Isomaltooligosaccharides Research Articles

Co‐immobilization of dextransucrase and dextranase for the facilitated synthesis of isomalto‐oligosaccharides: Preparation, characterization and modeling

Co-Immobilization of dextransucrase (DS) and dextranase (DN) into calcium alginate includes the co-entrapment of soluble DS and adsorbed DN. DS converts sucrose into dextran, which is the substrate for DN, so that isomalto-oligosaccharides (IMOs) are follow-up products of dextran hydrolysis. The boundary conditions for the successful preparation are investigated with respect to choice of DN adsorbate, surface modifications using blotting agents and optimal enzyme activity ratios. Further, repetitive batch experiments suggest the selection of medium activity ratios for continuous use (0.3 U(DN)U(-1) (DS), e.g.). Product formation at various cosubstrate:substrate concentrations as well as at different DN:DS ratios are discussed. Moreover, the complexity of the bi-enzymatic system can be reduced considering the molar ratios of cosubstrate:substrate (glucose:sucrose). Based on these factors, a mechanistic kinetic model is developed, which distinguishes the corresponding contributions of the two enzymes upon overall product formation. In general, at low glucose:sucrose ratios isomaltose synthesis is featured primarily by DN action. Yet with increasing amounts of glucose both the quantity and quality of DN substrate changes, so that its contribution to product formation decreases in an exponential manner; still the overall product yield continuously increases due to enhanced DS contribution.

In vitro study on gas generation and prebiotic effects of some carbohydrates and their mixtures

This study was carried out to examine the effect of inulin (IN), fructooligosaccharide (FOS), polydextrose (POL) and isomaltooligosaccharides (ISO), alone and in combination, on gas production, gas composition and prebiotic effects. Static batch culture fermentation was performed with faecal samples from three healthy volunteers to study the volume and composition of gas generated and changes in bacterial populations. Four carbohydrates alone or mixed with one another (50:50) were examined. Prebiotic index (PI) was calculated and used to compare the prebiotic effect. The high amount of gas produced by IN was reduced by mixing it with FOS. No reduction in gas generation was observed when POL and ISO mixed with other substrates. It was found that the mixture of IN and FOS was effective in reducing the amount of gas produced while augmenting or maintaining their potential to support the growth of bifidobacteria in faecal batch culture as the highest PI was achieved with FOS alone and a mixture of FOS and IN. It was also found that high volume of gas was generated in presence of POL and ISO and they had lower prebiotic effect. The results of this study imply that a mixture of prebiotics could prove effective in reducing the amount of gas generated by the gut microflora.

Development and Verification of an Optimum Composition Model for a Synbiotic Fermented Milk Using Sequential Quadratic Programming Techniques

The purpose of this research was to develop an optimum composition model for a new synbiotic fermented dairy product with high probiotic cell counts, and to experimentally verify this model. The optimum composition model indicated the growth promoter ratio that could provide the highest growth rate for probiotics in this fermented product. Different levels of growth promoters were first blended with milk to improve the growth rates of probiotics, and the optimum composition model was determined. The probiotic viabilities and chemical properties were analyzed for the samples made using the optimal formula. The optimal combination of the growth promoters for the synbiotic fermented milk product was 1.12% peptides, 3% fructooligosaccharides (FOS), and 1.87% isomaltooligosaccharides (IMO). A product manufactured according to the formula of the optimum model was analyzed, showing that the model was effective in improving the viability of both Lactobacillus spp. and Bifidobacterium spp.

