The C-Terminal Domain of Liquorilactobacillus nagelii Dextransucrase Mediates the Production of Larger Dextrans Compared to Liquorilactobacillus hordei.

Dextransucrases released by certain lactic acid bacteria form glucose polymers with predominantly α-1,6-linkages and may be exploited biotechnologically for the tailored production of polysaccharides with application potential. Despite releasing two closely related dextransucrases, previous studies showed that water kefir borne Liquorilactobacillus (L.) hordei TMW 1.1822 and L. nagelii TMW 1.1827 produce different amounts of polysaccharides with distinct particle sizes (molecular weight and radius of gyration) and molecular architectures. To investigate where these differences originate and thus to provide deeper insights into the functionally diverse nature of polysaccharide formation during water kefir fermentation, we constructed two variants of the L. nagelii dextransucrase-a full-length enzyme and a truncated variant, devoid of a C-terminal glucan-binding domain that reflects the domain architecture of the L. hordei dextransucrase-and applied them at various enzyme concentrations to form dextran over 24 h. The full-length enzyme exhibited a high activity, forming constant amounts of dextran until a four-fold dilution, whereas the truncated variant showed a gradual decrease in activity and dextran formation at an increasing dilution. The application of the full-length enzyme resulted in higher average particle sizes compared to the truncated variant. However, the dilution of the enzyme extracts also led to a slight increase in the average particle size in both enzymes. Neither the domain architecture nor the enzyme concentration had an impact on the structural architecture of the dextrans. The presented results thus suggest that the comparatively higher processivity of the L. nagelii dextransucrase is predominantly caused by the additional C-terminal glucan-binding domain, which is absent in the L. hordei dextransucrase. The average particle size may be influenced, to some extent, by the applied reaction conditions, whereas the structural architecture of the dextrans is most likely caused by differences in the amino acid sequence of the catalytic domain.

Bombella favorum sp. nov. and Bombella mellum sp. nov., two novel species isolated from the honeycombs of Apis mellifera.

As part of a study investigating the microbiome of bee hives and honey, two novel strains (TMW 2.1880T and TMW 2.1889T) of acetic acid bacteria were isolated and subsequently taxonomically characterized by a polyphasic approach, which revealed that they cannot be assigned to known species. The isolates are Gram-stain-negative, aerobic, pellicle-forming, catalase-positive and oxidase-negative. Cells of TMW 2.1880T are non-motile, thin/short rods, and cells of TMW 2.1889T are motile and occur as rods and long filaments. Morphological, physiological and phylogenetic analyses revealed a distinct lineage within the genus Bombella. Strain TMW 2.1880T is most closely related to the type strain of Bombella intestini with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 94.16 and 56.3 %, respectively. The genome of TMW 2.1880T has a size of 1.98 Mb and a G+C content of 55.3 mol%. Strain TMW 2.1889T is most closely related to the type strain of Bombella apis with a 16S rRNA gene sequence similarity of 99.5 %, and ANIb and in silico DDH values of 85.12 and 29.5 %, respectively. The genome of TMW 2.1889T has a size of 2.07 Mb and a G+C content of 60.4 mol%. Ubiquinone analysis revealed that both strains contained Q-10 as the main respiratory quinone. Major fatty acids for both strains were C16 : 0, C19 : 0 cyclo ω8c and summed feature 8, respectively, and additionally C14 : 0 2-OH only for TMW 2.1880T and C14 : 0 only for TMW 2.1889T. Based on polyphasic evidence, the two isolates from honeycombs of Apis mellifera represent two novel species of the genus Bombella, for which the names Bombella favorum sp. nov and Bombella mellum sp. nov. are proposed. The designated respective type strains are TMW 2.1880T (=LMG 31882T=CECT 30114T) and TMW 2.1889T (=LMG 31883T=CECT 30113T).

Insights into extracellular dextran formation by Liquorilactobacillus nagelii TMW 1.1827 using secretomes obtained in the presence or absence of sucrose.

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.

