Many sugars such as mono- and disaccharides, sugar alcohols and starch were heated at 180°C with asparagines (Asn) in a model food formula to investigate their effect on the formation of acrylamide (AA) in foods. The typical profile of AA formation from mono- and disaccharides was that the AA level increased after 5 min and reached the maximum value around 15 min and then decreased to a third value at 30 min. This profile was independent of pH and the kind of mono- or disaccharide. The amounts of AA from ketoses were much higher than those from aldoses and the contribution of their reducing character was not so great. Although the AA levels from sugar alcohols were quite low, the production of AA was obvious. In use of starch and α-cyclodextrin, the maximum of AA contents was comparable to that from glucose; however, their AA formations were relatively slow. Though the Maillard reaction between reducing sugars and Asn was widely accepted as a pathway to produce AA in processed foods, some of our results could not be explained by the theory. Consequently, it was suspected that some unknown compounds from decomposition of carbohydrates reacted with Asn to produce AA.
An α-amylase II (TVA II) produced by Thermoactinomyces vulgaris R-47 exhibits wide substrate specificity for starch, pullulan, cyclodextrins and isopanose. Asp465 and Arg469 are located at subsite (-2) and are observed among many α-amylase family enzymes. Here, we have investigated the relationship between the substrate specificity of TVA II and the substrate binding by Asp465 and Arg469 using site-directed mutagenesis and X-ray crystallographic analyses. The five mutated TVA IIs (D465N, D465E, D465Q, R469L and R469K) showed lower specific activities for all substrates tested here than the wild-type TVA II. In particular, the mutation of Arg469 significantly decreased the hydrolyzing activities compared to the mutation of Asp465. While the Km values for α-cyclodextrin were increased for all the mutants, large decreases of the kcat values were observed only for R469L and R469K. The side chain of Arg469 is close to that of Asp421, which is one of three catalytic residues, and the positive charge of Arg469 seems to affect the catalytic mechanism via Asp421. These findings suggest that Asp465 and Arg469 are important residues for the substrate binding and the catalytic reaction. Furthermore, the binding of the α-maltosyl unit by Asp465 and Arg469 is commonly observed among TVA II and other α-amylase family enzymes, which is thought to be the basic mechanism of substrate binding.
Long chains (LC) were fractionated by 1-butanol precipitation from isoamylase-debranched amylopectins of rice, maize, wheat, buckwheat, and sweet potato. The structure of the precipitate (LCppt) was characterized by high-performance size-exclusion chromatography (HPSEC) with pre-column labeling with 2-aminopyridine. HPSEC showed that LCppt corresponded to the LC fraction of unit-chain distribution of amylopectin. LCppt had a number-average degree of polymerization of 330-490 and an average-number of chains of 1.2-1.4, indicating that the structure of LCppt is very similar irrespective of its botanical sources and is distinct from amylose. HPSEC revealed that size distribution of LCppt was also similar among the specimens examined. The distribution was distinct from and much narrower than that of amylose. Still, LCppt consisted of a significant amount (by weight) of chains as long as amylose. LCppt contained 5.1-9.4 mol% of branched molecules, which had ∼5 chains per molecule (except for 10 of indica rice IR36), indicating incomplete debranching of the side chains on LC by isoamylase. The structure of the isoamylase-resistant branches is unknown. In this respect, LCppt and amylose appear to have a common branched structure, which is unusual in starch α-glucans.
