

Table 1 Glycosidic linkages (mol%) of the dextrans used in this study as determined by methylation analysis.

2.7. Mass spectrometry The purified oligosaccharides were analyzed for their molecular mass and their MS2 fragmentation as described previously (Maina et al., 2013). Briefly, an aliquot of a concentrated oligosaccharide solution was diluted with 400 μL of a methanol/water/formic acid mixture (50:49:1, v/v/v). After addition of 3 μL of an ammonium chloride solution (10 mg/mL), the sample was directly injected into an LXQ linear ion trap system (Thermo Fisher Scientific) by using a syringe pump (20–40 μL/min). Prior to analyzing the purified oligosaccharides, MS parameters were optimized for the detection of chloride adducts of isomalto-oligosaccharides in negative ionization mode by tuning with isomaltotriose. A normalized collision energy of 50 was applied for MS2 fragmentation (these conditions resulted in the highest yield of fragment ions).

Glycosidic linkage

L. hilgardii TMW 1.1828

L. hordei TMW 1.1822

L. curvatus TMW 1.624

L. reuteri TMW 1.106

t-Glcp 1,3-Glcp 1,4-Glcp 1,6-Glcp 1,3,6-Glcp 1,4,6-Glcp

11.3 3.2 – 79.7 5.8 –

6.3 0.7 – 90.1 2.9 –

5.3 – – 89.8 4.9 –

18.5 – 0.6 68.3 12.5

All analyses were performed in duplicate and relative half range uncertainties were mostly < 5%. t: terminal; Glc: glucose; p: pyranose. Numbers indicate the substituted positions of a sugar unit.

Furthermore, L. curvatus dextrans were in part insoluble, thus the signal integrals may not completely reflect the actual degree of branching. To obtain information on the structural composition of all dextrans used in this study, their glycosidic linkages were analyzed by methylation analysis (Table 1). In agreement with the results from NMR spectroscopy, all samples yielded solely glucose-derived PMAAs and 1,6-linked glucose units were the predominating glycosidic linkage. However, portions of terminal glucose units were higher than the portions of PMAAs representing branched backbone units in all samples. This is most likely derived from the presence of some low molecular weight dextrans or low amounts of residual sucrose. Nevertheless, valuable information on the structural composition of the dextrans can be derived from the ratio between linear and branched backbone units. For example, methylation analysis results suggested that dextrans produced by L. reuteri dextransucrase are exclusively O4-branched which is in good agreement with previous studies (Rühmkorf et al., 2013). From the ratios between the PMAAs derived from linear backbone units (1,6-Glcp) and branched backbone units (1,4,6-Glcp) a comparatively high degree of branching can be derived. For L. hilgardii, L. hordei and L. curvatus dextrans, methylation analysis confirmed that O3-branched dextrans are present. However, some structural differences can be derived from the portions of the glycosidic linkages. While dextrans from L. hordei yield the lowest portion of branched backbone residues (1,3,6-Glcp), L. hilgardii and L. curvatus dextrans show comparable portions of this PMAA. However, significant portions of 1,3-linked glucose units were detected in L. hilgardii dextrans. This is in good agreement with the insolubility of these dextrans and previous studies (Pidoux et al., 1990). Consequently, NMR spectroscopy and methylation analysis showed the same trend for the degree of branching of L. reuteri, L. curvatus, and L. hordei dextrans, but a higher portion of side chains was indicated by methylation analysis. However, it can be concluded that the dextrans used in this study have different structures with regards to their position and degree of branching. To gain insights into the oligosaccharides liberated from the dextrans, a small scale incubation with endo-dextranase from Chaetomium erraticum was conducted and the hydrolysates were analyzed by HPAEC-PAD (Fig. 1). As it would be expected from the glycosidic linkages, the endodextranase hydrolysate of L. reuteri dextrans yielded a completely different oligosaccharide profile than the hydrolysates of the other dextrans. Notably, relatively high portions of oligosaccharides with a high degree of polymerization were observed besides the four predominating, earlier eluting compounds D4-IV-VII. The high number of enzymatically liberated isomalto-oligosaccharides with a varying

