Characterization of a recombinant sucrose isomerase and its application to enzymatic production of isomaltulose.

OBJECTIVE: To characterize a recombinant isomerase that can catalyze the isomerization of sucrose into isomaltulose and investigate its application for the enzymatic production of isomaltulose. RESULTS: A sucrose isomerase gene from Erwinia sp. Ejp617 was synthesized and expressed in Escherichia coli BL21(DE3). The enzymatic characterization revealed that the optimal pH and temperature of the purified sucrose isomerase were 6.0 and 40 °C, respectively. The enzyme activity was slightly activated by Mn2+and Mg2+, but partially inhibited by Ca2+, Ba2+, Cu2+, Zn2+ and EDTA. The kinetic parameters of Km and Vmax for sucrose were 69.28 mM and 118.87 U/mg, respectively. The time course showed that 240.9 g/L of isomaltulose was produced from 300 g/L of sucrose, and the yield reached 80.3% after bioreaction for 180 min. CONCLUSIONS: This recombinant enzyme showed excellent capability for biotransforming sucrose to isomaltulose at the substrate concentration of 300 g/L. Further investigations should be carried out focusing on selection of suitable heterologous expression system with the aim to improve its expression level.

Green synthesis of isomaltulose from cane molasses by Bacillus subtilis WB800-pHA01-palI in a biologic membrane reactor.

A green process and environmentally benign process is highly desirable in the development of enzymatic catalysis. In this work, the shuttle plasmid pHA01 was constructed and the sucrose isomerase (SIase) was expressed in Bacillus subtilis WB800. The optimal nitrogen and carbon sources for SIase expression were yeast extract (15g/L) and un-pretreated cane molasses (UCM, 20g/L), respectively. After the UCM fed, the whole cell activity reached 5.2U/mL in a 7.5L fermentor. Optimum catalytic temperature and pH of whole cell were 35°C and 5.5, respectively. Although the biologic membrane reactor (BMR) system consecutively worked for 12 batches, the sucrose conversion remained higher than 90%, indicating the BMR system had a greater operational stability. Furthermore, isomaltulose production using the BMR system with low-cost cane molasses as its substrate not only reduces the production cost and mediates environmental pollution, but also solves the genetic background problem of the non-food-grade strains.

A Survey of Commercially Available Isomaltooligosaccharide-Based Food Ingredients.

Isomaltooligosaccharides (IMOs) are included in many commercially available food products including protein/fiber bars, shakes, and other dietary supplements. Marketed as "high fiber," "prebiotic soluble fiber," and/or as a "low-calorie, low glycemic sweetener," IMO may be present in significant amounts, for example, more than 15 g/item or serving. Herein, high-pressure anion exchange chromatography with pulsed amperometric detection and high-pressure liquid chromatography with differential refractive index detection are used to compare 7 commercially available IMO-containing bulk food ingredients. The ingredients are typical of those produced either (a) via bacterial fermentation ("fermented" IMO or MIMO) of sucrose in the presence of a maltose acceptor mediated by a glucosyltransferase enzyme (dextransucrase), or (b) via transglycosylation of hydrolyzed starch with α-glucosidase ("industrial" IMO). Analysis of the results with respect to digestibility suggests that the potential glycemic impact of the ingredients and products containing "industrial" IMO may be inconsistent with the product labeling and/or certificates of analysis with respect to overall fiber content, prebiotic fiber content, and glycemic response and are thus inappropriate for diabetic patients and those on low-carbohydrate (for example, ketogenic) diets.

Inaccuracy of AOAC method 2009.01 with amyloglucosidase for measuring non-digestible oligosaccharides and proposal for an improvement of the method.

We wished to clarify the inaccuracy of AOAC method 2009.01 for the measurement of non-digestible oligosaccharides and to propose an improved method using porcine intestinal enzymes. Amyloglucosidase used in AOAC method 2009.01 scarcely hydrolyses sucrose, palatinose and panose (which are readily digested by intestinal enzymes). Hence, oligosaccharides could not be measured accurately by AOAC method 2009.01. To confirm the inaccuracy of the method, we used porcine intestinal enzymes instead of amyloglucosidase. Using the improved method, fructooligosaccharide and galactooligosaccharide were measured accurately as non-digestible oligosaccharides, but sucrose, palatinose, panose and isomaltooligosaccharide were not. The improved method hydrolysed digestible oligosaccharides into monosaccharides. These results demonstrate that the inaccuracy of AOAC method 2009.01 for oligosaccharide measurement is due to incomplete hydrolysis by amyloglucosidase. We propose that amyloglucosidase should be replaced with porcine intestinal enzymes for such measurements.

