The exopolysaccharides produced by Leuconostoc mesenteroides XR1 and its effect on silk drawing phenomenon of yoghurt.

Yoghurt fermented by Leuconostoc mesenteroides XR1 from Kefir grains was found to produce a unique silk drawing phenomenon. This property was found to be associated with the exopolysaccharides (EPS), X-EPS, produced by strain XR1. In order to better understand the mechanism that produced this phenomenon, the X-EPS was extracted, purified and characterized. The molecular weight and monosaccharide composition were determined by size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and ion chromatography (IC) analysis, respectively. The results showed that its molecular weight was 4.183 × 106 g/mol and its monosaccharide composition was glucose, and glucuronic acid, with the contents of 567.6148 and 0.2096 µg/mg, respectively. FT-IR and NMR analyses showed that X-EPS was an α-pyranose polysaccharide and was composed of 92.22 % α-(1 â†’ 6) linked d-glucopyranose units and 7.77 % α-(1 â†’ 3) branching. Furthermore, it showed a chain-like microstructure with branches in atomic force microscopy (AFM) and scanning electron microscopy (SEM) experiments. These results suggested that the unique structure of X-EPS, gave the yoghurt a strong viscosity and cohesiveness, which resulted in the silk drawing phenomenon. This work suggested that X-EPS holds the potential for food and industrial applications.

Structural and functional characterization of exopolysaccharide from Leuconostoc citreum BH10 discovered in birch sap.

In this study, Leuconostoc citreum BH10, an endophytic strain, was isolated from aseptically collected xylem sap of birch for the first time, and its exopolysaccharide (LCEPS) production was up to 46.31 g/L in glucan producing medium. The produced LCEPS was purified to obtain two water-soluble fractions, named as LCEPS-1 and LCEPS-2, respectively. The major fraction LCEPS-1 was characterized to be comprised of glucose with average molecular weight of 6.34 × 106 Da. The structure of LCEPS-1 was investigated by spectroscopy analysis, which revealed that LCEPS-1 was identified with containing 90.45 % α-(1,6) linkages in the main chains and 9.55 % α-(1,3) branch linkages. The scanning electron microscope results demonstrated that the dried LCEPS-1 appeared porous surface overlaid with an irregular glittering. The water solubility index (WSI) and water holding capacity (WHC) of LCEPS-1 were 88.02 ± 1.69 % and 241.43 ± 6.38 %, respectively. Besides, it exhibited high thermal stability as well as fine antioxidant activities. Taken together, the results indicated that LCEPS-1 could have good potentiality to be applied in fields of foods, cosmetics, nutraceuticals and pharmaceutical industries as the natural agent.

Chemical Synthesis and Evaluation of Exopolysaccharide Fragments Produced by Leuconostoc mesenteroides Strain NTM048.

Exopolysaccharides (EPSs) occur widely in natural products made by bacteria, fungi and algae. Some EPSs have intriguing biological properties such as anticancer and immunomodulatory activities. Our group has recently found that EPSs generated from Leuconostoc mesenteroides ssp. mesenteroides strain NTM048 (NTM048 EPS) enhanced a production of mucosal immunoglobulin A (IgA) of mouse. Herein, we described the synthesis and evaluation of the tetrasaccharide fragments of NTM048 EPS to obtain information about the molecular mechanism responsible for the IgA-inducing activity.

Biosorption of Pb2+ and Zn2+ by Ca-alginate immobilized and free extracellular polysaccharides produced by Leuconostoc citreum B-2.

AIMS: Heavy metal pollution seriously threatens human health and ecological environment. Due to high efficiency and excellent development prospect, adsorption technology has attracted worldwide attention. It is significant to develop renewable adsorbents with excellent adsorption performance. SCOPE: In this study, the Pb2+ and Zn2+ adsorption capacity of Ca-alginate immobilized and free (without immobilization) Leu. citreum B-2 extracellular polysaccharides (EPS) was investigated. Isotherm and kinetic models were used to evaluate the adsorption performance. The adsorbents were characterized by SEM, FT-IR and XPS spectroscopy. CONCLUSIONS: The maximum biosorption of Pb2+ 269.54 and Zn2+ 49.88 mg/g was achieved with immobilized EPS. Thermodynamic studies showed that the adsorption of Pb2+ and Zn2+ on EPS was a spontaneous and feasible process, and the adsorption properties of EPS were exothermic for lead and endothermic for zinc. All the adsorption processes conformed to the pseudo-second-order model and Langmuir adsorption isotherm model, indicating that the adsorption was mainly chemisorption taken placed on single adsorption surface. SEM results showed that the surface of EPS become denser after adsorption. FTIR and XPS analysis indicated that the adsorption mechanism mainly involved the complexation reaction and ion exchange of functional groups such as CO, O-C-O, -COOH and C-OH.