2.8. NMR spectroscopy For NMR spectroscopic analyses, the purified and freeze-dried oligosaccharides were dissolved in 500 μL of D2O and acetone was added as an internal reference (2.22 ppm/30.89 ppm according to Gottlieb, Kotlyar, & Nudelman (1997)). Spectra were recorded on an Ascend 500 MHz spectrometer (Bruker, Rheinstetten, Germany) equipped with a Prodigy cryoprobe. Standard Bruker parameter sets were used to record proton, Heteronuclear Single Quantum Coherence (HSQC), Heteronuclear Two-bond Correlation (H2BC), H,H-Correlated Spectroscopy (COSY), Total Correlated Spectroscopy (TOCSY), Heteronuclear Multiple Bond Correlation (HMBC), and HSQC-TOCSY spectra. 3. Results and discussion 3.1. Characterization, hydrolysis, and fractionation For isolation and characterization of endo-dextranase liberated isomalto-oligosaccharides, L. hordei TMW 1.1822 and L. hilgardii TMW 1.1828 dextrans were produced by fermentation. Previous studies already suggested that these dextrans have important biological functions because they contribute to water kefir granule formation and yeast aggregation (Pidoux, 1989; Pidoux, Deruiter, Brooker, Colquhoun, & Morris, 1990; Waldherr, Doll, Meissner, & Vogel, 2010; Xu et al., 2018). Furthermore, L. hilgardii dextrans belong to the rather rare group of insoluble glucans and L. hordei dextrans showed a unique oligosaccharide profile in a previous study and preliminary works (Pidoux et al., 1990; Xu et al., 2018). Dextrans were also synthesized by using the recombinant dextransucrases from L. curvatus TMW 1.624 and L. reuteri TMW 1.106 described by Rühmkorf et al. (2013). In previous studies, it was demonstrated that dextrans synthesized by these enzymes have a high potential for application in gluten-free sourdough breads and that they are branched on position O3 (L. curvatus) and position O4 (L. reuteri) (Kaditzky et al., 2008; Rühmkorf et al., 2013; Rühmkorf et al., 2012). Dextrans synthesized by fermentation and recombinant dextransucrases were initially analyzed by one- and twodimensional NMR spectroscopy. This approach confirmed the presence of dextrans and the absence of major impurities. In addition, the signal integrals in the proton spectra indicated that the degree of branching decreases from L. reuteri dextrans (10.9% side chain derived residues) to L. curvatus dextrans (3.2% side chain derived residues) and L. hordei dextrans (2.3% side chain derived residues, Fig. S1). Due to their insolubility, dextrans from L. hilgardii did not yield any spectra.

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Fig. 1. HPAEC-PAD chromatograms of the endo-dextranase hydrolysates of dextrans produced by L. reuteri dextransucrase (A), L. curvatus dextransucrase, L. hilgardii, and L. hordei (B). The nomenclature of the indicated isolated oligosaccharides was derived from their structure (see text).

degree of polymerization indicates that the analyzed L. reuteri dextrans have a high complexity. In contrast, the hydrolysates of the O3-branched dextrans contain only up to four major oligosaccharides. Among the three dextrans, the one from L. hordei showed a unique oligosaccharide profile, because only three oligosaccharides and comparably high portions of oligosaccharide D3-VI were detected. In contrast, comparable portions of all four oligosaccharides were liberated from the L. curvatus and L. hilgardii dextrans. However, the hydrolysate of the L. hilgardii dextran additionally contains low portions of oligosaccharides with a higher degree of polymerization. The differences in the oligosaccharide profiles are in agreement with methylation analysis results which showed an increase of structural complexity from L. hordei dextrans to L. curvatus and L. hilgardii dextrans. To purify and determine the structure of the enzymatically liberated isomalto-oligosaccharides, dextrans were produced in a larger scale and hydrolyzed by using Chaetomium erraticum endo-dextranase. Because of their varying abundance and the partially low degree of hydrolysis, the O3branched oligosaccharides were purified from different dextrans (D3IV: L. hordei/L. hilgardii, D3-Va: L. hordei, D3-Vb: L. curvatus, D3-VI: L. hordei). For purification of oligosaccharide D3-Vb, Penicillium sp. endodextranase in combination with oligo-1,6-glucosidase was used, because this enzyme blend gave a higher yield of this otherwise lowly abundant oligosaccharide. By using a combination of gel permeation chromatography and semipreparative HPLC, it was possible to purify the oligosaccharides marked in Fig. 1. The purity of all oligosaccharides as determined by HPAEC-PAD was mostly higher than 75%. Only oligosaccharides with a high degree of polymerization showed a lower purity (around 60%). However, the impurities were mostly caused by several lowly abundant oligosaccharides which yielded only a low signal intensity. Therefore, it was possible to analyze the structure of the main compounds by mass spectrometry and two-dimensional NMR spectroscopy.

