Experimental and theoretical study on the intermolecular complex formation between trehalose and benzene compounds in aqueous solution.

The uniqueness of trehalose as a stress protectant may exist in its potential amphiphilic character capable of interacting with both hydrophilic and hydrophobic partners in aqueous solution. To address this issue, we here investigated the interaction between trehalose and aromatic compounds. NMR measurements, including (1)H-(1)H NOESY spectra, provide direct evidence for the formation of stable intermolecular complexes of trehalose with benzene (or p-cresol) in aqueous solution. In addition, corresponding theoretical evidence is provided by calculating the potential mean force as a function of the distance between trehalose and benzene. In the energy minimum structure, the benzene molecule is located only around the hydrophobic side of trehalose where the first hydration shell is not formed. Therefore, it can be concluded that benzene binds to trehalose in a fashion in which dehydration penalty is minimized. Finally, we discuss the possible biological roles of the trehalose-benzene interaction discovered here.

Lactosucrose inhibits body fat accumulation in rats by decreasing intestinal lipid absorption.

Lactosucrose (LS, 4(G)-beta-D-galactosylsucrose) is a non-digestible oligosaccharide, and the consumption of LS selectively increases the proportion of intestinal bifidobacteria. We examined in this study the hypolipidemic potential of LS. An oral triolein tolerance test on rats indicated that LS reduced the elevation of serum triglyceride (TG) and free fatty acids (FFA). Furthermore, LS inhibited the enzymatic digestion of triolein by pancreatic lipase in vitro. NMR spectroscopy showed that LS formed an intermolecular complex with triolein. The long-term consumption of a diet containing 5% LS for 8 weeks significantly decreased the weight of abdominal adipose tissue when compared with that of the control group. Thus, LS may reduce adipose tissue accumulation by inhibiting intestinal lipid absorption via a direct interaction with TG.

Thermodynamic, hydration and structural characteristics of alpha,alpha-trehalose.

A nonreducing disaccharide, alpha,alpha-trehalose, accumulates endogenously in diverse anhydrobiotic organisms in their dehydrating process or prior to their desiccation, being thought to have a protective function either as a water replacement molecule or as a vitrification agent in the dry state. Trehalose acts also as a protectant against physiological stress, including freezing, ethanol and oxidation. To elucidate the origin of these different functions of this sugar, it is necessary to obtain a deep insight into the physicochemical properties of trehalose at the molecular level. In this review, we focus our attention on the thermodynamic, hydration and structural properties of carbohydrates, and extract the characteristic feature of trehalose. On the basis of these findings, we subsequently discuss the underlying mechanism for protein stabilization by trehalose in solution and for its anitoxidant function on unsaturated fatty acids.

Effects of dietary lactosucrose (4G-beta-D-galactosylsucrose) on the IgE response in mice.

In this study, we examined the effects of dietary lactosucrose (LS, a non-digestible oligosaccharide) on the IgE response in mice immunized with ovalbumin (OVA)/alum. In addition to IgG1 and IgG2a responses, the anti-OVA IgE response in mice fed LS diets was dose-dependently suppressed, as compared with the control mice, while the serum total IgG levels were comparable. Moreover, dietary LS feeding inhibited antigen-specific IgE and IgG1 productions even after a second immunization. Regarding with cytokine production, when stimulated in vitro with OVA, splenocytes obtained from LS-fed mice produced a similar level of IFN-gamma, and lower levels of IL-4 and IL-5, as compared with the control mice. But IL-10 production by OVA-stimulated splenocytes was augmented in LS-fed mice, suggesting that IL-10 producing cells are responsible for the immunoregulatory effect of LS. Our findings indicate the further possibility that dietary LS supplementation can be used to prevent IgE-mediated allergic diseases.

Long-term administration of 4G-beta-D-galactosylsucrose (lactosucrose) enhances intestinal calcium absorption in young women: a randomized, placebo-controlled 96-wk study.