Bacteriocin production by Leuconostoc citreum ST110LD isolated from organic farm soil, a promising biopreservative.

AIMS: The objective of this study was to isolate a bacteriocin-producing strain and to characterize the expressed bacteriocin for the control of Listeria monocytogenes with aim of biopreservation application. METHODS AND RESULTS: Soil samples from a Korean organic farm were subjected to microbiological analysis for isolation of potential bacteriocinogenic LAB, based on a three-level approach, using L. monocytogenes ATCC 15313 as an indicator test micro-organism. From a total of 17 isolates with inhibitory potential, seven were confirmed to be bacteriocin producers. The selected isolates were differentiated based on their morphology, catalase reaction, sugar fermentation profile obtained by API50CHL and by RAPD-PCR generating two unique profiles. One of the isolates, ST110LD, a specific strong producer of anti-Listeria bacteriocins (12 800 AU ml-1 ) was identified as Leuconostoc citreum. The proteinaceous nature of the inhibitory compound produced by Leuc. citreum ST110LD was confirmed through treatment with pepsin and α-chymotrypsin. Bacteriocin activity was observed to be not affected by the presence of milk, NaCl, SDS, Tween 80 or glycerol. Bacteriocin ST110LD effectively inhibited the growth of exponentially growing L. monocytogenes ATCC 15313 during a 10-h incubation period in BHI at 37°C. In addition, this bacteriocin showed specific inhibition of only Listeria spp., but did not inhibit the growth of beneficial cultures included in the microbial test panel for assessment of the spectrum of activity. CONCLUSIONS: Leuconostoc citreum ST110LD was evaluated as safe bacterium strain, producing bacteriocin with high specificity against listerial and enterococcal species. Specificity of producer strain and expressed bacteriocin can be explored in biopreservation of different fermented food products or applied in biotherapy of antibiotic resistant listerial or enterococcal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of bacteriocin produced by Leuc. citreum strain with highly specific antimicrobial activity against Listeria sp. and Enterococcus sp.

Rheological and emulsifying properties of an exopolysaccharide produced by potential probiotic Leuconostoc citreum-BMS strain.

EPS-BMS, is to our knowledge, the first high molecular weight exopolysaccharide from potential probiotic Leuconostoc citreum-BMS strain that consists on a mixture of α-(1,6)-dextran branched at the third position and ß-(2,6)-levan. This sample exhibited interesting rheological and emulsifying properties under different conditions. Steady shear experiments proved that EPS-BMS had a pseudoplastic behavior without thixotropic properties. Interestingly, pseudoplasticity was maintained even under stress conditions of temperature, pH and salts, which could provide some sensory properties for food products such as mouth feel. Dynamic oscillatory measurements reflected a liquid-like behavior of the sample regardless of the studied EPS concentration, pH, temperature and ionic force. Results related to the emulsifying as well as interfacial properties showed that EPS-BMS had great potential to be applied as emulsifier and/or emulsion stabilizer in both neutral and acidic conditions. Based on the properties reported in this work, EPS-BMS could be potentially applied in the food industry.

Carboxymethylation of (1 → 6)-α-dextran from Leuconostoc spp.: Effects on microstructural, thermal and antioxidant properties.

The carboxymethylated (1 â†’ 6)-α-dextran (CM-dex) was synthesized by introducing carboxymethyl groups at different degrees of substitution (DS). The resulting dex1-1, dex2-1, dex3-1, and dex4-1 products had degrees of substitution of 0.57, 0.78, 1.13, and 1.25, respectively. The dex3-1 showed the highest glass transition temperature (Tg) of 215.96 °C, whereas Tg of pure dextran was 149.83 °C. TGA results indicated that the residual loss was reduced along with the increase of DS in the high-temperature region (450-600 °C). Besides, the CM-dex had stronger scavenging capacity against OH radicals but lower scavenging capacity for DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals compared to that of pure dextran. The carboxymethylation of (1 â†’ 6)-α-dextran will extend the applications for modified dextran.

