Fourier-transform,infrared difference-spectrometry for structural analysis of dextrans

The Fourier-transform (F.t.), infrared (i.r.) spectra of a series of branched dextrans were examined. The dextrans studied were those from the N R R L collection designated Leuconostoc mesenteroides B-1142, B-1191, B-1299 fraction S, B-1355 fraction S, B-1402, and B-1422, and Streptobacterium dextranicum B-1254 fractions S[L] and L[S]. The spectrum of a levan, N R R L L. mesenteroides B-523 fraction M, was also examined, for comparison with the spectra of the dextrans. Meaningful results were obtained by “weight-normalizing” the spectral absorbance to that of the dextran of very low degree of branching (dextran B-1254 fraction L[S]), and then subtracting this spectrum of linear dextran from each of the other polysaccharide spectra. The resulting i.r.-absorbance difference-spectra were plotted, at uniform scale-expansion across the 1800-400-cm^?1 region, resulting in difference-absorbance features at ≈ 1100 and ≈ 800 cm^?1 for all branched dextrans. These absorbance differences could be correlated to the type and degree of dextran branching, which had previously been established by permethylation analysis. It was concluded that such F.t.-i.r. difference-spectra have general application for the structural analysis of polysaccharides.     

An improved synthesis of 1,6-anhydro-2,4-dideoxy-β-D-threo-hexopyranose

Dextran fractions from NRRL strains Leuconostoc mesenteroides B-1299 and B-1399, and the native, structurally homogeneous dextrans from L. mesenteroides. B-640, B-1396, B-1422, and B-1424, were examined by ¹³C-n.m.r. spectroscopy at 34 and at 90°, and by g.l.c.-m.s. The ¹³C-n.m.r. data indicate that the dextrans of this series branch exclusively through α-d-(1→2)-linkages, and differ from one another only in degree of linearity. Diagnostic, ¹³C-n.m.r resonances, correlating with 2,6-di-O-substituted α-d-glucosyl residues at branch points, have chemical shifts that are independent of the degree of linearity of the dextran. The intensities of these diagnostic resonances from branching residues, compared to the resonances associated with linear dextran (low degree of branching), are generally proportional to the degree of branching established by methylation-fragmentation analysis. The validity of assignment of the diagnostic, ¹³C-n. m.r. resonances is substantiated by a critical review of methods previously used to provide structural information on dextrans having α-d-(1→2)-linkages, and by evaluation of the corresponding results on the basis of the ultimate standard-methylation structural analysis.     

High-temperature enhancement of 13C-N.M.R. chemical-shifts of unusual dextrans,and correlation with methylation structural analysis

Dextran fractions from NRRL strains Leuconostoc mesenteroides B-742, B-1299, B-1355, and Streptobacterium dextranicum B-1254 were examined by ¹³C-n.m.r. spectroscopy at 34 and 90°, and by methylation structural analysis. The native, structurally homogeneous dextran from L. mesenteroides NRRL B-1402 was also examined. The data allow correlations to be made between the structure and physical properties of the S (soluble) and L (less-soluble) fraction pairs of dextrans B-742, B-1254, B-1299, and B-1355. For the dextrans under consideration here, increasing solubility of the dextran (both in water and in aqueous ethanol) was found to correlate with decreasing percentages of α-d-(1→6)-linked d-glucopyranosyl residues. Both the diagnostic nature of the 70–75-p.p.m. spectral region with regard to type of dextran branching, and the increase in resolution of the polysaccharide spectra at higher temperatures, have been further confirmed.     