Starch granules were prepared from mature grains of 75 cultivars (23 indica, 27 Chinese indica, 6 japonica and 19 javanica) of rice originating in Asia and the other countries, including Brazil (4), China (25), India (10), Indonesia (3), Japan (8), Korea (2), Laos (3), Myanmar (3), Nepal (4), Pakistan (1), the Philippines (1), Russia (1), Taiwan (3), Thailand (1) and the USA (6). They were cultivated and harvested in the paddy field of Prefectural University of Hiroshima in 2001. We showed that starches of non-waxy cultivars of the indica and Chinese indica, in general, had higher contents of the apparent amylose (AAM) and super-long chains (SLC) of amylopectin by GPC of Pseudomonas isoamylase-debranched starches and amylopectins through Toyopearl columns. High performance anion exchange chromatography with a pulsed amperometric detection (HPAEC-PAD) of isoamylase-debranched starches showed that the starches of non-waxy cultivars of the indica and Chinese indica, in general, had decreased amounts of branch chains with DP 6-12 (Fr. A). The Fr. A contents correlated positively with the alkali spreading score (ASS) of rice grains and negatively with the peak temperature (Tp) of gelatinization of the rice starches. Among the pasting characteristics of the starches measured using a Rapid Visco Analyser (RVA), setback (SB) and breakdown (BD) showed high positive and negative correlations with SLC contents, respectively, and both peak top viscosity (PV) and BD negatively correlated to AAM contents. There was a high positive relationship between amounts of Waxy (Wx) protein and SLC contents in starch. This appears to show that Wx protein is concerned with synthesis of SLC. SLC contents in starches of rice originating in Asia and the other countries were evenly observed in the range of 0.0-13.4%.
Mogroside V (MV), a main sweet triterpene glycoside in the extract of the fruit of Luo-han-guo (Siraitia grosvenori Swingle), was transglycosylated by cyclodextrin glucanotransferases (CGTases) using starch as a donor substrate. CGTases from Bacillus macerans, B. circulans, B. stearothermophilus and Thermoanaerobacter sp. were effective for the transglycosylation of MV. It was appropriate for the production of glycosylated MV to react 9% (w/v) MV with 20% (w/v) tapioca starch and B. macerans enzyme (30 U/g-starch) at 50°C and pH 6.5. Under these conditions, the reaction finished within 24 h and more than 90% of a glycosylation yield was attained. Three products that have 1-3 additional glucose residues were identified as transglycosylation products by mass spectrometry. The greater number of glucose residues introduced, the less intensity of sweetness compared to MV was estimated by sensory evaluation. However the qualities of sweetness such as bitterness, after-taste, and peculiarity were improved by transglycosylation.
Slightly acid-treated potato starch (ATS) granules were conjugated with ε-poly(L-lysine) (PL) by using the Maillard reaction. Coomassie Brilliant Blue staining indicated the conjugation of PL to ATS. The PL content of the ATS-PL conjugate was estimated to be in the rage of about 1.7-2.5%. Conjugation with PL increased the gelatinization temperature, and reduced the swelling, solubility, retrogradation, and digestibility with α- or β-amylase. The ATS-PL conjugate exhibited 1/4-1/2 lower antibacterial activity toward Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, and Candida utilis than free PL did.
An exo-1,5-α-L-arabinanase was purified as an electrophoretically homogenous protein from a liquid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 kDa by gel filtration chromatography. The isoelectric point of the enzyme was 3.7. The maximum velocity of carboxymethyl (CM)-linear arabinan degradation by the exo-arabinanase was attained at 50°C and at pH 5.0. The purified enzyme was stable in a range from pH 6.0 to 8.0 and up to 45°C. The activity of the enzyme was significantly inhibited by Ag+ (1 mM) and Cr2+ (1 mM), and stimulated by SDS (5 mM). The Km value for the 1,5-arabinan from beet was 5.8 mg/mL. The sequence of amino-terminus (25 residues) of the exo-arabinanase from A. sojae exhibits extensive identity (69%) with that of Penicillium chrysogenum. After the hydrolysis of 1,5-arabinan from beet, the major product was arabinobiose, and no liberation of arabinose was observed in the reaction mixture.