Metabolism of isomalto-oligosaccharides by Lactobacillus reuteri and bifidobacteria.

UNLABELLED: Commercial isomalto-oligosaccharides (IMO) are functional food ingredients. They are composed of α(1→6)- and α(1→4)-linked oligosaccharides. IMO are partially indigestible, and dietary IMO stimulate beneficial members of intestinal microbiota, including lactobacilli and bifidobacteria. However, data on IMO metabolism by lactobacilli are not available. It was the aim of this study to identify metabolic pathways of IMO metabolism in lactobacilli. This study focused on the host-adapted species Lactobacillus reuteri. Metabolism of bifidobacteria was analysed for comparison. Commercial IMO contained IMO with a degree of polymerization (DP) of up to four and panose-series oligosaccharides (POS) with a DP of up to 5. Lactobacilli metabolized isomaltose preferentially over oligosaccharides with higher DP. Bifidobacteria preferentially metabolized oligosaccharides with higher DP and accumulated glucose. Metabolism of IMO and POS by L. reuteri was attributed to α(1→6)-specific glucanase DexB and maltose phosphorylase. Contribution of maltose phosphorylase was verified by quantification of IMO and POS phosphorolysis in crude cellular extracts of L. reuteri 100-23. In conclusion, metabolism of IMO by lactobacilli is limited to short-chain oligosaccharides, while bifidobacteria preferentially metabolize oligosaccharides with higher DP. The functionality of commercial IMO can thus be modified by degree of polymerization. SIGNIFICANCE AND IMPACT OF THE STUDY: Isomalto-oligosaccharides (IMO) are applied as functional food ingredients, but the composition and biological functionality of current commercial products are poorly documented. This study is the first to analyse IMO metabolism by Lactobacillus reuteri. Bifidobacteria were used for comparison. Commercial IMO contained IMO with degree of polymerization (DP) of up to four and panose-series oligosaccharides with DP of up to 5. L. reuteri preferentially metabolized short-chain oligosaccharides, whereas bifidobacteria preferentially metabolized higher oligosaccharides. Results of this study allow the modification of the biological and technological functionality of commercial IMO by adjustment of the degree of polymerization and will thus facilitate the application development for IMO.

Influence of processing on the volatile profile of strawberry spreads made with isomaltulose.

A new strawberry spread formulated with fructose and isomaltulose (replacing sucrose partially or totally) and a high percentage of fruit was developed in line with the new trend of healthier products. This work studies the influence of some process variables (percentage of sugar, pectin and citric acid, and time of thermal treatment) on the volatile profile of these spreads with different formulations. The ripeness of the raw strawberries influences the concentrations of some of the compounds in the spreads, such as isobutyl acetate, butyl butyrate, 3-hexen-1-yl acetate or propan-2-ol. The process conditions have an important effect on the volatile profiles. Most of the esters and alcohols decreased whereas 13 new compounds appear, mostly furans (furfural, 2-acetylfurane, 5-methyl furfural, mesifurane) and aldehydes (octanal, nonanal, decanal and benzaldeyhde). In general, the spreads formulated with sucrose-isomaltulose that contained higher levels of pectin and citric acid gave better results in the preservation of the original aromatic compounds in raw strawberries.

Analysis of sugars in Chinese rice wine by Fourier transform near-infrared spectroscopy with partial least-squares regression.

The feasibility of rapid analysis for oligosaccharides, including isomaltose, isomaltotriose, maltose, and panose, in Chinese rice wine by Fourier transform near-infrared (FT-NIR) spectroscopy together with partial least-squares regression (PLSR) was studied in this work. Forty samples of five brewing years (1996, 1998, 2001, 2003, and 2005) were analyzed by NIR transmission spectroscopy with seven optical path lengths (0.5, 1, 1.5, 2, 2.5, 3, and 5 mm) between 800 and 2500 nm. Calibration models were established by PLSR with full cross-validation and using high-performance anion-exchange chromatography coupled with pulsed amperometric detection as a reference method. The optimal models were obtained through wavelength selection, in which the correlation coefficients of calibration (r(cal)) for the four sugars were 0.911, 0.938, 0.925, and 0.966, and the root-mean-square errors of calibrations were 0.157, 0.147, 0.358, and 0.355 g/L, respectively. The validation accuracy of the four models, with correlation coefficients of cross-validation (r(cv)) being 0.718, 0.793, 0.681, and 0.873, were not very satisfactory. This might be due to the low concentrations of the four sugars in Chinese rice wine and the influence of some components having structures similar to those of the four sugars. The results obtained in this study indicated that the NIR spectroscopy technique offers screening capability for isomaltose, isomaltotriose, maltose, and panose in Chinese rice wine. Further studies with a larger Chinese rice wine sample should be done to improve the specificity, prediction accuracy, and robustness of the models.