This study determined the effect of long-term administration of 4(G)-beta-D-galactosylsucrose (lactosucrose; LS) on intestinal calcium absorption. In a randomized, single-blind, parallel-group study, LS (n=9, 6.0 g twice daily) or a placebo (maltose; n=8, 6.0 g twice daily) was administered to healthy young women for 92 wk: the study also included a 4-wk post-administration period. All participants completed the study. Dietary nutrient intake; fecal weight, pH, and moisture content; fecal concentrations of short-chain fatty acids (SCFA), putrefactive products, ammonia, and minerals (calcium, magnesium, phosphorus, and iron); and serum calcium and osteocalcin concentrations were measured every 24 wk. Urinary pyridinoline (PYR) and deoxypyridinoline (DPD), and urinary calcium excretion were measured every 12 wk. Significant effects of oligosaccharide treatment, time, and the interaction between oligosaccharide treatment and time were observed for fecal pH, SCFA, ammonia, and putrefactive product values (p<0.05). Fecal pH, ammonia, and putrefactive product values decreased in the LS group, and the fecal SCFA concentration significantly increased during the administration period; these changes were not observed 4 wk post-administration. To examine the mineral balance of calcium, magnesium, and phosphorus in detail, all the participants completed a 6-d mineral balance study, sometime between week 56 and 60 of the longer study. During the mineral balance study, the daily calcium intake was set at 400 mg; all feces and urine were collected each day for 6 d after an 8-d acclimation period. In the balance study, fecal calcium excretion was significantly lower in the LS group than in the placebo group (p<0.05), and apparent calcium absorption and retention, apparent magnesium and phosphorus absorption, and magnesium retention were significantly higher in the LS group than in the placebo group (p<0.05). Our results suggest that the administration of LS produces a long-term enhancement of intestinal calcium absorption in healthy young women with lower than recommended calcium intakes.

Production of isocyclomaltopentaose from starch using isocyclomaltooligosaccharide glucanotransferase.

Production of a novel cyclomaltopentaose cyclized by an alpha-1,6-linkage, [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch was performed using isocyclomaltooligosaccharide glucanotransferase (IGTase) derived from Bacillus circulans AM7. The optimal conditions for ICG5-production from partially hydrolyzed starch were as follows: substrate concentration, 1.0% (w/v); pH, 5.5; temperature, 45 degrees C; reaction time, 24 h, IGTase, 1.0 unit/g-dry solid (DS); isoamylase, 2,500 units/g-DS. The yield of ICG5 reached 25.9% under optimal conditions. ICG5-production was achieved from partially hydrolyzed starch using a crude enzyme preparation containing IGTase. Finally, ICG5 was obtained in a yield of 17.9% (99.3% purity, 2,681 g-DS). A digestive test with a human salivary amylase, an artificial gastric juice, a pancreatic amylase, and small intestinal enzymes showed that ICG5 was an indigestible oligosaccharide.

Effect of dietary cyclic nigerosylnigerose on intestinal immune functions in mice.

We examined the dietary effects of cyclic nigerosylnigerose (CNN), a dietary indigestible oligosaccharide with four D-glucopyranosyl residues linked by alternating alpha-(1-->3)- and alpha-(1-->6) glucosidic linkages, on the intestinal immune function of mice, and the effects were compared with those of alpha-(1-->3)-linked oligosaccharide (nigerooligosaccharides, NOS) or alpha-(1-->6)-linked oligosaccharide (isomaltooligosaccharides, IMO). BALB/c mice were fed with 1-5% CNN, 5% IMO, or 12.5% NOS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 1-5% CNN fed groups, the amounts of IgA in feces increased significantly. In addition, IgA, transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) secretion by Peyer's patch (PP) cells were enhanced in CNN fed mice. In the 5% CNN group, pH in the cecum decreased, and the amounts of lactic acid and butyric acid increased. These findings were not observed in the NOS- or IMO-fed group of mice. They suggest that CNN supplementation changes the intestinal environment of microflora and indirectly enhances the immune function in the gut.

Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens.

Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475-483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G6-alpha-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells.

Suppressive effect of trehalose on acrylamide formation from asparagine and reducing saccharides.

The influence of saccharides on the formation of acrylamide (AcA) was investigated. The reducing saccharides reacted with asaparagine to form AcA, but the non-reducing saccharides, except sucrose, gave no AcA. AcA formation from a mixture containing glucose and asaparagaine was suppressed by the non-reducing saccharides, especially trehalose (76% suppression) and neotrehalose (75% suppression). Glucose is heat-degraded into pyruvaldehyde and 5-hydroxymethyl-2-furfural in the water system. The degradation products react with asparagines to generate AcA. Trehalose appears to inhibit not only the formation of these intermediates and asparagines for AcA, but also the AcA formation from these intermediates.