This paper reviews the present status for the development of functional oligosaccharides in Japan and looks over the future prospects of these saccharides. Since 1970, several novel microbial enzymes producing specific oligosaccharides have been discovered. Using these new enzymes, it is now possible to produce on an industrial scale various oligosaccharides such as glycosylsucrose, fructooligosaccharides, maltooligosaccharides, isomaltooligosaccharides (branched-oligosaccharides), galactooligosaccharides, xylooligosaccharides, palatinose (isomaltulose), lactosucrose and so on. Recent developments in industrial enzyme-utilization technology have made possible a series of new oligosaccharides such as β-1,6-linked gentiooligosaccharides, α,α-1,1-linked trehalose, α-1,3-linked nigerooligosaccharides, branched-cyclodextrins, maltosyltrehalose, cyclic difructose and cyclic tetrasaccharide. The development of novel and highly functional oligosaccharides with physiological properties is now continuing and the market is expanding gradually. Recent human intervention and animal studies have revealed that foods are able to modulate the functions of innate or aquired immunity. In the near future, the development of oligosaccharides as immune system modulators including prebiotics is expected, and these saccharides may play an important role, especially in the reduction of lifestyle-related diseases as well as the maintenance and improvement of human health.
We found here that ω-epoxyalkyl α-D-glucopyranosides consisting of three, four and five alkyl carbons (α-E3G, α-E4G and α-E5G, respectively), which are known to be affinity-labeling reagents of β-amylase, had the effect of inactivating two pullulan-hydrolyzing α-amylases from Thermoactinomyces vulgaris R-47, TVA I and TVA II, at high concentration (ca. 0.1-1.5 M). The inactivation exhibited saturation kinetics of a two-step mechanism, and an inactivation rate constant, k, and equilibrium dissociation constant, KR, of α-E5G were calculated. The k/KR values of α-E5G for TVA I and TVA II were 13.1 × 10-4 and 6.41 × 10-4 M-1 · S-1 respectively. In terms of the power of inactivation, the orders for TVA I and TVA II were α-E5G>α-E3G≈α-E4G, and α-E5G>α-E3G>α-E4G, respectively. The findings indicated that the relation between the lengths of the alkyl carbons and the inactivation of TVA I and TVA II differs from that for β-amylase and isomalto-dextranase.
In Aspergillus awamori glucoamylase, the optimal pH has been reported to increase to maintain activity by a mutation of Ser411 which forms a hydrogen-bond with a catalytic base (Fang and Ford, Protein Eng., 11, 383-388 (1998)). Most glucoamylases have either Ser or Gly at this position, whereas only Thermoactinomyces vulgaris R-47 glucoamylase (TGA) and two putative glucoamylases have Trp. We focused on Trp622 in TGA and examined the pH optima of five mutants, W622C, W622D, W622G, W622H and W622S. The pH optima of these mutants were 6.2-6.8, which was identical to or slightly lower than that of the wild-type enzyme. However, the activities of these mutants at pH optima decreased to 4.3-52% of that of wild-type enzyme. From these results and information on the crystal structures of glucoamylases, Trp622 in TGA is suggested to be an important residue for substrate binding rather than for determination of optimal pH.
Production of L-arabinose and xylose from arabinoxylan in corn hull and bagasse was investigated using dilute hydrochloric acid treatment at 125 or 130°C. In the corn hull arabinoxylan, which has a high L-arabinose content (Ara/Xyl=0.590), L-arabinose was more preferentially released than xylose as a result of less than 5 min treatment with 0.2 M HCl at 125°C. The yield from arabinoxylan was 73% after 5 min treatment, but high L-arabinose ratio of 60% was obtained in the released xylose and L-arabinose mixture. After 20 min treatment, the amounts of L-arabinose and xylose were increased to 162 and 290 mg/g corn hull, and their yields from the corn hull arabinoxylan were 89 and 99%, respectively. Furthermore, during enzymatic hydrolysis using cellulase, glucose was significantly released from the HCl-treated corn hull. On the other hand, in the bagasse arabinoxylan which has a low L-arabinose content (Ara/Xyl=0.073), the L-arabinose yield from arabinoxylan was 99%; however, the xylose yield was smaller (63%) and glucose release was not enhanced with enzymatic hydrolysis because of higher lignin content.