標準試薬を用いた検量線による市販イソマルトオリゴ糖製品中のイソマルトオリゴ糖の定量改良法

市販イソマルトオリゴ糖製品中のオリゴ糖の定量法としてポリマー担体のアミノカラムを用いて，改良定量法を開発した．改良定量法では従来使用していたシリカ担体のアミノカラムよりも耐久性の高いカラムを用いた．本カラムを使用することにより市販イソマルトオリゴ糖製品中の各標準オリゴ糖の定量がRI検出による絶対検量線法で可能となった．グルコース，マルトース，コージビオース，ニゲロース，イソマルトース，マルトトリオース，パノース，イソマルトトリオース，マルトテトラオース，イソマルトテトラオースの濃度とピーク高の検量線を作成したところ17 mg/mLまで直線を示し，最小二乗法で相関係数0.999以上の高い相関性が認められた．各糖類ごとに直線の勾配は異なっており，グルコースが最も高く，イソマルトテトラオースが最も低くなった．グルコースの勾配に対して各糖類の相対勾配(各糖類の勾配/グルコースの勾配)を求め，変換ファクターとした．イソマルトオリゴ糖工業製品中の各糖類の濃度定量は液クロ分析で得られたピーク高より以下の式に従い算出した： (糖類Aの濃度，mg/mL)=(グルコース標準の濃度，mg/mL)×(糖類Aのピーク高)/(糖類Aの変換ファクター)/(グルコース標準のピーク高) 改良定量法を用いて市販イソマルトオリゴ糖製品を分析した結果，製品100 g(糖固形分75.6 g含有)当たりに標準イソマルトオリゴ糖は42.7 gであった．その構成糖はイソマルトース19.2 g，イソマルトトリオース10.3 gを主成分として，パノース4.9 g，ニゲロース，コージビオース，イソマルトテトラオースが各々2.0，3.5，2.8 gであった．これら標準オリゴ糖の含有量は従来法と比較して高くなったが，これは従来定量法が各オリゴ糖の検量線法を用いて定量できなかったことに起因するものと考えられた．更に改良定量法ではニゲロース，コージビオース，4種類の未知のオリゴ糖の分離も可能となった．4種類の未知オリゴ糖の中で最も多い成分を単離・精製し，1Hおよび13C-NMR解析したところイソマルトトリオシルグルコースであった．

Isomaltose formation by free and immobilized dextransucrase

The acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL-B512F with glucose as acceptor is of technical interest for isomaltooligosaccharide (IMOs) synthesis. Different experimental conditions were investigated for free and immobilized enzyme. The data for oligosaccharide formation up to a degree of polymerization 4 were correlated with a model developed earlier, and optimal reaction conditions for immobilized dextransucrase design and application were identified for later continuous application. Furthermore, stability was investigated for free and immobilized enzyme including stabilization by sugars.

Isomalto-Oligosaccharides Polarize Th1-Like Responses in Intestinal and Systemic Immunity in Mice

Isomalto-oligosaccharides (IMO) belong to a group of prebiotics that significantly increase the number of protective gut microflora. In the present study, we investigated the effects of IMO on intestinal and systemic immunity in mice. When mice were fed a diet supplemented with 20% IMO for 4 wk, the number of lactobacilli and the levels of IgA in feces were greater than those of mice fed the control diet (P < 0.05). Interferon-gamma (IFN-gamma) production by intestinal intraepithelial lymphocytes (i-IEL) in response to T-cell receptor (TCR) triggering was greater in mice fed IMO than in controls (P < 0.05), indicating T helper-1 (Th1) polarization of intestinal immunity by IMO. The proportion of natural killer (NK) T cells in the liver mononuclear cells (MNC), and the production of IFN-gamma by the liver MNC in response to TCR triggering were greater in mice fed IMO than in controls (P < 0.05), suggesting that the Th1/Th2 balance was shifted toward the Th1 lineage by IMO in systemic immunity. Furthermore, the proportion and activity of NK cells were greater in the spleens of the mice fed IMO than in the controls. Dietary IMO protected the mice from gamma-irradiation-induced lethality, accompanied by an inhibition of the translocation of Enterobacteriaceae. Notably, when mouse macrophage-like J774.1 cells were cultured with Lactobacillus gasseri in the presence of IMO, interleukin (IL)-12 production was greater than in the absence of IMO. These results suggest that IMO, in synergy with lactobacilli, upregulate the Th1 response and beneficially modulate host defense.

Optimal combination of the encapsulating materials for probiotic microcapsules and its experimental verification (R1)

The purpose of this research is to develop an optimal composition model for the encapsulating materials of probiotic microcapsules after one-week storage, with prebiotics and alginate as the encapsulating materials in given ranges of concentrations. The procedure includes three phases: building the response surface models, finding the optimal composition model with global optimization, and verifying the optimal model. The response surface method and the sequential quadratic programming (SQP) technique were employed to determine the optimal ratio and evaluate the tolerance of the microencapsulated probiotics to gastric conditions. Results indicated that the optimized microcapsules could increase the viability of probiotic bacteria under the simulated gastric fluid test (SGFT). The optimal combination of encapsulating materials for the probiotic microcapsules was found to be 3% sodium alginate blended with 1% peptides, 3% fructooligosaccharides (FOS), and 0% isomaltooligosaccharides (IMO). Verification experiments confirmed the SQP-based prediction with no apparent significant differences ( P > 0.05).