Application of response surface methodology to cell immobilization for the production of palatinose.

Response surface methodology (RSM), based on multivariate non-linear model, was applied to study the interactions and optimization of the immobilization parameters for cell entrapment, namely alginate concentration, cell loading and bead diameter using Erwinia rhapontici NCPPB 1578 that produced palatinose. ANOVA analysis and statistical parameters calculations showed that RSM could be used effectively to model and improve a complex system like cell immobilization. Palatinose yield was increased by 40%. The maximum yield of 140 mg/ml was achieved in a batch of 1h at alginate concentration of 5% w/v, cell loading of 5 g l(-1) and 2.25 mm bead diameter. Thus, the E. rhapontici NCPPB 1578 immobilization in alginate bead and subsequent palatinose yield was successfully improved by application of RSM technique.

Application of a fast high-performance liquid chromatography method for simultaneous determination of furanic compounds and glucosylisomaltol in breakfast cereals.

The high-performance liquid chormatography method reported in the present paper provides a fast, low-cost, precise, and simple technique to analyze simultaneously 3 different indicators of nonenzymatic browning, i.e., hydroxymethylfurfural (HMF), furfural, and glucosylisomaltol (GIM), in breakfast cereals. These compounds were extracted in aqueous solution and separated on a reversed-phase C18 column with water-acetonitrile (95 + 5, v/v) mobile phase under isocratic conditions. Average recovery rates for HMF, furfural, and GIM were 99.1, 98.4, and 99.4%, respectively. The coefficients of variation were 3.67, 2.42, and 1.59% for HMF, furfural, and GIM, respectively. The detection limit was 0.01 mg/kg, and the quantitation limit was 0.05 mg/kg for the 3 studied compounds.

Identification and quantification of the plasma volume expander dextran in human urine by liquid chromatography-tandem mass spectrometry of enzymatically derived isomaltose.

Plasma volume expanders are used in sports in order to control haematological parameters and/or to mask erythropoietin (EPO) misuse. A reliable method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for doping control purposes, enabling the identification and quantification of the plasma volume expander dextran in human urine. The dextran polymer was enzymatically hydrolysed by alpha-1,6-glucosidase (dextranase) followed by acetylation of the generated isomaltose subunits, allowing the chromatographic separation of different disaccharides, such as lactose, saccharose and isomaltose, as well as the identification and quantification of the analyte in human urine. The method was used to determine the basal concentration of isomaltose resulting from the enzymatic hydrolysis of polymeric 1,6-linked glucose in 238 routine doping control samples. In addition the concentration of dextran measured as isomaltose was estimated in seven urine specimens obtained from patients treated with dextran. Calibration curves for dextran were linear and reproducible. The inter- and intra-assay coefficients of variation for dextran ranged from 4.9 to 7.3% at three concentration levels between 53 and 1186 microg/mL. Recovery ranged from 97 to 112% (mean 106.9%). The assay limit of detection was 3.8 microg/mL and the lower limit of quantification was 12.5 microg/mL. In 96% of the investigated doping control samples, the concentrations of isomaltose were below the LLOQ of 12.5 microg/mL. Even the highest concentrations were approximately 100-300-fold lower than concentrations found in urine samples of patients after intravenous application of dextran. The presented results demonstrate the capability and reliability of the developed LC-MS/MS method for the identification and quantification of dextran in human urine and can be regarded as a method revealing the misuse of dextran in sports.

Glucosylisomaltol, a new indicator of browning reaction in baby cereals and bread.