Development of Slowly Digestible Starch Derived α-Glucans with 4,6-α-Glucanotransferase and Branching Sucrase Enzymes.

Previously, we have identified and characterized 4,6-α-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (α1→4)-linkages in amylose and introduce (α1→6)-linkages in linear chains. The 4,6-α-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (α1→6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1→2,6)-α-d-Glcp-(1→6)- (Leuconostoc citreum enzyme) or -(1→3,6)-α-d-Glcp-(1→6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1→3,6)-α-d-Glcp-(1→6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (α1→3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various α-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (α1→3)-linkages present. The treatment of amylose with 4,6-α-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived α-glucans with markedly reduced digestibility. These starch derived α-glucans may find applications in the food industry.

Novel amphiphilic dextran esters with antimicrobial activity.

New amphiphilic dextran esters were obtained by polysaccharide functionalization with different substituted 1,2,3-triazoles-4-carboxylic acid via in situ activation with N, N'-carbonyldiimidazole. Nitrogen-containing heterocyclic derivatives were achieved by copper(I)-catalyzed cycloaddition reaction between organic azides and ethyl propiolate. Structural characteristics of the compounds were studied by elemental analysis, Fourier transform infrared and nuclear magnetic resonance spectroscopy (1H and 13C-NMR). Thermogravimetric analysis, differential scanning calorimetry and wide-angle X-ray diffraction were used for esters characterization. Properties of polymeric self-associates, formed in aqueous solution, were studied by dynamic light scattering and transmission electron microscopy. The critical aggregation concentration values for dextran esters, determined by fluorescence spectroscopy, were in the range of 4.1-9.5 mg/dL. Antimicrobial activity, investigated for some of the polymers by disc-diffusion method, pointed out that polysaccharide esters were active.

Purification and characterization of an exopolysaccharide from Leuconostoc lactis L2.

An exopolysaccharide (EPS)-producing bacterium was isolated from the homemade fermentation product of hami melon (Cucumis melo var. saccharinus) and identified as Leuconostoc lactis L2 mainly based on 16S rDNA sequence analysis and the API 50 CHL test. The monosaccharide composition of Leu. lactis L2 EPS was determined to be glucose by gas chromatography (GC), and its molecular weight was 3.7 × 103 kDa by high-performance size exclusion chromatography (HPSEC). The EPS microscopic characteristics were compact, branched and tubular under scanning electron microscopy (SEM). Atomic force microscopy (AFM) showed a smooth and consistent polymeric matrix. The EPS was a linear polymer composed of α-(1 → 6)-linked D-pyranose residues, based on the Fourier transform infrared (FT-IR), 1H, 13C nuclear magnetic resonance (NMR) and heteronuclear single quantum coherence (HSQC) spectra. This EPS exhibited a high water solubility index (WSI) and water holding capacity (WHC) (91.90 ±â€¯2.45% and 509.45 ±â€¯28.59%, respectively), as well as good emulsifying properties with many organic solvents. These results collectively suggest the applicable potential of this EPS in the food, feed and pharmaceutical fields.

Spray-drying microencapsulation of nisin by complexation with exopolysaccharides produced by probiotic Bacillus tequilensis-GM and Leuconostoc citreum-BMS.

This work aims to encapsulate nisin by complexation with exopolysaccharides (EPS), produced by Bacillus tequilensis-GM and Leuconostoc citreum-BMS, namely EPS-GM and EPS-BMS, respectively, using spray-drying technique, and to evaluate the effect of this encapsulation on the structure of nisin. Results related to suspensions turbidity showed that EPS/nisin complexes were formed through electrostatic attractions. These interactions were confirmed by Fourier transform infrared (FTIR) analysis. Scanning electron microscopy (SEM) micrographs of the spray-dried complexes revealed the presence of well-separated spherical microcapsules. Besides, results obtained by UV spectra showed that no significant changes occurred on EPS-GM/nisin microcapsules suggesting that this EPS may act as protective agent of nisin structure against spray-drying conditions.

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.

Extraction and characterization of dextran from Leuconostoc pseudomesenteroides YB-2 isolated from mango juice.