Six unusual dextrans: methylation structural analysis by combined g.l.c.—m.s. of per-O-acetyl-aldononitriles

Six bacterial dextrans from NRRL strains Leuconostoc mesenteroides B-1299, B-1303. B-1355, and B-1399; Streptobacterium dextranicum B-1254; andA g.l.c. procedure permitted, for the first time. separation of the 2,3,4- from the 2.3.6-tri-O-methyl derivative of d-glucose. Deuteriomethyla     

Structural analysis of dextrans containing 4-O-α-d-glucosylated α-d-glucopyranosyl residues at the branch points,by use of 13c-nuclear magnetic resonance spectroscopy and gas-liquid chromatography-mass spectrometry

Dextran fractions from NRRL strain Streptococcus sp. B-1526 and the native, structurally homogeneous dextrans from Acetobacter capsulatum B-1225, Leuconostoc mesenteroides B-1307, and L. dextranicum B-1420 were examined by ¹³C-n.m.r. spectroscopy at 90°. Dextran B-1526 fraction I and dextran B-1420 were also examined by g.l.c:-m.s., methylation-structural analysis. All of these dextrans and dextran fractions branch, either primarily or exclusively, through α-d-(1→4)-glucopyranosyl linkages; however, their degrees of branching differ. Several ¹³C-n.m.r. resonances that are diagnostic for 4,6-di-O-substituted α-d-glucopyranosyl residues have been identified. Comparison was made with dextrans from L. mesenteroides B-742 fraction L and Streptobacterium dextranicum B-1254 fraction S[L], for which previously published, methylation-structural analyses had established the presence of 4,6-di-O-substituted α-d-glucopyranosyl residues at the branch points. These fermentation culture, and in a sedimented gum-phase (fraction I). The product from the soluble phase is designated here as fraction S in order to simplify the terminology. Originally⁷, this product was not designated a fraction, because it was, by definition⁸, the main dextran product. The same distinction also applies to the pairs of products from strains B-1380, B-1420, and b-1394 (see ref. 7). The attempts thus made to establish the significance of the phase separation were indeterminant.Methods.— Methods previously described were used for the mythylation⁹ of the dextrans and for structural analysis^6.38 by combined g.l.c-electron-impact mass spectrometry of the aldononitriles. For each permethylation, three successive Hakomori³⁹ methylations were employed on an initial, 40-mg sample, with ～80% (final weight) recovery of each permethylated dextran. Successive formolysis and acetic acid hydrolysis were employed, and, after each step, the resulting solutions were clear, colorless, and free from suspended material. All mass spectra were recorded with a Hewlett-Packard 5980A GC/MS integrated g.l.c.-m.s.-computer system. The g.l.c. peak-integrals reported in Table II were obtained with a Barber-Coleman Series 5000 g.l.c. instrument equipped with hydrogen-flame detectors. On-column injection with glass columns (2mmi.d. x 1.23m) was employed for all chromatograhy.The ¹³C-n.m.r. conditions and the methods for the preparation of dextran samples have been described⁴. In general, a Varian XL-100-15 spectrometer equipped with a Nicolet TT-100 system was employed in the Fourier-transform mode. The dextran samples, ～0.3g/4 mL of deuterium oxide, were maintained at 90°. Chemical shifts are expressed in p.p.m. relative to external tetramethylsilane, but were actually calculated by reference to the solvent lock-signal. The convolution-difference resolution-enhancement (c.d.r.e.) technique has been described⁴⁰.     

Stepwise synthesis of methyl 4-O-[3-O-(β-d-xylopyranosyl)-β-d-xylopyranosyl]-β-d-xylopyranoside

The general properties and specificity of a dextran α-(1→2)-debranching enzyme from Flavobacterium have been examined in order to apply this enzyme to the structural analysis of highly branched dextrans. The optimum pH range and temperature were pH 5.5–6.5, and 45°, respectively. The enzyme was stable up to 40° on heating for 10 min, and over a pH range of 6.5–9.0 on incubation at 4° for 24 h. The effects of various metal ions and chemical reagents have also been examined. The debranching enzyme has a strict specificity for the (1→2)-α-d-glucosidic linkage at branch points of dextrans and related branched oligosaccharides, and produces d-glucose as the only reducing sugar. The degree of hydrolysis of the dextrans by this enzyme and the K_m value (mg/mL) were as follows: B-1298 soluble, 25.2%, 0.21; B-1299 soluble, 31.5%, 0.27; and B-1397, 11.8%, 0.91. The debranching enzyme thus has a novel type of specificity as a dextranhydrolase. We have termed this enzyme as dextran α-(1→2)-debranching enzyme, and its systematic name is also discussed.     