Glucosylisomaltol is proposed as a new indicator of the browning reaction in baby cereals and bread. The glucosylisomaltol was synthesized from maltose and proline, purified by semipreparative HPLC, and characterized by NMR, high-resolution mass spectrometry, and GC-MS analysis. Analysis of glucosylisomaltol, previously separated from cereals by centrifugation, was carried out by reversed-phase HPLC with UV detection in isocratic elution with water/acetonitrile (95:5). Mean recovery of glucosylisomaltol by the standard addition method was 96.9%. The relative standard deviation and detection limit were 1.56% and 0.14 mg/kg, respectively. This compound was identified in samples by the similarity of the t(R) and UV spectra to those of synthesized glucosylisomaltol. Moreover, the glucosylisomaltol from samples, previously separated by semipreparative HPLC, was acetylated and then separated and confirmed by GC-MS. Glucosylisomaltol was determined in baby cereals stored at 32 and 55 degrees C for 1 year and at 25 and 55 degrees C for 1 month at a water activity of 0.65. The amount of this indicator increased during storage from 0.48 to 7.7 mg/kg. The glucosylisomaltol was also determined in prebaked bread by heating at 190 degrees C for 30 min. The amount of this compound increased from nondetectable to 20.9 mg/kg after 30 min of baking. Glucosylisomaltol is a useful indicator to control the browning reaction during baby cereal storage and the baking of bread.

Application of two-dimensional mapping for an analysis of galactosyllactoses in yogurt.

A two-dimensional mapping analysis was performed by HPLC for 4 kinds of standard galactosyllactoses (GLs, trisaccharide) which were assumed to be produced from lactose (galactopyranosyl beta 1-->4 glucopyranose) in yogurt during the fermentation of lactic acid bacteria. After the pyridylamination of GLs, they were analyzed by HPLC in the reverse-phase (RP) and anion-exchange (AE) modes. The retention times of each peak obtained were converted to glucose units (GU) in RP mode for the pyridylaminated isomaltooligosaccharides (G1-3) and to relative retention time (RRT) in AE mode against pyridylaminated-isomaltotriose, and then the address data [GU, RRT] were plotted on a graph. This two-dimensional mapping method was found useful for a rapid qualitative evaluation of the chemical structure of trisaccharides formed in yogurt.

Total intestinal lactase and sucrase activities are reduced in aged rats.

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestinal microvillus membrane hydrolases that play important roles in carbohydrate digestion. Although the expression of these enzymes during postnatal development has been characterized, the effect of old age on disaccharidase activity is poorly understood. In the present investigation, we examined the effect of aging on lactase and sucrase activities and their mRNA levels in the small intestines of 3-, 12- and 24- mo-old rats by sampling from nine equidistant segments of small intestine. Total intestinal disaccharidase activity or mRNA abundance was determined from areas under the proximal-to-distal curves. Rats 24 mo of age had total intestinal lactase and sucrase activities that were 12 and 38% lower, respectively, than the 3-mo-old animals (P < 0.05). In contrast, total LPH and SI mRNA abundance did not change significantly. Thus, total intestinal lactase and sucrase activities decrease with age in a manner that likely involves a posttranscriptional process. The age-related decline in disaccharidase activity, if extrapolated to humans, may have important implications for the digestion of carbohydrate contained in the diet of the elderly.

Enzymically produced cyclic alpha-1,3-linked and alpha-1,6-linked oligosaccharides of D-glucose.

A new type of bacterial enzyme hydrolyzed alternan (Leuconostoc mesenteroides NRRL B-1355 fraction S dextran, an alternating alpha-1,3-alpha-1,6-D-glucan) to give rise to a series of oligosaccharides. The oligosaccharide formed in the greatest proportion was a cyclic tetrasaccharide of D-glucosyl residues linked in an alternating alpha-1,3-alpha-1,6 fashion. Other saccharide products included isomaltose and alpha-D-glucopyranosyl-1,3-alpha-D-glucopyranosyl-1,6-D-glucose. Oligosaccharides of higher degrees of polymerization were also formed, and included alpha-D-glucosylated derivatives of the cyclic tetrasaccharide. This is the first report of a naturally produced cyclic tetrasaccharide.

Distribution of three microvillar enzymes along the small intestinal crypt-villus axis.

Aminopeptidase N (ApN), dipeptidyl peptidase IV (DPP IV) and sucrose-isomaltase (S-I) are differentially expressed along the pig jejunum crypt-villus axis. Quantitative immunoelectron microscopy and enzyme cytochemistry show that DPP IV and S-I are expressed in enterocytes along the entire length of the axis, whereas ApN is found mainly in the villar and upper crypt enterocytes. All three enzymes are detected in the basolateral membrane at all levels along the crypt-villus axis, although ApN and S-I only occurred at low intensities in the villus region. The microvillar/basolateral labelling ratio for the three enzymes increases to a varying degree for the three enzymes along the axis suggesting that the sorting efficiency to the apical membrane improves at least for ApN and S-I as the cells mature. These findings might indicate that the enterocytes change from a transcytotic to a direct apical transport as the enterocytes mature.