An exopolysaccharide (EPS)-producing strain of YB-2 isolated from mango juice was identified as Leuconostoc pseudomesenteroides. The molecular weight (Mw) of this EPS was 7.67×105 Da. Gas chromatography (GC) analysis confirmed the presence of only glucose monomers. Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectra displayed the glucan nature of the EPS with 96.8% α-(1→6) and 3.2% branching α-(1→3) linkages. Scanning electron microscopy (SEM) showed smooth surfaces and compact structure. The water solubility index (WSI) and water-holding capacity (WHC) of dextran were 97.48±2.46% and 287.51±7.93%, respectively. The rheological analysis of dextran elucidated a non-Newtonian pseudoplastic behavior. The dextran revealed an inhibitory activity against Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) of 2.0 mg/mL and 3.0 mg/mL, respectively.

Combining multi-scale modelling methods to decipher molecular motions of a branching sucrase from glycoside-hydrolase family 70.

Among α-transglucosylases from Glycoside-Hydrolase family 70, the ΔN123-GB-CD2 enzyme derived from the bifunctional DSR-E from L. citreum NRRL B-1299 is particularly interesting as it was the first described engineered Branching Sucrase, not able to elongate glucan polymers from sucrose substrate. The previously reported overall structural organization of this multi-domain enzyme is an intricate U-shape fold conserved among GH70 enzymes which showed a certain conformational variability of the so-called domain V, assumed to play a role in the control of product structures, in available X-ray structures. Understanding the role of functional dynamics on enzyme reaction and substrate recognition is of utmost interest although it remains a challenge for biophysical methods. By combining long molecular dynamics simulation (1µs) and multiple analyses (NMA, PCA, Morelet Continuous Wavelet Transform and Cross Correlations Dynamics), we investigated here the dynamics of ΔN123-GB-CD2 alone and in interaction with sucrose substrate. Overall, our results provide the detailed picture at atomic level of the hierarchy of motions occurring along different timescales and how they are correlated, in agreement with experimental structural data. In particular, detailed analysis of the different structural domains revealed cooperative dynamic behaviors such as twisting, bending and wobbling through anti- and correlated motions, and also two structural hinge regions, of which one was unreported. Several highly flexible loops surrounding the catalytic pocket were also highlighted, suggesting a potential role in the acceptor promiscuity of ΔN123-GBD-CD2. Normal modes and essential dynamics underlined an interesting two-fold dynamic of the catalytic domain A, pivoting about an axis splitting the catalytic gorge in two parts. The comparison of the conformational free energy landscapes using principal component analysis of the enzyme in absence or in presence of sucrose, also revealed a more harmonic basin when sucrose is bound with a shift population of the bending mode, consistent with the substrate binding event.

Investigation of microorganisms involved in kefir biofilm formation.

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.

Response of Leuconostoc strains against technological stress factors: Growth performance and volatile profiles.

The ability of twelve strains belonging to three Leuconostoc species (Leuconostoc mesenteroides, Leuconostoc lactis and Leuconostoc pseudomesenteroides) to grow under diverse sub-lethal technological stress conditions (cold, acidic, alkaline and osmotic) was evaluated in MRS broth. Two strains, Leuconostoc lactis Ln N6 and Leuconostoc mesenteroides Ln MB7, were selected based on their growth under sub-lethal conditions, and volatile profiles in RSM (reconstituted skim milk) at optimal and under stress conditions were analyzed. Growth rates under sub-lethal conditions were strain- and not species-dependent. Volatilomes obtained from the two strains studied were rather diverse. Particularly, Ln N6 (Ln. lactis) produced more ethanol and acetic acid than Ln MB7 (Ln. mesenteroides) and higher amounts and diversity of the rest of volatile compounds as well, at all times of incubation. For the two strains studied, most of stress conditions applied diminished the amounts of ethanol and acetic acid produced and the diversity and levels of the rest of volatile compounds. These results were consequence of the different capacity of the strains to grow under each stress condition tested.

Isolation, purification and characterization of exopolysaccharide produced by Leuconostoc pseudomesenteroides YF32 from soybean paste.