possible to reliably extract information on substitution type and position under the conditions used in this study. However, this study was focused on the NMR spectroscopic characterization which can be used for complete and unambiguous structural elucidation of complex oligosaccharides. In general, all purified isomalto-oligosaccharides yielded complex proton spectra, therefore, several two dimensional NMR experiments were carried out for structural characterization of the complex dextran oligosaccharides. Among these, the HSQC spectra which yield one-bond couplings between protons and carbons provide a good resolution of signals overlapping in one of the two dimensions. Beginning with the (in most cases) well-resolved anomeric HSQC signals (4.5–5.5 ppm/90–105 ppm), it was possible to assign the remaining HSQC signals of the single sugar units by using HSQC-TOCSY, COSY, and H2BC experiments. Eventually, inter-glycosidic linkages were evaluated by HMBC correlations and, if possible, confirmed by using standard compounds (isomaltotriose, maltotriose) and literature data (Dobruchowska et al., 2013; Maina et al., 2011). As it would be expected for endo-dextranase liberated oligosaccharides, all compounds yielded two sets of HSQC signals which represent an O6-substituted, reducing glucose unit (anomeric signals at 4.67/96.7 ppm and 5.24/92.8 ppm). This is also in good agreement with the MS2 results. Because these signals were present in all samples, they will not be discussed any further in the following sections. The nomenclature of the oligosaccharides was derived from the ramification position (D4: O4-branched oligosaccharides; D3: O3-branched oligosaccharides) as well as the degree of polymerization (Roman numerals, determined by mass spectrometry). 3.2.1. Oligosaccharides from L. reuteri TMW 1.106 dextran Besides the anomeric signals of an O6-substituted, reducing glucose unit, the HSQC spectrum of oligosaccharide D4-IV only contained one (broadened) signal at 4.96/98.5 ppm and one signal at 5.38/100.4 ppm. The HSQC-TOCSY spectrum (Fig. 2) clearly demonstrated that the latter HSQC signal (assigned as unit a) results from a terminal glucose unit. This can be derived from the 13C chemical shift of C6 (61.1 ppm) which does not show the characteristic downfield shift of O6-substituted glucose residues. The HSQC-TOCSY spectrum (as well as the signal integrals) also clearly demonstrated that the second HSQC signal at 4.96/98.5 ppm represents two anomeric protons/carbons (unit A and B). From the correlation signals along the F1 dimension it was possible to detect 13C chemical shifts characteristic for terminal (61.1 ppm), O6substituted (66.3 ppm), and O4-substituted glucose units (77.6 ppm). In addition, the signals along the F2 dimension were well-resolved and

3.2. Oligosaccharide characterization To determine the molecular mass of the purified oligosaccharides, their chloride adduct ions were analyzed by ESI-MS in negative mode according to Maina et al. (2013). In addition, MS2 spectra were recorded to obtain initial information on the oligosaccharide structures. All oligosaccharides showed neutral losses of 18, 60, 90, and 120 for the first two hexoses which indicates one reducing O6-substitued and one 1,6-linked glucose unit (Maina et al., 2013). Because fragments with a lower molecular mass were absent or of low abundance, it was not

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Fig. 2. HSQC-TOCSY spectra of the anomeric region of oligosaccharides D4-IV, D4-V, and D4-VI. The structures as well as the descriptors of the sugar units are shown in Fig. 3.

allowed for differentiation and assignment of the two sugar units based on slightly different 13C chemical shifts of the anomeric carbon (Fig. S2). The two different substitution patterns of the monomeric units were identified based on the chemical shifts of some characteristic HSQC signals (Table 2) as well as some diagnostic inter-glycosidic HMBC correlations (Fig. S3). The C4H4 and C5H5 signals of unit A were in good agreement with data for α-1,6-substituted glucose units (derived from isomaltose and literature data (Dobruchowska et al., 2013; Maina et al., 2011)). In addition, an HMBC correlation between H4 of unit A and the characteristically downfield shifted C6 carbons at 66.3 ppm confirmed this assignment. For unit B, a characteristic downfield shift was observed for C4H4 (3.65/77.6 ppm) which suggests substitution at this position. From an HMBC correlation between the H4 proton and a terminal C6 at 3.65/61.1 ppm it was furthermore concluded that unit B is solely α-1,4linked. Eventually, the diagnostic HMBC correlation between H1 of unit a and C4 of unit B demonstrated that unit a is attached to O4 of unit B, leading to the structure shown in Fig. 3. The assignments were also confirmed by the comparison of the 1H and 13C chemical shifts of unit B and a (Table 2) with literature data for →4)-α-Glcp-(1→6)- and α-Glcp(1→4) units (Dobruchowska et al., 2013). For oligosaccharide D4-V, the HSQC-TOCSY spectrum (Fig. 2) as well as the other two-dimensional experiments allowed for the assignment of the 1H and 13C chemical shifts to the five sugar units (Table 2). From the results it becomes obvious that unit a again represents a terminal, O4-bound glucose unit. However, the chemical shift of the C1H1 signal of unit a (5.32/100.5 ppm) was clearly different from the C1H1 signal in D4-IV (5.38/100.4 ppm) which indicates a different