Radical scavenger activity of the heated, aqueous solution of 1,5-anhydro-D-fructose was higher than that of non-heated one. The reason was ascopyrone P, which had 500-fold stronger radical-scavenger activity than 1,5-anhydro-D-fructose, was derived from heat treatment. Gradual conversion of 1,5-anhydro-D-fructose into ascopyrone P seemed one of the key for the long-lasting, antioxidative action of 1,5-anhydro-D-fructose preparation. Efficient production of ascopyrone P was achieved by heat treatment, namely, 50% of 1,5-anhydro-D-fructose was converted by the reaction at 155°C for 5 min. In foods, ascopyrone P was produced by retort cooking of the materials containing 1,5-anhydro-D-fructose, such as truffle and red seaweed Gracilaria verrucosa. Alternatively, the derivative (approximately 20 μg) was synthesized on baking or frying of foods (1 g) containing glucans, starch or cellulose.
Early stage (within 24 h storage) retrogradation of cooked rice stored at 4°C or 15°C were evaluated by instrumental methods, that is, differential scanning calorimetry (DSC), whiteness, hardness and adhesiveness of rice grain under 25% or 80% deformation, and sensory test. The rate of retrogradation of cooked rice measured by instrumental methods increased after 6 h storage at 4°C, although the rate was low and the increase in retrogradation was slow in the case of storage at 15°C. The retrogradation score for cooked rice stored at 4°C by sensory evaluation increased earlier than that by instrumental measurements. Coefficients of correlation between instrumental measurements and sensory evaluation indicated that storage temperature influenced physical properties of cooked rice, and as a result, the index for evaluating cooked rice stored at 15°C was different from that for stored at 4°C. Concretely, whiteness, DSC and hardness under 25% deformation were closely related to sensory evaluation in the oral cavity for cooked rice stored at 4°C, while adhesiveness under 25% and 80% deformation correlated to sensory evaluation in the oral cavity for cooked rice stored at 15°C.
We investigated the effect of multiple treatments (10, 20 and 30 times, one cycle of treatment: 24 h) of freeze-thaw (-30°C) or low temperature (4°C) on wheat starch gel for elucidation of the formation mechanism of Furunori (the traditional paste for decoration of Japanese painting). Furunori needs over ten years maturation for its formation. Hence, the development of a scientific method for making Furunori in a short term is very much in demand. Our report of the molecular characteristics of Furunori was previously published, and it was suggested that the main factor of Furunori formation might be temperature change with transition of season during maturation. The Furunori model by the above treatment was set for elucidation of this factor effect. The results are as follows: 1) The SEM image of the materials in the 4°C treatment shows a pebble-like mass and fairly closely resembling Furunori, but the SEM image the materials in the -30°C treatment shows a flat flaky mass. The size of both the 4°C treatment and -30°C treatment masses becomes smaller with each treatment. 2) The peak of X-ray diffraction pattern for the -30°C treatment is higher than that for the 4°C treatment and Furunori. 3) The gelatinization degree in the 4°C treatment is far larger than in the -30°C treatment measured by the BAP method, but that in the -30°C treatment is larger than in the 4°C treatment measured by the glucoamylase method. 4) All GPC patterns of both the -30°C treatment and 4°C treatment on Toyopearl HW 75 F have an amylopectin peak which Furunori does not have. 5) DSC results show that the -30°C treatment has a clear endothermal peak which becomes larger with each treatment whereas the 4°C treatment scarcely has an peak. In conclusion, the -30°C treatment is more retrograded than the 4°C treatment, but the mechanism of retrogradation might be different. The formation mechanism of Furunori might resemble that of the 4°C treatment, but we can not explain the disagreement between the two GPC patterns. Probably, the special gelatinization process of Furunori (heating and stirring over 24 h) may hold key to the explanation of its GPC pattern.