A water-soluble exopolysaccharide (EPS)-producing strain YF32 was isolated from soybean paste, which was then identified as Leuconostoc pseudomesenteroides. After culturing the strain in Man-Rogosa-Sharpe (MRS) medium containing 5% sucrose at 30°C for 48h, the EPS was purified, and a yield of 12.5g/L was achieved. The weight-average molecular weight (Mw) was 5.54×106Da by high-performance size-exclusion chromatography (HPSEC). The structural characterization of the purified EPS was determined by gas chromatography (GC), Fourier transform infrared (FT-IR), 1H, 13C nuclear magnetic resonance (NMR) spectroscopy. The results demonstrated that the exopolysaccharide was glucan with a peak, a linear backbone composed of consecutive α-(1→6)-linked d-glucopyranose units. No branching was observed in the dextran structure. The degradation temperature (Td) of EPS was 307.62°C, which suggested that dextran exhibited high thermal stability. YF32 dextran also showed high water solubility and emulsibility. All results suggested that dextran has the potential to be applied in food fields as a food additive.

Purification of leucocin A for use on wieners to inhibit Listeria monocytogenes in the presence of spoilage organisms.

The aims of this study were to improve the method for purification of leucocin A to increase yield of peptide and to evaluate the efficacy of leucocin A and an analogue of leucocin A (leucocin N17L) to inhibit the growth of Listeria monocytogenes on wieners in the presence of spoilage organisms. Leucocin A was produced by Leuconostoc gelidum UAL187 and purified with a five-fold increase in yield; leucocin N17L was synthesized replacing asparagine at residue 17 with leucine. Five strains of L. monocytogenes associated with foodborne illness were used to assess bacteriocin efficacy in vitro and in situ. Minimum inhibitory concentrations could not be determined in broth; however, on agar the minimum inhibitory concentrations ranged from 11.7-62.5µM and 62.5->500µM for leucocin A and leucocin N17L, respectively. Leucocin N17L was less effective than the native bacteriocin at controlling the growth of L. monocytogenes. The inactivation profiles of L. monocytogenes in broth in the presence of leucocin A suggested each isolate had different levels of resistance to the bacteriocin as determined by the initial bactericidal effect. The formation of spontaneously resistance subpopulations were also observed for each strain of L. monocytogenes. In situ, wieners were inoculated with the spoilage organisms, Carnobacterium divergens and Brochothrix thermosphacta, followed by surface application of purified leucocin A, and inoculated with a cocktail of L. monocytogenes. Wieners were vacuum packaged and stored at 7°C for 16d. Leucocin A reduced the counts L. monocytogenes on wieners during storage, regardless of the presence of C. divergens. B. thermosphacta was unaffected by the presence of leucocin A on wieners over the duration of storage. This study suggests that leucocin A may be beneficial to industry as a surface application on wieners to help reduce L. monocytogenes counts due to post-processing contamination even in the presence of spoilage organisms. However, further investigation on the ability of L. monocytogenes to form spontaneous resistance to class II bacteriocins on food matrices during prolonged storage is warranted.

Efficient Production of Prebiotic Gluco-oligosaccharides in Orange Juice Using Immobilized and Co-immobilized Dextransucrase.

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was subjected to immobilization and co-immobilization with dextranase from Chaetomium erraticum. Immobilization has enhanced the operational and storage stability of dextransucrase. Two hundred milligrammes (2.4 IU/mg) of alginate beads (immobilized and co-immobilized) were found to be optimum for the production of gluco-oligosaccharides (GOS) in orange juice with a high degree of polymerization. The pulp of the orange juice did not interfere in the reaction. In the batch process, co-immobilized dextransucrase (41 g/L) produced a significantly higher amount of GOS than immobilized dextransucrase (37 g/L). Alginate entrapment enhanced the thermal stability of dextransucrase for up to 3 days in orange juice at 30 °C. The production of GOS in semi-continuous process was 39 g/L in co-immobilized dextransucrase and 33 g/L in immobilized dextransucrase. Thus, immobilization technology offers a great scope in terms of reusability and efficient production of a value added functional health drink.

Isomelezitose formation by glucansucrases.

Several glucansucrases were surveyed for their ability to produce isomelezitose, a trisaccharide with the structure α-D-glucopyranosyl (1 â†’ 6) ß-D-fructofuranosyl (2 â†” 1) α-D-glucopyranoside. Nearly all strains tested, with one exception, produced at least trace levels of isomelezitose. Yields were low but significant, ranging from less than 1% to approximately 5% based on sucrose. This trisaccharide may arise in either of two ways: glucopyranosyl transfer to the 6Fru-OH position of sucrose, or to the anomeric OH position of isomaltulose. This study indicates that isomelezitose formation may be a general phenomenon of many glucansucrase reactions.