ß-glucan formation is a selective advantage for beer-spoiling Levilactobacillus brevis TMW 1.2112 during planktonic growth.

Some lactic acid bacteria (LAB) isolated from beer or wine produce capsular ß-glucans from UDP-glucose via the membrane-anchored glycosyltransferase GTF-2. This phenomenon is feared in breweries, because the viscosity of the affected liquids drastically increases due to the ß-glucan and concomitant pellicle formation of these LAB. Currently it is unknown if this type of polysaccharide formation provides any advantage for the producing LAB during the colonization of (ethanol-containing) liquids. We thus used the ß-glucan producer Levilactobacillus (L.) brevis TMW 1.2112 and its ß-glucan-deficient transposon mutant (Δ gtf-2), and compared their growth at different ethanol concentrations and their competitiveness during co-cultivation. No significant inhibition in growth and differences in acidification were observed for both strains up to ethanol concentrations of 8% (v/v). At 10 % ethanol, the ß-glucan forming wildtype increased its cell number and produced more acid in comparison to the mutant strain, which settled at the bottom of the fermentation tubes at any tested condition. At higher ethanol concentrations (12-18 % v/v) both strains failed to grow, while a higher viability of the wildtype strain was observed. After co-cultivation of both strains for up to 72 h in liquid nutrient medium (without ethanol), significantly more ropy wildtype colonies were detected, if the wildtype had been initially applied in similar cell counts or in excess. By contrast, the number of smooth mutant colonies was solely significantly higher after 24 h of growth, if the mutant strain had been initially inoculated in excess. These results indicate that the ß-glucan-mediated pellicle formation by L. brevis TMW 1.2112 is its dominant phenotype and a selective advantage during colonization of liquids.

Analysis of Structural and Functional Differences of Glucans Produced by the Natively Released Dextransucrase of Liquorilactobacillus hordei TMW 1.1822.

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.

Living the Sweet Life: How Liquorilactobacillus hordei TMW 1.1822 Changes Its Behavior in the Presence of Sucrose in Comparison to Glucose.

Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei) is one of the dominating lactic acid bacteria within the water kefir consortium, being highly adapted to survive in this environment, while producing high molecular weight dextrans from sucrose. In this work, we extensively studied the physiological response of L. hordei TMW 1.1822 to sucrose compared to glucose, applying label-free, quantitative proteomics of cell lysates and exoproteomes. This revealed the differential expression of 53 proteins within cellular proteomes, mostly associated with carbohydrate uptake and metabolism. Supported by growth experiments, this suggests that L. hordei TMW 1.1822 favors fructose over other sugars. The dextransucrase was expressed irrespectively of the present carbon source, while it was significantly more released in the presence of sucrose (log2FC = 3.09), being among the most abundant proteins within exoproteomes of sucrose-treated cells. Still, L. hordei TMW 1.1822 expressed other sucrose active enzymes, predictively competing with the dextransucrase reaction. While osmolysis appeared to be unlikely, sucrose led to increased release of a multitude of cytoplasmic proteins, suggesting that biofilm formation in L. hordei is not only composed of a polysaccharide matrix but is also of proteinaceous nature. Therefore, our study highlights the intrinsic adaptation of water kefir-borne L. hordei to sucrose-rich habitats and provides fundamental knowledge for its use as a starter culture in plant-based food fermentations with in situ dextran formation.

High yield production of levan-type fructans by Gluconobacter japonicus LMG 1417.

Levan, a ß-2,6-glycosidic linked fructan, is a promising alternative for the inulin dominated fructan market. Although levan is already used in some cosmetic products, the commercial availability of the fructan is still limited. Here we show that Gluconobacter japonicus LMG 1417 is a potent levan-forming organism and a promising platform for the industrial production of levan. The levansucrase LevS1417, which is produced by G. japonicus LMG 1417 and secreted by a signal-peptide-independent pathway, exhibited extraordinary high activity (4726 ±â€¯821 U mg-1 at 50 °C). A cell-free levan production based on the supernatant of the investigated strain led to a final levan yield of 157.9 ±â€¯7.6 g L-1. The amount of secreted levansucrase was more than doubled by plasmid-mediated homologous overproduction of LevS1417 in G. japonicus LMG 1417. Accordingly, the space-time yield of cell-free levan production was doubled using the plasmid-bearing mutant.