chemical environment. Accordingly, the C6H6 signals of the O4-substituted glucose unit B (which can be identified in the HSQC-TOCSY spectrum by the characteristically downfield shifted C4H4 signal at 3.67/78.9 ppm) were shifted downfield in the carbon dimension to 67.2 ppm (Fig. 2 and Table 2). Therefore, this glucose unit is additionally substituted at position O6. Just as for D4-IV, the C1H1 signal of unit B overlapped with the signal of an α-1,6-linked glucose (unit A) which can also be derived from the corresponding HSQC-TOCSY signals (Fig. 2). The remaining glucose unit T yielded an additional C1H1 HSQC signal at 4.99/98.9 ppm. From the HSQC-TOCSY spectrum and the resulting 1H/13C chemical shifts (C6 at 61.0 ppm, C5H5 at 3.75/ 72.6 ppm), it could be deduced that this signal corresponds to a terminal, O6-bound glucose unit. Furthermore, an HMBC correlation between C6 of unit B and H1 of unit T (and other diagnostic HMBC correlations, Fig. S4) demonstrated the interconnection between these two monosaccharide units and that the attachment of unit T to unit B is the only difference between D4-IV and D4-V (Fig. 3). For oligosaccharide D4-VI, the HSQC-TOCSY spectrum (in combination with other experiments) again allowed for complete assignment of all signals. Just as for the other oligosaccharides, the substitution of the sugar units can be seen from the correlations along the F1 dimension. Unit a was again identified as a terminal, O4-bound glucose unit. However, the C1H1 HSQC signal of this sugar unit showed a slight upfield shift which indicates some structural modification of its chemical environment. The HSQC signal at 4.96/98.4 ppm again represents one α-1,6-linked and one α-1,4,6-linked glucose unit. However, an additional terminal, O6-bound glucose unit was identified from the characteristically shifted C6 at 61.2 ppm. In contrast, the HSQC-TOCSY

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correlations and the 13C chemical shifts of unit C clearly demonstrate that this unit is substituted at position O6. Because the C1H1 HSQC signal of unit C is only slightly shifted compared to unit T of oligosaccharide D4-V, it can be concluded that it is also bound to the 1,4,6substituted glucose (unit B). This was confirmed by an HMBC correlation between C6 of unit B (characteristic 13C chemical shift at 67.4 ppm) and H1 of unit C (Fig. S5). Therefore, it can be concluded that two glucose units are attached to the O4-substituted glucose unit in oligosaccharide D4-VI, leading to the structure shown in Fig. 3. For the three oligosaccharides with a higher degree of polymerization (D4-VII, D4-IX, and D4-X), the same anomeric HSQC signals than for D4-VI were obtained. Therefore, it is likely that varying portions of the same structural elements are present. This was confirmed by the HSQC-TOCSY spectra which clearly showed that e.g. terminal, O4bound glucose units are abundant and that no side chain elongation is present in the oligosaccharides analyzed. In addition, varying volume integrals were obtained for the C1H1 signals (Fig. 4). The volume integrals can be used to obtain information on the abundance of the structural elements represented by the individual C1H1 HSQC signals. In addition, information on the portions of terminal, linear, and branched glucose units can be obtained from volume integration of the C4H4 HSQC signals which show a clearly different chemical shift for terminal (3.43/70.1 ppm), 1,4,6-linked (3.64/ 79.2 ppm), and O6-substituted/1,6-linked glucose units (3.51–3.57/ 70.1 ppm). Because all anomeric HSQC signals showed identical chemical shifts than the corresponding signals of oligosaccharide D4-VI, it can further be assumed that at least two 1,6-linked glucose units are present on the reducing and non-reducing end of the oligosaccharides. Based on this assumption and the signal integrals, the oligosaccharide structures shown in Fig. 4 can be proposed. The C1H1 signal integrals demonstrated that oligosaccharide D4-VII contains only one terminal, O4-bound glucose unit and one glucose unit bound to an 1,4,6-linked glucose unit. Furthermore, the presence of one 1,4,6-linked glucose unit, one terminal, O6-bound glucose unit and two 1,6-linked glucose units could be derived from volume integration of the C4H4 signals. Because of the complete overlap of the corresponding HSQC signals, it is not possible to unambiguously determine whether the additional 1,6-linked glucose unit is located before or after the 1,4,6-linked glucose unit by using NMR spectroscopy. However, further structural information was derived from an enzymatic hydrolysis of D4-