Hydrolysis of fourteen native dextrans by arthrobacter isomaltodextranase and correlation with dextran structure

The isomaltodextranase (EC 3.2.1.94) from Arthrobacter globiformis T6 hydrolysed thirteen dextrans to various extents (11?64% after 13 days) at initially large but gradually decreasing rates. Dextran B-1355 fraction S was, unlike the other dextrans, hydrolysed by the dextranase initially at the lowest rate among the dextrans used, but the rate was maintained for a long period with little decrease, so that the hydrolysis reached as high as 85% after 13 days. Paper chromatography of these dextran digests revealed that this dextranase produces in addition to isomaltose, one or two trisaccharides [isomaltose residues substituted by (1 →2)-, (1→3)-, or (1→4)-α-D-glucopyranosyl groups at the non-reducing D-glucopyranosyl residues] from every dextran used. It is evident that the non-(1→6)-linkages of these trisaccharide products constitute the “anomalous” linkages of the corresponding dextrans. The relative amounts of these trisaccharide products appear to indicate the approxima te relative amounts of a particular linkage among the dextrans, or the relative amounts of two kinds of linkages of each dextran. The kinds and the relative amounts of “anomalous” linkages of some dextrans were established on the basis of the trisaccharides produced by isomaltodextranase.     

Structural analysis of leuconostoc dextrans containing 3-O-α-d-glucosylated α-d-glucosyl residues in both linear-chain and branch-point positions,or only in branch-point positions,by methylation and by 13C-N.M.R. spectroscopy

It had been established by methylation-structural analysis that dextran fraction S from Leuconostoc mesenteroides NRRL B-1355 has two types of α-d-glucopyranosyl residues that are linked through O-3, i.e., 35% of the residues carry a (1→3)-bond, and ～10% carry a (1→6)-bond in addition to a (1→3)-bond. Two similarly constituted dextrans have now been identified by methylation-structural analysis, namely, the S-type fractions from L. mesenteroides strains NRRL B-1498 and B-1501. The S-type fractions from L. mesenteroides strains B-1355, B-1498, and B-1501 are structurally differentiated from the α-d-glucans (characteristically insoluble) of certain cariogenic Streptococci which also contain both 3-O- and 3,6-di-O-substituted α-d-glucopyranosyl residues. ¹³C-N.m.r. spectra have been recorded at 90° for both the S- and L-type fractions of strains B-1355, b-1498, and B-1501. The L-type fractions have a low degree of branching through 3,6-di-O-substituted αd-glucopyranosyl residues, but no 3-mono-O-substituted residues. (Dextran fraction S of Streptococcus 5000 g.l.c. instrument equipped with hydrogen-flame detectors. On-column injection of glass columns (2 mm i.d. x 1.23 m) was employed for all such chromatography.The ¹³C-n.m.r. conditions and methods for preparation of dextran samples have been described(su4). In general, a Varian XL-100-15 spectrometer equipped with a Nicolet TT-100 system was employed in the Fourier-transform mode. Chemical shifts are expressed in p.p.m. relative to external tetramethylsilane, but were actually calculated by reference to the lock signal.     

