Ndegwa Henry Maina

In situ production and analysis of Weissella confusa dextran in wheat sourdough

Several lactic acid bacteria belonging to the genera Leuconostoc, Lactobacillus, and Weissella have been introduced to wheat sourdough baking for in situ production of exopolysaccharides. This is considered a novel method for improving the shelf-life, volume and nutritional value of bread without additives. However, in situ production of exopolysaccharides during sourdough fermentation is challenged by simultaneous acidification due to metabolic activities of the bacteria, which may significantly diminish the positive technological impact of exopolysaccharides. In this study, the growth, activity and in situ production of dextran by Weissella confusa VTT E-90392 in wheat sourdoughs were investigated. Furthermore, the influence of dextran-enriched sourdoughs, at the addition level of 43%, on the subsequent bread quality was established. W. confusa efficiently produced dextran from the added sucrose in wheat sourdough without strong acid production. A new specific enzyme-assisted method for in situ analysis of dextran in sourdoughs was developed. With this method, we could for the first time proof significant (11-16 g/kg DW) production of polymeric dextran in sourdoughs. Concomitant formation of shorter isomaltooligosaccharides by W. confusa was also detected. The produced dextran significantly increased the viscosity of the sourdoughs. Application of dextran-enriched sourdoughs in bread baking provided mildly acidic wheat bread with improved volume (up to 10%) and crumb softness (25-40%) during 6 days of storage. Hence, W. confusa is a promising new strain for efficient in situ production of dextrans and isomaltooligosaccharides in sourdoughs without strong acidification.

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa.

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.

Weissella confusa Cab3 dextransucrase: Properties and in vitro synthesis of dextran and glucooligosaccharides

Food-derived Weissella spp. have gained attention during recent years as efficient dextran producers. Weissella confusa Cab3 dextransucrase (WcCab3-DSR) was isolated applying PEG fractionation and used for in vitro synthesis of dextran and glucooligosaccharides. WcCab3-DSR had a molar mass of 178 kDa and was activated by Co(2+) and Ca(2+) ions. Glycerol and Tween 80 enhanced enzyme stability, and its half-life at 30°C increased from 10h to 74 h and 59 h, respectively. The (1)H and (13)C NMR spectral analysis of the produced dextran confirmed the presence of main chain α-(1→6) linkages with only 3.0% of α-(1→3) branching, of which some were elongated. An HPSEC analysis in DMSO revealed a high molecular weight of 1.8 × 10(7)g/mol. Glucooligosaccarides produced through the acceptor reaction with maltose, were analyzed with HPAEC-PAD and ESI-MS/MS. They were a homologous series of isomaltooligosaccharides with reducing end maltose units. To the best of our knowledge, this is a first report on native W. confusa dextransucrase.

Structural Analysis of Enzyme-Resistant Isomaltooligosaccharides Reveals the Elongation of α-(1→3)-Linked Branches in Weissella confusa Dextran

Weissella confusa VTT E-90392 is an efficient producer of a dextran that is mainly composed of α-(1→6)-linked D-glucosyl units and very few α-(1→3) branch linkages. A mixture of the Chaetomium erraticum endodextranase and the Aspergillus niger α-glucosidase was used to hydrolyze W. confusa dextran to glucose and a set of enzyme-resistant isomaltooligosaccharides. Two of the oligosaccharides (tetra- and hexasaccharide) were isolated in pure form and their structures elucidated. The tetrasaccharide had a nonreducing end terminal α-(1→3)-linked glucosyl unit (α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc), whereas the hexasaccharide had an α-(1→3)-linked isomaltosyl side group (α-D-Glcp-(1→6)[α-D-Glcp-(1→6)-α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). A mixture of two isomeric oligosaccharides was also obtained in the pentasaccharide fraction, which were identified as (α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc) and (α-D-Glcp-(1→6)[α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). The structures of the oligosaccharides indicated that W. confusa dextran contains both terminal and elongated α-(1→3)-branches. This is the first report evidencing the presence of elongated branches in W. confusa dextran. The (1)H and (13)C NMR spectroscopic data on the enzyme-resistant isomaltooligosaccharides with α-(1→3)-linked glucosyl and isomaltosyl groups are published here for the first time.

The impact of fermentation with exopolysaccharide producing lactic acid bacteria on rheological, chemical and sensory properties of pureed carrots (Daucus carota L.)

Fermentation with lactic acid bacteria (LAB) offers a natural means to modify technological and nutritional properties of foods and food ingredients. This study explored the impact of fermentation with different exopolysaccharide (EPS) producing LAB on rheological, chemical and sensory properties of puréed carrots in water, as a vegetable model, with the focus on texture formation. The screening of 37 LAB strains for starter selection revealed 16 Lactobacillus, Leuconostoc and Weissella strains capable of EPS (dextran, levan, and/or β-glucan) production in the carrot raw material. Fermentations with five out of six selected EPS producers modified perceived texture of the liquid carrot model (p<0.05). The formation of low-branched dextran correlated with perceived thickness, whereas the production of β-glucan correlated with perceived elasticity. Low-branched dextran producing Weissella confusa and Leuconostoc lactis strains produced thick texture accompanied by pleasant odour and flavour. The fermentation with the selected EPS-producing LAB strains is a promising clean label approach to replace hydrocolloid additives as texturizers in vegetable containing products, not only carrot.