Persistence and ß-glucan formation of beer-spoiling lactic acid bacteria in wheat and rye sourdoughs.

Some beverage-spoiling lactic acid bacteria (LAB) produce capsular ß-glucans from UDP-glucose, which is accompanied by cell network formation causing viscosity increases of liquids. This feature of certain LAB is feared in breweries but could be useful for structural and nutritional improvement of baked goods, provided that these LAB are suited for the manufacture of sourdoughs. The aim of this study was to investigate the persistence and ß-glucan formation of the brewery isolates Levilactobacillus (L.) brevis TMW 1.2112 and Pediococcus (P.) claussenii TMW 2.340 in wheat and rye sourdoughs. Both the wild-type strains and the respective ß-glucan-deficient mutants were dominant in wheat and rye sourdoughs and acidified them to characteristic pH ranges. The formation of ß-glucan capsules during sourdough fermentations was stable in L. brevis TMW 1.2112 in contrast to P. claussenii TMW 2.340. Wheat sourdoughs fermented with the ß-glucan producing L. brevis TMW 1.2112 cells were significantly more viscous than doughs fermented by the P. claussenii TMW 2.340 cells and the applied mutant strains. In conclusion, L. brevis TMW 1.2112 and P. claussenii TMW 2.340 were suited and persistent wheat and rye sourdough starters, while the in situ ß-glucan formation in sourdoughs was hardly detectable in case of P. claussenii.

Insights into the pH-dependent, extracellular sucrose utilization and concomitant levan formation by Gluconobacter albidus TMW 2.1191.

Many bacteria and archaea produce the polydisperse fructose polymer levan from sucrose upon biofilm formation via extracellular levansucrases (EC 2.4.1.10). We have investigated levansucrase-release and -activities as well as molecular size of the levan formed by the acetic acid bacterium Gluconobacter albidus TMW 2.1191 at varying environmental pH conditions to obtain insight in the ecological role of its constitutively expressed levansucrase and the produced levan. A buffer system was established enabling the recovery of levansucrase-containing supernatants from preincubated cell suspensions at pH 4.3-pH 5.7. The enzyme solutions were used to produce levans at different pH values and sucrose concentrations. Finally, the amounts and size distributions of the produced levans as well as the corresponding levansucrase activities were determined and correlated with each other. The data revealed that the levansucrase was released into the environment independently of its substrate sucrose, and that more levansucrase was released at pH ≥ 5.0. The glucose release and formation of high molecular weight levans (> 3.5 kDa) from 0.1 M initial sucrose was comparable between pH ~ 4.3-5.7 using equal amounts of released levansucrase. Hence, this type of levansucrase appears to be structurally adapted to changes in the extracellular pH and to exhibit a similar total activity over a wide acidic pH range, while it produced higher amounts of larger levan molecules at higher production pH and sucrose concentrations. These findings indicate the physiological adaptation of G. albidus TMW 2.1191 to efficient colonisation of sucrose-rich habitats via released levansucrases despite changing extracellular pH conditions in course of acid formation.

Size-Dependent Variability in Flow and Viscoelastic Behavior of Levan Produced by Gluconobacter albidus TMW 2.1191.

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 (<107 Da), produced without adjustment of the pH, exhibited Newton-like flow behavior up to a specific concentration of 25% (w/v). By contrast, larger levans (>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.

A systematic approach to study the pH-dependent release, productivity and product specificity of dextransucrases.