Table 2 1 H and 13C chemical shifts of the structural units of the isomalto-oligosaccharides isolated from the endo-dextranase hydrolysate of L. reuteri dextran. The structures of the oligosaccharides are shown in Fig. 3. Compound/Structural unit

1

2

3

4

5

6-1

6-2

R-a

5.24 92.8 4.67 96.7 4.96 98.5 4.96 98.1 5.38 100.4

D4-IV 3.53 72.1 3.25 74.7 3.58 72.1 3.58 72.1 3.58 72.1

3.70 73.6 3.48 76.7 3.71 73.6 3.99 74.1 3.67 73.5

3.51 70.1 3.51 70.1 3.51 70.1 3.65 77.6 3.41 69.9

4.01 70.6 3.64 74.9 3.91 70.9 3.85 70.9 3.72 73.6

3.70 66.4 3.76 66.4 3.76 66.3 3.85 61.1 3.76 61.1

4.01 66.4 3.98 66.4 3.95 66.3 3.85 61.1 3.85 61.1

5.24 92.8 4.67 96.7 4.96 98.4 4.95 98.0 4.99 98.9 5.32 100.5

D4-V 3.53 72.1 3.25 74.7 3.58 72.2 3.62 71.8 3.53 72.2 3.58 72.2

3.70 73.6 3.48 76.6 3.71 73.7 4.01 73.8 3.71 73.8 3.67 73.6

3.51 70.2 3.51 70.2 3.50 70.2 3.67 78.9 3.43 70.1 3.41 70.1

4.01 70.7 3.63 74.8 3.92 70.9 4.02 69.8 3.75 72.6 3.75 73.5

3.70 66.4 3.76 66.4 3.76 66.3 3.87 67.2 3.77 61.1 3.76 61.1

4.01 66.4 3.98 66.4 3.96 66.3 3.96 67.2 3.84 61.1 3.86 61.1

5.24 93.0 4.67 96.8 4.96 98.4 4.96 98.0 5.00 99.1 4.96 98.4 5.31 100.8

D4-VI 3.53 72.1 3.25 74.8 3.58 72.3 3.62 71.8 3.55 72.2 3.55 72.2 3.58 72.3

3.70 73.8 3.48 76.7 3.71 73.9 4.01 74.0 3.73 73.9 3.71 73.9 3.67 73.7

3.51 70.2 3.51 70.2 3.51 70.2 3.64 79.2 3.57 70.0 3.43 70.1 3.43 70.1

4.01 70.6 3.64 74.9 3.91 71.0 4.03 69.8 3.93 71.0 3.70 72.6 3.75 73.7

3.70 66.4 3.76 66.4 3.76 66.4 3.90 67.4 3.72 65.9 3.77 61.2 3.77 61.2

4.01 66.4 3.98 66.4 3.97 66.4 3.95 67.4 4.02 65.9 3.84 61.2 3.86 61.2

R-b A B a

R-a R-b A B T a

R-a R-b A B C T a

Fig. 3. Structures of isomalto-oligosaccharides isolated from the endo-dextranase hydrolysate of L. reuteri dextran. The italic letters are used to describe the sugar units in Fig. 2 and Table 2.

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Fig. 4. Anomeric HSQC signals and proposed structures of oligosaccharides D4-VII, D-IX, and D4-X. The proposed structures are based on the signals integrals (and enzymatic digestion, see text). The anomeric HSQC signals are colored according to the structural elements they represent and numbers show the approximate integrals obtained from volume integration. The two black signals represent the reducing, O6-substituted glucose unit which was present in all oligosaccharides analyzed in this study. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

is able to hydrolyze sections with multiple branched residues in close proximity, if the branched sugar units are adjacent to at least four 1,6linked glucose units. Consequently, neighboring branched backbone units should also result in corresponding oligosaccharides. However, it cannot be excluded that some of the lowly abundant oligosaccharides possess elongated side chains or neighboring 1,4,6-linked glucose units.