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Basic structure.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 and of DSM 20068 was isolated by phenol/water extraction and hydrophobic-interaction chromatography. The preparations from both strains have an identical structure: a 1,3-linked poly(glycerophosphate) chain phosphodiester-linked to Glc-(alpha 1-2)Glc(alpha 1-3)acyl2Gro as the lipid anchor. The chain is substituted with D-alanine ester and glycosyl residues which comprise mono-, di-, tri- and tetra-alpha-D-glucopyranosyl residues with (1-6) interglycosidic linkages. The glycosylglycerols were released with 48% (by mass) hydrofluoric acid, separated and characterized by a combination of chemical procedures and modern techniques of 1H-NMR and 13C-NMR spectroscopy. The alpha-isomalto-oligosaccharides add a novel motif to lipoteichoic-acid chain substituents. 1H-NMR and 13C-NMR spectroscopy also provided a detailed picture of the basic glycosylated poly(1,3-glycerophosphate) diglucosylglycerol. It proved a single unbranched chain structure, provided evidence for the chain length, the extent of glycosylation, the structure of the lipid anchor and the site of attachment of the poly(glycerophosphate) chain on the lipid anchor. Owing to its unique glycosyl substituents the lipoteichoic acid may serve as a taxonomic marker for the redefined species S. sanguis (formerly S. sanguis type I).

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Microheterogeneity and distribution of chain substituents.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 contains a poly(glycerophosphate) chain, with 49% of the glycerophosphate residues being substituted with D-alanine ester, 35% with alpha-D-glucopyranosyl and alpha-isomalto-oligosaccharide residues. Analysis of molecular species by affinity chromatography on concanavalin A showed all chains to be substituted and alanine ester and glycosyl residues to be present on the same rather than on separate chains. Molecular species varied in the length of the poly(glycerophosphate) chain, the extent of glycosylation, and had a constant alanine-ester content. An alkali-hydrolysis procedure revealed a distribution pattern between random and regular for the glycosyl substituents and suggested a similar distribution for the alanyl residues which occupy the free positions between the glycosyl substituents.

A novel method for differentiating dextran sulfate from related sulfated polysaccharides.

A method is described for unequivocal identification of dextran sulfate, based on combined chemical desulfation and dextranase enzymolysis of dextran sulfate moieties to isomaltose, a specific indicator of dextran-type precursors. The method was developed using high-resolution (300 MHz) 1H NMR spectroscopy for assurance of the molecular transformations, identification, and estimation of the hydrolysis products. Overall conversion of approximately 80% of highly sulfated and moderately sulfated dextran sulfates was realized. Both 2-D 1H and 13C NMR spectra of a dextran sulfate (MW 500,000) clarified the extent of sulfation (75%) at C-4 and confirmed that sulfation at positions C-2 and C-3 was virtually complete. Estimation of the hydrolysis products (isomaltose, major; alpha-D-glucose, minor) is not restricted to 1H NMR now that the desulfation-enzymolysis methodology has been established; rather, it can be performed using HPLC or GLC (with derivatization).

Intestinal lactase (beta-galactosidase) and other disaccharidase activities of suckling and adult common brushtail possums, Trichosurus vulpecula (Marsupialia:Phalangeridae).

Small-intestinal disaccharidase activities of eight suckling T. vulpecula, aged from 34 to 150 days, and of two adult animals were investigated. Intestinal maltase, isomaltase and sucrase activities increased with age, whereas lactase activities decreased. Trehalase activities were relatively high in all animals and showed no obvious age-related changes. Three separate beta-galactosidase activities, one neutral and two acid, acted on lactose. The neutral beta-galactosidase activity appeared to be due to a brush border enzyme similar to that of eutherian mammals, whereas the acid beta-galactosidases were soluble and probably of lysosomal origin. One of these, acid beta-galactosidase-1, had similar properties to the sole intestinal beta-galactosidase of macropodid marsupials, whereas the other, acid beta-galactosidase-2, has not previously been described. Galactosyl oligosaccharides isolated from macropodid milk were readily hydrolysed by both acid beta-galactosidases but not by the neutral beta-galactosidase. The total intestinal lactase activity in animals aged up to 125 days was due mainly to acid beta-galactosidase-1, whereas in older animals it was due mostly to the neutral beta-galactosidase; this suggests that late in lactation the young T. vulpecula change from a macropodid mode of digestion of galactosyl oligosaccharides to a eutherian mechanism for the digestion of lactose. These findings may have implications for the hand-rearing of orphaned T. vulpecula.