BACKGROUND: Dextransucrases are extracellular enzymes, which catalyze the formation of α-1→6-linked glucose polymers from sucrose. These enzymes are exclusively expressed by lactic acid bacteria, which commonly acidify the extracellular environment due to their physiology. Dextransucrases are thus confronted with steadily changing reaction conditions in regards to the environmental pH, which can further affect the amount of released dextransucrases. In this work, we studied the effect of the environmental pH on the release, the productivity and the product specificity of the dextransucrase expressed by Lactobacillus (L.) hordei TMW 1.1822. Dextransucrases were recovered as crude extracts at pH 3.5-pH 6.5 and then again used to produce dextrans at these pH values. The respectively produced dextran amounts and sizes were determined and the obtained results finally systematically correlated. RESULTS: Maximum dextran amounts were produced at pH 4.0 and pH 4.5, while the productivity of the dextransucrases significantly decreased at pH 3.5 and pH 6.5. The distribution of dextran amounts produced at different pH most likely reflects the pH dependent activity of the dextransucrases released by L. hordei, since different transglycosylation rates were determined at different pH using the same dextransucrase amounts. Moreover, similar hydrolysis activities were detected at all tested conditions despite significant losses of transglycosylation activities indicating initial hydrolysis prior to transglycosylation reactions. The molar masses and rms radii of dextrans increased up to pH 5.5 independently of the stability of the enzyme. The gelling properties of dextrans produced at pH 4.0 and pH 5.5 were different. CONCLUSIONS: The presented methodological approach allows the controlled production of dextrans with varying properties and could be transferred and adapted to other microbes for systematic studies on the release and functionality of native sucrases or other extracellular enzymes.

Acetic acid bacteria encode two levansucrase types of different ecological relationship.

Acetic acid bacteria (AAB) are associated with plants and insects. Determinants for the targeting and occupation of these widely different environments are unknown. However, most of these natural habitats share plant-derived sucrose, which can be metabolized by some AAB via polyfructose building levansucrases (LS) known to be involved in biofilm formation. Here, we propose two LS types (T) encoded by AAB as determinants for habitat selection, which emerged from vertical (T1) and horizontal (T2) lines of evolution and differ in their genetic organization, structural features and secretion mechanism, as well as their occurrence in proteobacteria. T1-LS are secreted by plant-pathogenic α- and γ-proteobacteria, while T2-LS genes are common in diazotrophic, plant-growth-promoting α-, ß- and γ-proteobacteria. This knowledge may be exploited for a better understanding of microbial ecology, plant health and biofilm formation by sucrase-secreting proteobacteria in eukaryotic hosts.

In situ production and characterization of cloud forming dextrans in fruit-juices.

Turbidity in beverages is typically achieved by addition of emulsion based cloud systems. Their intrinsic instability necessitates the widespread use of technological measures and use of food additives to prevent emulsion decay. In this work, we explored the possibility to establish a new generation of natural, stable clouding systems based on bacterial dextrans. Lactobacillus hordei TMW 1.1907 originating from water kefir was used to produce dextrans in sucrose supplemented apple or grape juices. By varying the fermentation conditions, two distinct types of dextran molecules could be produced at yields ranging from 2.5 to 8.5 g/L. The dextran-containing fermentates showed an unchanged turbidity after pasteurization at acidic pH and subsequent storage for three months. No sedimentation of particles occurred upon storage. Neutralization of the acidic fruit juices to pH 7 prior to fermentation significantly increased the dextran yields. The molecular weight, rms radii and turbidity of dextrans produced at 20 °C were higher than those produced at 30 °C. Characterization of the isolated dextrans by asymmetric flow field-flow fractionation coupled to multi-angle laser light scattering revealed a random-coil like structure and rms radii ranging from 66.0 to 87.4 nm. The averaged molar masses of the cloud forming dextrans were in the approximate range of 103.1 to 141.6 MDa. In conclusion, our results demonstrate the possibility to ferment fruit juices for in situ production of dextrans exhibiting novel techno-functional properties beyond gelling and thickening.

Identification and comparison of two closely related dextransucrases released by water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827.

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 (Mw) of 7.9×107 Da, while those produced by the dextransucrase released by L. hordei exhibited an Mw 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.

Detailed Structural Characterization of Glucans Produced by Glucansucrases from Leuconostoc citreum TMW 2.1194.