An enzymatically produced novel cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->} (cyclic maltosyl-(1-->6)-maltose), from starch.

A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.

Interaction between trehalose and alkaline-earth metal ions.

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.

Combined NMR and quantum chemical studies on the interaction between trehalose and dienes relevant to the antioxidant function of trehalose.

In a previous study (Oku, K.; Watanabe, H.; Kubota, M.; Fukuda, S.; Kurimoto, M.; Tujisaka, Y.; Komori, M.; Inoue, Y.; Sakurai, M. J. Am. Chem. Soc. 2003, 125, 12739), we investigated the mechanism of the antioxidant function of trehalose against unsaturated fatty acids (UFAs) and revealed that the key factor relevant to the function is the formation of OH...pi and CH...O hydrogen bonds between trehalose and the cis double bonds of the UFA. Here, we investigate whether such intriguing interactions also occur between this sugar and cis double bonds in other unsaturated compounds. For this purpose, we selected various diene compounds (1,3-butadiene, 1,3-pentadiene, 1,4-pentadiene, and 2,5-heptadiene) as interaction partners. All NMR experiments performed, including 1H-1H NOESY measurements, indicated that trehalose selectively interacts with the cis-olefin proton pair in the above diene with a 1:1 stoichiometry, and the C-3 (C-3') and C-6' (C-6) sites of the sugar are responsible for the interaction. Similar interactions were not observed for the mixtures of the diene and other saccharides (neotrehalose, kojibiose, nigerose, maltose, isomaltose, sucrose, maltitol, and sorbitol). Quantum chemical calculations revealed that the OH-3 and OH-6 groups bind to the olefin double bonds of the diene through OH...pi and CH...O types of hydrogen bonds, respectively, and the stabilization energy of the resulting complex is 5-6 kcal mol(-1). These results strongly support the above NMR results. Finally, the activation energies were calculated for the hydrogen abstraction reactions from the activated methylene group of heptadiene. In particular, when the reaction was initiated by a methyl radical, the activation energy was only 10 kcal mol(-1) for the free heptadiene, but on complexation with trehalose it drastically increased to ca. 40 kcal mol(-1). This indicates that trehalose has a significant depression effect on the oxidation of the diene compounds. These results strongly support the antioxidant mechanism deduced in the previous study and indicate that the formation of unique multiple hydrogen bonds between trehalose and cis-olefin bonds is rather a general event not confined to the case of UFA.

NMR and quantum chemical study on the OH...pi and CH...O interactions between trehalose and unsaturated fatty acids: implication for the mechanism of antioxidant function of trehalose.

Trehalose is a disaccharide that attracts much attention as a stress protectant. In this study, we investigated the mechanism of the antioxidant function of trehalose. The spin-lattice relaxation times (T(1)) of (1)H and (13)C NMR spectra were measured to investigate the interaction between trehalose and unsaturated fatty acid (UFA). We selected several kinds of UFA that differ in the number of double bonds and in their configurations (cis or trans). Several other disaccharides (sucrose, maltose, neotrehalose, maltitol, and sorbitol) were also analyzed by NMR. The T(1) values for the (1)H and (13)C signals assigned to the olefin double bonds in UFA decrease with increasing concentration of trehalose and the changes reaches plateaus at integer ratios of trehalose to UFA. The characteristic T(1) change is observed only for the combination of trehalose and UFA with cis double bond(s). On the other hand, from the (13)C-T(1) measurements for trehalose, the T(1) values of the C-3 (C-3') and C-6' (C-6) are found to change remarkably by addition of UFA. (1)H[bond](1)H NOESY measurements provide direct evidence for complexation of trehalose with linoleic acid. These results indicate that one trehalose molecule stoichiometrically interacts with one cis-olefin double bond of UFA. Computer modeling study indicates that trehalose forms a stable complex with an olefin double bond through OH...pi and CH...O types of hydrogen bonding. Furthermore, a significant increase in the activation energy is found for hydrogen abstraction reaction from the methylene group located between the double bonds that are both interacting with the trehalose molecules. Therefore, trehalose has a significant depression effect on the oxidation of UFA through the weak interaction with the double bond(s). This is the first study to elucidate the antioxidant function of trehalose.