VII with oligo-α-1,6-glucosidase. This enzyme specifically cleaves off α1,6-linked glucose units from the non-reducing end of oligosaccharides. Because D4-VII was hydrolyzed to D4-V, it can be concluded that the additional 1,6-linked glucose unit is located after the 1,4,6-linked glucose unit as shown in Fig. 4. The C1H1 and C4H4 signal integrals of oligosaccharide D4-IX showed that one terminal O6-bound, two terminal O4-bound glucose units, and thus also two 1,4,6-linked glucose units are present. Furthermore, the presence of two glucose units bound to an 1,4,6linked glucose unit demonstrated that the two O4-branched residues are not directly linked with each other. Under the assumption that the remaining 1,6-linked glucose unit is located after the reducing end and before the first 1,4,6-linked glucose unit, the structure shown in Fig. 4 can be derived. Oligosaccharide D4-X yielded the same signal integrals than D4-IX, except for the HSQC signal at 4.96/98.5 ppm which represented five anomeric signals. From the number of terminal, O6-bound glucose units (one) and 1,4,6-linked glucose units (two) it can be derived that an additional 1,6-linked glucose unit is present compared to D4-IX. Although this residue cannot be unambiguously located due to signal overlap, the predominant appearance of oligosaccharides with two 1,6linked glucose units on the reducing and non-reducing end strongly suggest that the additional 1,6-linked glucose unit is located between the two 1,4,6-linked glucose units (Fig. 4). The characterized oligosaccharides clearly demonstrate that the L. reuteri dextran is mainly branched with single glucose units which are bound to position O4. In addition, characterization of the nona- and decasaccharides D4-IX and D4-X suggested that at least one 1,6-linked glucose unit is located between most branched backbone residues. The structures of these two oligosaccharides also show that endo-dextranase

3.2.2. Oligosaccharides from O3-branched dextrans Just as for oligosaccharide D4-IV, two anomeric HSQC signals (besides the signals of the reducing, O6-substituted glucose unit) were observed for oligosaccharide D3-IV. The HSQC volume integrals and the HSQC-TOCSY correlations along F1 showed that the signal at 5.35/ 99.8 ppm represents one terminal glucose unit (a) and that the signal at 4.97/98.5 ppm represents one O6-substituted (A) and one terminal (B) glucose unit with almost identical carbon shifts (Table 3). However, the assignment of unit A and unit B was possible due to the HSQC-TOCSY correlations starting from the isolated signals at 3.92/ 70.7 ppm and 3.85/80.4 ppm (Fig. 5). By using the COSY and HMBC spectra it was furthermore possible to assign all other signals of the two sugar units and to identify the two signals as C5H5 of unit A and C3H3 of unit B. Because of the characteristically shifted C6H6 of unit A and because the 1H and 13C chemical shifts of unit A were comparable to unit A of D4-IV, it can be concluded that this glucose moiety is 1,6-linked. Furthermore, the characteristic downfield shift of BC3H3 of the terminal unit B suggests substitution at position O3. This was confirmed by an HMBC correlation between H1 of the remaining terminal unit a and C3 of unit B. Thus, the structure shown in Fig. 6 was determined for oligosaccharide D3-IV. An identical oligosaccharide was already characterized by Maina et al. (2011) and the 1H and 13C chemical shifts were in good

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correlation between C3 of unit B and H1 of unit a confirmed the presence of an O3-bound side chain. However, the C3H3 signal of unit B was slightly shifted in both dimensions, indicating modification of the side chain. This assumption was confirmed by the clearly different chemical shifts of unit a. Especially the clearly downfield shifted C6H6 signals showed that the side chain of this oligosaccharide is elongated at position O6. In addition, the C5H5 correlation was characteristically downfield shifted in the proton dimension which is in good agreement with the data provided by Maina et al. (2011). Therefore, it can be concluded that the additional glucose unit in oligosaccharide D3-Va (unit b) is bound to position O6 of unit a. The chemical shifts of unit b, which were assigned by using the distinct anomeric HSQC(-TOCSY) signal at 4.96/98.4 ppm confirmed the presence of an O6-bound, terminal glucose unit and led to the structure shown in Fig. 6. The anomeric HSQC signals of the second pentasaccharide D3-Vb were located at the same position than in D3-Va. However, it was again possible to assign the remaining signals by using the HSQC-TOCSY, COSY, H2BC, and HMBC spectra. Because unit A yielded the same chemical shifts than unit A from oligosaccharide D3-Va, it can be concluded that D3-Vb also contains a 1,6-linked disaccharide at the reducing end. Unit T showed identical chemical shifts than unit b from oligosaccharide D3Va and was therefore identified as a terminal O6-bound glucose unit. Only unit B and unit a showed some deviating and characteristic signals which allowed for the identification of the structural difference between the two pentasaccharides: The chemical shifts of the C6H6 (3.76/ 3.80/60.8 ppm) and C5H5 (4.00/72.4 ppm) signals of unit a demonstrated that a terminal side chain is present in D3-Vb. In contrast, the chemical shifts of the C6H6 (3.76/3.97/66.2 ppm) and C5H5 (3.92/ 70.7 ppm) signals of unit B were characteristic for an O6-substituted glucose unit. Therefore, it was concluded that unit T is bound to position O6 of unit B (which was also demonstrated by a corresponding HMBC signal), leading to the structure shown in Fig. 6. A mixture of the two pentasaccharides D3-Va and D3-Vb was also characterized by Maina et al. (2011) which suggests that these two hydrolysis products are widely distributed among dextrans. Apart from one HSQC signal at 3.88/70.8 ppm (Fig. S6), the HSQC spectrum of oligosaccharide D3-VI and oligosaccharide D3-Va were identical. The presence of a terminal, O3-branched glucose unit and an elongated side chain was additionally confirmed by the diagnostic HSQC signals and the HSQC-TOCSY correlations (Fig. S6). Furthermore, volume integration and analysis of the other two-dimensional NMR spectra demonstrated that the signal at 3.88/70.8 ppm results from the C5H5 correlation of an additional 1,6-linked glucose unit. Because of the complete overlap of all other signals of this unit, it was not possible to unambiguously determine the location of this unit by NMR spectroscopy. To evaluate the location of the additional 1,6-linked glucose unit, D3-VI was hydrolyzed by oligo-α-1,6-glucosidase which resulted in the formation of D3-IV. Therefore, it can be concluded that D3-VI contains a trimeric side chain as showed in Fig. 6. These results are also in good agreement with the characterization of D4-VII which also carried a trimeric 1,6-linked residue on the non-reducing end. Maina et al. (2011) also isolated a hexasaccharide with an elongated O3bound side chain. However, the oligosaccharide characterized in their study carried a dimeric side chain and an additional glucose unit bound to position O6 of the O3-branched glucose unit. Therefore, oligosaccharide D3-VI may be specific for the dextrans investigated in this study. The oligosaccharides isolated from the endo-dextranase hydrolysates of the O3-branched dextrans show that all of the dextrans used