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.

Sucrose-Induced Proteomic Response and Carbohydrate Utilization of Lactobacillus sakei TMW 1.411 During Dextran Formation.

Lactobacillus (L.) sakei belongs to the dominating lactic acid bacteria in indigenous meat fermentations, while diverse strains of this species have also been isolated from plant fermentations. We could recently show, that L. sakei TMW 1.411 produces a high molecular weight dextran from sucrose, indicating its potential use as a dextran forming starter culture. However, the general physiological response of L. sakei to sucrose as carbohydrate source has not been investigated yet, especially upon simultaneous dextran formation. To address this lack of knowledge, we sequenced the genome of L. sakei TMW 1.411 and performed a label-free, quantitative proteomics approach to investigate the sucrose-induced changes in the proteomic profile of this strain in comparison to its proteomic response to glucose. In total, 21 proteins were found to be differentially expressed at the applied significance criteria (FDR ≤ 0.01). Among these, 14 were associated with the carbohydrate metabolism including several enzymes, which enable sucrose and fructose uptake, as well as, their subsequent intracellular metabolization, respectively. The plasmid-encoded, extracellular dextransucrase of L. sakei TMW 1.411 was expressed at high levels irrespective of the present carbohydrate and was predominantly responsible for sucrose consumption in growth experiments using sucrose as sole carbohydrate source, while the released fructose from the dextransucrase reaction was more preferably taken up and intracellularly metabolized than sucrose. Genomic comparisons revealed, that operons coding for uptake and intracellular metabolism of sucrose and fructose are chromosomally conserved among L. sakei, while plasmid-located dextransucrase genes are present only in few strains. In accordance with these findings, all 59 different L. sakei strains of our strain collection were able to grow on sucrose as sole carbohydrate source, while eight of them exhibited a mucous phenotype on agar plates indicating dextran formation from sucrose. Our study therefore highlights the intrinsic adaption of L. sakei to plant environments, where sucrose is abundant, and provides fundamental knowledge regarding the use of L. sakei as starter culture for sucrose-based food fermentation processes with in-situ dextran formation.

Structural characterization of the surface-associated heteropolysaccharide of Lactobacillus plantarum TMW 1.1478 and genetic analysis of its putative biosynthesis cluster.

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.

Lactobacillus hordei dextrans induce Saccharomyces cerevisiae aggregation and network formation on hydrophilic surfaces.

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.

Structural characterization of the exopolysaccharides from water kefir.

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.

Influence of Different Sugars and Initial pH on ß-Glucan Formation by Lactobacillus brevis TMW 1.2112.

We wanted to identify key factors influencing the extent of ß-glucan production by Lactobacillus brevis TMW 1.2112, which has been isolated from viscous, spoiled beer and which could contribute to viscosity increases of spoiled beverages via exopolysaccharide (EPS) production. In this way, we analyzed the influence of different initial pH values and carbohydrate sources on growth of and slime/ß-glucan formation by this strain. In a screening of 48 carbohydrates, 14 fermentable sugars which enabled growth were identified. These sugars were further investigated regarding their EPS formation-promoting properties. The hexose-based mono- and di-saccharides enabled slime formation, while all pentoses failed to cause any thickening effect. The strongest slime formation was observed upon growth on D-maltose, the weakest on D-fructose. A lower initial pH (4.3) caused significant higher viscosities than an initially higher one (pH 6.2). This effect was independent from the carbohydrate supplied. Although the thickening of nutrient media by L. brevis TMW 1.2112 strongly depended on the initial pH and the available carbon source, all isolated polysaccharides were exclusively composed of glucose moieties and exhibited highly similar elution profiles after separation via asymmetric flow field-flow fractionation independently of the provided carbon source. Our results suggest that the extent of ß-glucan/slime formation by special L. brevis strains isolated from the brewery environment is strongly influenced by the initial pH and the availability of certain EPS formation-promoting sugars with maltose being the most favored carbohydrate for the regulated and directive biosynthesis of capsular ß-glucan.