Challenges in analysis of high-molar mass dextrans: Comparison of HPSEC, AsFlFFF and DOSY NMR spectroscopy

Dilute solutions of various dextran standards, a high-molar mass (HMM) commercial dextran from Leuconostoc spp., and HMM dextrans isolated from Weissella confusa and Leuconostoc citreum were analyzed with high-performance size-exclusion chromatography (HPSEC), asymmetric flow field-flow fractionation (AsFlFFF), and diffusion-ordered NMR spectroscopy (DOSY). HPSEC analyses were performed in aqueous and dimethyl sulfoxide (DMSO) solutions, while only aqueous solutions were utilized in AsFlFFF and DOSY. The study showed that all methods were applicable to dextran analysis, but differences between the aqueous and DMSO-based solutions were obtained for HMM samples. These differences were attributed to the presence of aggregates in aqueous solution that were less prevalent in DMSO. The study showed that DOSY provides an estimate of the size of HMM dextrans, though calibration standards may be required for each experimental set-up. To our knowledge, this is the first study utilizing these three methods in analyzing HMM dextrans.

Cloning and Characterization of a Weissella confusa Dextransucrase and Its Application in High Fibre Baking

Wheat bran offers health benefits as a baking ingredient, but is detrimental to bread textural quality. Dextran production by microbial fermentation improves sourdough bread volume and freshness, but extensive acid production during fermentation may negate this effect. Enzymatic production of dextran in wheat bran was tested to determine if dextran-containing bran could be used in baking without disrupting bread texture. The Weissella confusa VTT E-90392 dextransucrase gene was sequenced and His-tagged dextransucrase Wc392-rDSR was produced in Lactococcus lactis. Purified enzyme was characterized using 14C-sucrose radioisotope and reducing value-based assays, the former yielding K m and V max values of 14.7 mM and 8.2 μmol/(mg∙min), respectively, at the pH optimum of 5.4. The structure and size of in vitro dextran product was similar to dextran produced in vivo. Dextran (8.1% dry weight) was produced in wheat bran in 6 h using Wc392-rDSR. Bran with and without dextran was used in wheat baking at 20% supplementation level. Dextran presence improved bread softness and neutralized bran-induced volume loss, clearly demonstrating the potential of using dextransucrases in bran bioprocessing for use in baking.

The oxidative degradation of barley β-glucan in the presence of ascorbic acid or hydrogen peroxide

Cereal β-glucans are polysaccharides with health benefits that have been linked to their ability to increase luminal viscosity. However, the functional properties of cereal β-glucans may be diminished by the susceptibility of this polysaccharide to oxidative degradation. In this study, barley β-glucan was oxidised with hydrogen peroxide or ascorbic acid and the oxidative degradation of β-glucan was investigated using both asymmetrical flow field-flow fractionation (AsFlFFF) with aqueous solvent and high performance size exclusion chromatography (HPSEC) with LiBr in DMSO as the solvent. Oxidation was shown to cause degradation of β-glucan, the reaction being faster when oxidised with hydrogen peroxide compared with ascorbic acid. Both HPSEC and AsFlFFF showed comparable results as long as aggregates (only observed in AsFlFFF) were not included in the integration. The compact aggregates observed in oxidised samples suggest oxidation driven interactions between β-glucan molecules.

Structural analysis of linear mixed-linkage glucooligosaccharides by tandem mass spectrometry.

Dextrans and glucooligosaccharides (GLOS) are produced by lactic acid bacteria (LAB) during sourdough fermentation. The dextrans can act as hydrocolloids in sourdough bread, while the GLOS may have antistaling and prebiotic properties, depending on their structure. Development of high-throughput methods for screening the structural properties of dextrans and GLOS produced by different LAB in varying fermentation conditions is therefore of interest. In this study we explored the possibility of using electrospray ionisation tandem mass spectroscopy (ESI-MS/MS) to unequivocally determine the structures of underivatised GLOS. The emphasis was on linear mixed linked model GLOS, especially those containing (1→3) linkages that are common in dextrans. After evaluation of the model GLOS, the ESI-MS/MS method was used to determine the linkage positions of two mixed-linked tetrasaccharides obtained by hydrolysis of Weissella confusa and Leuconostoc citreum dextrans. In positive mode, only the reducing end linkage could be determined because isomeric fragment ions, present in subsequent MS(n) cycles, hindered assignment of the remaining linkages. By contrast, it was possible to unambiguously assign all the linkages in each GLOS using the negative mode spectra. The present study thus shows that negative mode is the preferred method for ESI-MS/MS structural analysis of underivatised GLOS. In combination with liquid chromatography this method will enable rapid profiling of the structural variation of dextrans and prebiotic GLOS.

Lactose- and cellobiose-derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase

Dextran-producing Weissella have received significant attention. However, except for maltose, the acceptor reactions of Weissella dextransucrases with different sugars have not been investigated. The action of recombinant Weissella confusa VTT E-90392 dextransucrase was tested with several potential acceptors, particularly, analogs lactose and cellobiose. The major acceptor products of both disaccharides were identified as branched trisaccharides, with a glucosyl residue α-(1 → 2)-linked to the acceptors reducing end. An additional product, isomelezitose (6(Fru)-α-Glcp-sucrose), was also produced when using lactose as an acceptor. This is the first report of the synthesis of isomelezitose by a dextransucrase. The NMR spectra of the three trisaccharides were fully assigned, and their structures were confirmed by selective enzymatic hydrolysis. The trisaccharides prepared from (13)C6(glc) sucrose and lactose were analyzed by ESI-MS(n), and the fragmentation patterns of these compounds were characterized.