Table 3 1 H and 13C chemical shifts of the structural units of the isomalto-oligosaccharides isolated from the endo-dextranase hydrolysate of L. curvatus, L. hilgardii, and L. hordei dextran. The structures of the oligosaccharides are shown in Fig. 6. Compound/Structural unit

1

2

3

4

5

6-1

6-2

R-α

5.24 92.8 4.67 96.8 4.97 98.5 4.97 98.5 5.35 99.8

D3-IV 3.53 72.1 3.25 74.7 3.56 72.2 3.66 70.8 3.56 72.2

3.70 73.7 3.48 76.7 3.72 73.7 3.85 80.4 3.74 73.7

3.51 70.1 3.51 70.1 3.53 70.1 3.66 70.8 3.45 70.0

4.01 70.7 3.64 74.9 3.92 70.7 3.72 72.4 4.01 72.3

3.70 66.4 3.76 66.4 3.74 66.0 3.77 60.9 3.76 60.7

4.01 66.4 3.98 66.4 3.99 66.0 3.85 60.9 3.80 60.7

5.24 92.8 4.67 96.7 4.98 98.5 4.98 98.4 5.33 100.0 4.96 98.4

D3-Va 3.53 72.1 3.25 74.7 3.56 72.1 3.65 70.7 3.56 72.2 3.56 72.2

3.70 73.8 3.47 76.6 3.72 73.8 3.82 80.9 3.72 73.8 3.72 73.8

3.51 70.1 3.51 70.1 3.53 70.1 3.65 70.7 3.53 70.0 3.42 70.2

4.01 70.7 3.64 74.8 3.92 70.6 3.72 72.2 4.20 70.9 3.70 72.4

3.70 66.4 3.76 66.4 3.74 66.0 3.76 60.9 3.67 65.8 3.77 61.1

4.01 66.4 3.98 66.4 3.99 66.0 3.83 60.9 4.01 65.8 3.84 61.1

5.24 92.8 4.67 96.8 4.98 98.4 4.98 98.4 4.96 98.5 5.32 100.0

D3-Vb 3.53 72.1 3.25 74.8 3.56 72.2 3.67 70.8 3.54 72.2 3.56 72.2

3.70 73.7 3.48 76.7 3.72 73.9 3.84 81.1 3.72 73.7 3.74 73.7

3.51 70.1 3.51 70.1 3.53 70.1 3.76 70.5 3.42 70.2 3.45 70.0

4.01 70.7 3.64 74.9 3.92 70.7 3.92 70.7 3.71 72.6 4.00 72.4

3.70 66.4 3.76 66.4 3.74 66.0 3.76 66.2 3.77 61.0 3.76 60.8

4.01 66.4 3.98 66.4 3.99 66.0 3.97 66.2 3.84 61.0 3.80 60.8

5.24 92.8 4.67 96.7 4.98 98.4 4.98 98.3 5.33 100.0 4.98 98.3 4.96 98.3

D3-VI 3.53 72.1 3.25 74.7 3.56 72.1 3.66 70.6 3.56 72.1 3.56 72.0 3.56 72.1

3.70 73.7 3.47 76.6 3.72 73.7 3.83 80.9 3.72 73.7 3.72 73.7 3.72 73.7

3.51 70.1 3.51 70.1 3.53 70.1 3.66 70.6 3.53 70.0 3.53 70.0 3.42 70.1

4.01 70.6 3.64 74.8 3.92 70.6 3.72 72.2 4.21 70.9 3.88 70.8 3.72 72.3

3.70 66.4 3.76 66.4 3.74 66.0 3.76 60.9 3.67 65.8 3.74 65.8 3.77 61.0

4.01 66.4 3.98 66.4 3.99 66.0 3.83 60.9 4.04 65.8 3.99 65.8 3.84 61.0

R-β A B a

R-α R-β A B a b

R-α R-β A B T a

R-α R-β A B a b c

agreement. Therefore, this oligosaccharide is a major hydrolysis product from the hydrolysis of structurally different dextrans with endodextranase. For oligosaccharide D3-Va, the HSQC-TOCSY spectrum also allowed for the assignment of unit A and B which showed almost identical chemical shifts than in oligosaccharide D3-IV. Furthermore, an HMBC

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Fig. 5. HSQC and HSQC-TOCSY spectra of the oligosaccharide D3-IV. The signals used to assign the remaining signals of unit A and unit B are marked in blue (A) and red (B). The structure of D3-IV is shown in Fig. 6. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