Isomaltooligosaccharide increases cecal Bifidobacterium population in young broiler chickens

A newly developed compound derived by fermentation, isomaltooligosaccharide (IMO), was hypothesized to enrich cecal bifidobacterial populations and reduce colonization levels of Salmonella in the ceca of broiler chickens. Broiler starter diets were prepared with final IMO concentrations of 1% (wt/wt), 2% (wt/wt), and 4% (wt/wt) and a control diet without IMO supplementation. Chickens were divided into 4 groups and challenged with 108 cell of Salmonella enterica ser. Typhimurium with 200μg/mL nalidixic acid resistant (S. Typhimurium Nalr) after 7 d of placement. The experiment was done in 3 replications. IMO-supplemented diets resulted in significantly higher cecal bifidobacteria compared with the control diet (P < 0.05). However, there was no significant difference in bifidobacteria counts among the treatment groups. Chickens fed diets with 1% IMO had a significant 2-log reduction in the level of inoculated S. Typhimurium Nalr (P < 0.05) present in, the ceca compared with the control group, but no differences were found between the control group and the groups fed 2 or 4% IMO for S. Typhimurium Nalr. No differences in feed consumption, feed conversion, or feed efficiency compared with the control group were observed; however, the result showed a significant reduction in weight for birds fed 1% IMO diet compared with those fed the control diet.

Production of high‐purity isomalto‐oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity.

Optimization on response surface models for the optimal manufacturing conditions of dairy tofu

The purpose of this research was to create response surface models through regression on experimental data and to apply the Sequential Quadratic Programming (SQP) and Genetic Algorithms (GAs) on the models to obtain optimal processing conditions for dairy tofu. The two-stage effort of obtaining a surface model using response surface methodology (RSM), and optimizing this model using GAs or SQP techniques was demonstrated to be an effective approach. Both SQP and GAs techniques were able to determine the optimal conditions for manufacturing the probiotic dairy tofu. The conditions were 1% of glucono-delta-lactone (GDL), 0% of peptides level, 3% of isomaltooligosaccharides (IMO) and 18% of milk concentrations, and they were confirmed by verification experiments. Among the SQP and two GAs employed, the SQP, modified with the multi-start capability, is the most efficient one.

Sequential Quadratic Programming for Development of a New Probiotic Dairy Tofu with Glucono‐δ‐Lactone

ABSTRACT: The purpose of this research was to evaluate the effects of various concentrations of glucono‐δ‐lactone (GDL) and skim milk powder, as well as the addition of prebiotics, on the rheology and probiotic viabilities of dairy tofu. Additionally, modern optimization techniques were applied to attempt to determine the optimal processing conditions and growth rate for the selected probiotics (Lactobacillus. acidophilus, L. casei, Bifidobacteria bifidum, and B. longum). There were 2 stages in this research to accomplish the goal. The 1st stage was to derive surface models using response surface methodology (RSM); the 2nd stage performed optimization on the models using sequential quadratic programming (SQP) techniques. The results were demonstrated to be effective. The most favorable production conditions of dairy tofu were 1% GDL, 0% peptides, 3% isomaltooligosaccharides (IMO), and 18% milk, as confirmed by subsequent verification experiments. Analysis of the sensory evaluation results revealed no significant difference between the probiotic dairy tofu and the GDL analog in terms of texture and appearance (P &lt; 0.05). The viable numbers of probiotics were well above the recommended limit of 106 CFU/g for the probiotic dairy tofu throughout the tested storage period.

Efficacy of Leuconostoc mesenteroides (ATCC 13146) isomaltooligosaccharides as a poultry prebiotic

The complex dietary carbohydrates, called prebiotics, have been used to control Salmonella and improve intestinal bacterial balance in broilers. Leuconostoc mesenteroides (ATCC 13146) isomaltooligosaccharides (IMO) stimulate growth of Bifidobacterium and Lactobacillus and are not used by Salmonella or Escherichia coli. We tested the efficacy of these IMO as a prebiotic. IMO, compared with fructooligosaccharides (FOS) as sole carbon source, promoted growth of chicken cecal isolates and Bifidobacterium. Cecal isolates and Salmonella typhimurium grown in mixed culture on IMO reduced the Salmonella population. Cecal isolates grown on IMO showed higher viable counts and faster growth than Salmonella, indicating a potential value for these oligomers for poultry intestinal microflora modification.

Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase and dextranase

Batch syntheses of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase were performed with the aim of understanding the reaction mechanism and the parameters which affect product characteristics and molecular size. Both activities described for dextransucrase (dextran formation and acceptor reaction) achieved synthesis whilst the hydrolytic activity of dextranase regulated the product molecular size and acceptor availability. Depending on the reaction conditions, the product oligosaccharide mixtures contained mainly sugars (up to 36%) with degrees of polymerization (DP) varying between 10 and 60 together with lower concentrations of both lower and higher molecular weight sugars. Alterations in substrate and dextranase concentrations (50–400 mg ml −1 and 2.5–46 U ml −1, respectively) affected the molecular weight of IMO, the reaction rate and the formation of leucrose. This permitted manipulation of the product characteristics. It was found that higher substrate and dextranase concentrations gave rise to products with lower molecular sizes and a dextransucrase:dextranase ratio of 1:1 or 1:2 appeared to produce a polymer with a molecular weight which is desirable for prebiotic use.

Effect of pH and Dose on the Growth of Gut Bacteria on Prebiotic Carbohydrates in vitro

The effect of pH and substrate dose on the fermentation profile of a number of commercial prebiotics was analysed in triplicate using stirred, pH and temperature controlled anaerobic batch culture fermentations, inoculated with a fresh faecal slurry from one of three healthy volunteers. Bacterial numbers were enumerated using fluorescence in situ hybridisation. The commercial prebiotics investigated were fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), isomaltooligosaccharides (IMO) and lactulose. Two pH values were investigated, i.e. pH 6 and 6.8. Doses of 1% and 2% (w/v) were investigated, equivalent to approximately 4 and 8 g per day, respectively, in an adult diet. It was found that both pH and dose altered the bacterial composition. It was observed that FOS and inulin demonstrated the greatest bifidogenic effect at pH 6.8 and 1% (w/v) carbohydrate, whereas GOS, IMO and lactulose demonstrated their greatest bifidogenic effect at pH 6 and 2% (w/v) carbohydrate. From this we can conclude that various prebiotics demonstrate differing bifidogenic effects at different conditions in vitro.

Cooperative action of alpha-glucanotransferase and maltogenic amylase for an improved process of isomaltooligosaccharide (IMO) production.

Maltogenic amylase and alpha-glucanotransferase (alpha-GTase) were employed in an effort to develop an efficient process for the production of isomaltooligosaccharides (IMOs). Bacillus stearothermophilus maltogenic amylase (BSMA) and alpha-GTase from Thermotoga maritima were overexpressed in Escherichia coli using overexpression vectors. An IMO mixture containing 58% of various IMOs was produced from liquefied corn syrup by the hydrolyzing and transglycosylation activities of BSMA alone. When BSMA and alpha-GTase were reacted simultaneously, the IMO content increased to 68% and contained relatively larger IMOs compared with the products obtained by the reaction without alpha-GTase. Time course analysis of the IMO production suggested that BSMA hydrolyzed maltopentaose and maltohexaose most favorably into maltose and maltotriose and transferred the resulting molecules simultaneously to acceptor molecules to form IMOs. alpha-GTase transferred donor sugar molecules to the hydrolysis products such as maltose and maltotriose to form maltopentaose, which was then rehydrolyzed by BSMA as a favorable substrate.

Ａｒｔｈｒｏｂａｃｔｅｒ ｇｌｏｂｉｆｏｒｍｉｓのＩｓｏｍａｌｔｏ‐ｄｅｘｔｒａｎａｓｅに関する酵素化学的研究

Isomalto-dextranase (EC 3.2.1.94) was purified from the culture of a soil bacterium, Arthrobacter globiformis T 6 by successive chromatographies on CM-cellulose and CM-sepharose to a homoge neous state as confirmed by PAGE. The molecular weight of the enzyme was estimated to be about 69 kDa by SDS-PAGE. The enzyme hydrolyzed α-1, 6-glucosidic linkages of dextran or isomalto oligosaccharides to release exolytically α-isomaltose from the non-reducing ends . The optimum pH and temperature of the enzyme were pH 5.3 and 65°C, respectively . The enzyme showed a weak isopullulanase activity, an endo-type attack on pullulan to produce isopanose. The isomalto dextranase expressed by the recombinant E. coli cells also produced isopanose from pullulan . The enzyme hydrolyzed α-1, 4-glucosidic linkage of panose as well as α-1, 6-glucosidic linkage of isomaltotriose. The kinetic features of the experiments with the mixed substrates of isomaltotriose and panose were in good agreement with those expected for a single catalytic site mechanism. The ionization constants, pKel and pKe2, of the essential ionizable groups 1 and 2 of the enzyme were 3.3 and 6.3 for dextran T2000 and 3.5 and 6.1 for isomaltotriose. The heats of ionization for groups 1 and 2 were 0 kcal/mol or less with both the substrates. These kinetic results suggested that the ionizable groups essential for the enzyme activity were carboxyl and carboxylate . Modification experiments with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), modifying carboxyl residues specifically, also indicated that the carboxyl groups were essential to the enzyme activity . The subsite affinities of the enzyme were culculated to be >7.3, <-7.2, 6.7, 0.74 and 0.18 kcal/mol for subsites 1, 2, 3, 4 and 5, respectively, from the rate parameters (Km and 10) for the hydrolysis of isomaltooligosaccharides. Subsites 1 and 3, showing large affinity values, were thought to attract the substrates and form the productive bindings. A new method for preparation of isomaltose was developed by using the enzyme and an acid-treated dextran. The branch points of dextran were selectively hydrolyzed by a mild acid pretreatment. When the acid-treated dextran was acted on by the enzyme, the maximal degree of hydrolysis went up to over 90%.

The effect of a commercial high-fibre diet and an iso-malto-oligosaccharide-supplemented diet on post-prandial glucose concentrations in dogs.

In order to evaluate the effect of insoluble and soluble fibre on the levels of post-prandial glycaemia, six healthy dogs were fed three different diets: a low-fibre control diet, a high-fibre diet (HF; mainly insoluble fibre) and the control diet with 10% iso-malto-oligosaccharides (IMO) added. The diets were fed for 2 days before the blood collections were started on the third day. Serial blood samples were taken 20, 60, 90, 150, 180, 240 and 360 min after feeding and one sample was taken just before the feeding after a fasting period of at least 20 h. There were no problems concerning the faecal consistency. The post-prandial glycaemia curve was significantly lower in the HF and IMO group in comparison with the control group. At 20 and 60 min the glucose concentration was significantly lower in the HF and IMO groups. At 90 and 150 min only the IMO group had a significantly lower glucose concentration. At 360 min there was a trend for a lower glucose concentration in the IMO group. The results show that both the HF and the IMO diets had a beneficial effect on post-prandial hyperglycaemia. Substitution of IMO may have the same or a slightly better effect, but this has to be confirmed in diabetic dogs and the effect may depend on the composition of the basal diet.

Effects of Isomalto-Oligosaccharides on Bowel Functions and Indicators of Nutritional Status in Constipated Elderly Men

Objectives: To evaluate effects of isomalto-oligosaccharides (IO) on the bowel function and nutritional status of elderly men.Methods: Seven older male subjects participated in this study that consisted of a 30-day control low fiber period followed by a 30-day IO-supplemented (10 g active components) experimental period. Bowel functions such as defecation, enema use and bloating were monitored daily. Fecal characteristics such as wet and dry stool weights, stool moisture, pH and short-chain fatty acid contents were determined on five-day fecal composites collected in each period. Feces were further fractionated into plant, bacterial and soluble fractions to determine the bases for the increase in stool weight. Nutritional status of subjects was assessed with anthropometric parameters, nutrient intake and biochemical measurements.Results: Incorporation of IO significantly increased the defecation frequency, wet stool output and dry stool weight by twofold, 70% and 55%, respectively. Fecal acetate and propionate concentrations significantly increased by nearly two and a half fold with IO supplement. The increase in stool bulk was mainly attributed by increased bacterial mass. Mean serum sodium concentration decreased in the experimental period while other blood characteristics did not change significantly. Anthropometric parameters and nutrient intake remained constant throughout the study.Conclusions: Consumption of IO effectively improved bowel movement, stool output and microbial fermentation in the colon without any adverse effect observed in this study. Therefore, supplementation of IO into ordinary low fiber diets may be practical in relieving constipation in the elderly population.

Isomaltooligosaccharide inhibits the crystallization of sorbitol humectant and glucan formation by Streptococcus mutans

The addition of 20% (w/w) isomaltooligosaccharide (IMO) to sorbitol humectant inhibited crystallization for up to 30 days at −15°C. IMO also decreased glucan formation by Streptococcus mutans KCTC 3065 by 70%, when mixed with sorbitol humectant. Furthermore, IMO increased the moisture content and viscosity of the mixture by up to 5% and 50%, respectively. © Rapid Science Ltd. 1998