contain both monomeric as well as oligomeric (and possibly polymeric) side chains. However, in case of the L. hilgardii dextran it has to be considered that the 1,3-linked glucose units detected by methylation analysis are not (completely) hydrolyzed by endo-dextranase. Therefore, different structural elements may be present in the polysaccharide areas which contain these glycosidic linkages. Nevertheless, the ratios of the endo-dextranase liberated oligosaccharides show that the α-1,6-linked areas of L. hilgardii and L. curvatus dextrans most likely have a comparable structural architecture. In contrast, dextrans from L. hordei show a significantly higher abundance of D3-Va and D3-VI (and a comparably low abundance of D3-IV). Consequently, this dextran mainly contains elongated side chains, an information which can only be assessed by chromatographic analyses of the endo-dextranase hydrolysates. Furthermore, only very low portions of D3-Vb were detected in the L. hordei dextran suggesting that the liberation of this oligosaccharide is not random but depends on the architecture of the dextrans (e.g. the distance between branched backbone units). Consequently, the oligosaccharide portions in the hydrolysate of the L. reuteri dextran may also be a result of the distribution of branched backbone units within the dextran.

used in this study mainly contains monomeric side chains. Because all predominantly abundant oligosaccharides contained at least one 1,6linked glucose unit adjacent to a 1,4,6-linked glucose unit and because no oligosaccharides with neighboring branched backbone residues could be isolated, it is likely that neighboring branched backbone residues are absent or rather rare. In case of the O3-branched dextrans from L. hordei, L. hilgardii and L. curvatus, the characterized oligosaccharides showed that side chain elongation occurs in all of these polysaccharides to some extent. However, a clearly higher portion of elongated side chains were detected in L. hordei dextrans (derived from the high relative abundance of oligosaccharide D3-Va and D3-VI), whereas L. hilgardii and L. curvatus dextran hydrolysates contained an additional oligosaccharide and higher portions of oligosaccharides with monomeric side chains. Overall, the oligosaccharide profiles are an ideal supplemental method to methylation analysis because detailed information can be obtained in a comparably short time. Funding sources This work was supported by the Carl-Zeiss Foundation.

4. Conclusions Acknowledgements

Overall, the isolation and characterization of endo-dextranase liberated isomalto-oligosaccharides from different dextrans yielded valuable information on the polysaccharide fine structures. From the oligosaccharide structures and their abundance in the enzymatic hydrolysate, it could be derived that the O4-branched L. reuteri dextran

The authors are very grateful to Frank Jakob and Rudi Vogel from the Chair of Technical Microbiology (TU Munich) for providing the E. coli clones and the fermentatively produced dextrans.

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Fig. 6. Structures of isomalto-oligosaccharides isolated from the endo-dextranase hydrolysate of L. curvatus, L. hilgardii, and L. hordei dextran. The italic letters are used to describe the sugar units in Table 3.

Appendix A. Supplementary data

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