Dextransucrase constitutive mutants of Leuconostoc mesenteroides B-1299

Leuconostoc mesenteroides B-1299 dextrans are separated into two kinds: fraction L, which is precipitated by an ethanol concentration of 38%, and fraction S, which is precipitated at an ethanol concentration of 40%. Fraction S dextran contained 35% of -1,2 branch linkages, and fraction L contained 27% -1,2 branch linkage with 1% -1,3 branch linkages. We have isolated mutants constitutive for dextransucrase from L. mesenteroides NRRL B-1299 using ethyl methane sulfonate. The mutants produced extracellular as well as cell-associated dextransucrases on glucose media with higher activities (2.5–4.5 times) than what the parental strain produced on sucrose. Based on Penicillium endo-dextranase hydrolysis, mutant B-1299C dextransucrases produced slightly different dextrans when they were elaborated on a glucose medium and on a sucrose medium. Mutant B-1299CA dextransucrase elaborated on a glucose medium and on a sucrose medium synthesized the same dextran, although the dextran was different from those of other mutants and the parental strain. Mutant B-1299CB dextransucrase, elaborated on a glucose medium and on a sucrose medium, formed different dextrans. Differences in water solubility, susceptibility to endo-dextranase hydrolysis, and the physical appearance of the ethanol precipitated dextrans elaborated by different mutants grown on glucose media and sucrose media were found. All mutant dextransucrases elaborated on a glucose medium bound to Sephadex G-200. After activity staining of nondenaturing sodium dodecyl sulfate—polyacrylamide gel electrophoresis activity bands, 184 and 240 Kd for each enzyme preparation, although each dextransucrase formed different dextran(s).     

Purification and some properties of the isomaltodextranase of Actinomadura strain r10 and comparison with that of Arthrobacter globiformis t6

A newly isolated soil-actinomycete, Actinomadura strain R10 (NRRL B-11411), produces an extracellular isomaltodextranase (optinal pH, 5.0) that was purified to homogeneity. It exolytically releases isomaltose and a minor trisaccharide product,α-d-Glcp-(1→3)-α-d-Glcp, from dextran B-512 and, in addition, forms transient transisomaltosylation products. This pattern of products is qualitatively similar to that previously reported for the isomaltodextranase (EC 3.2.1.94, optimal pH, 4-0) of Arthrobacter globiformis T6 (NRRL B-4425). The Arthrobacter isomaltodextranase is most active on the (1→6)-α-d-glucopyranosidic linkage, but the relative activity increases with the degrees of polymerization of isomalto-oligosaccharide substrates. In contrast, the relative activity of Actinomadura isomaltodextranase is almost constant throughout the same series of substrates, and is much higher on 3 O- and 4-O-α-isomaltosyl-oligosaccharides than that exhibited by the Arthrobacter enzyme; the activity of Actinomadura isomaltodextranase on the α-(1→4) linkage is 3-4 times greater than on the α-(1→6). These results indicate that, generically, the bacterial isomaltodextranase is a glycanase, whereas the actinomycetal enzyme is a glycosidase. This difference is reflected in the hydrolysis of dextrans, especially of dextran B-1355 (fraction S), which has a high content of unbranched α-(1→3) linked residues. In the digest of this dextran with Arthrobacter isomaltodextranse, short-chain fragments accumulated that were absent when the Actinomadura enzyme was employed.     

Studies on dextrans and dextranases. X. Types and percentages of secondary linkages in the dextrans elaborated by Leuconostoc mesenteroides NRRL B-1299

The water-soluble (dextran S) and less water-soluble (dextran L) dextrans elaborated by Leuconostoc mesenteroides NRRL B-1299 contain α-d-glucopyranose residues linked through positions 1 and 6, 1 and 3, as well as 1, 2, and 6. The approximate number of terminal non-reducing d-glucose residues and those linked through positions 1 and 6, 1 and 3, as well as 1, 2, and 6 in the average repeating-unit of dextran S are 5, 4, 1, and 5. The corresponding figures for dextran L are 5, 4, 3, and 5.     

Glucosyltransferase Mutants of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides NRRL B-1355 produces dextrans and alternan from sucrose. Alternan is an unusual dextran-like polymer containing alternating α(1→6)/α(1→3) glucosidic bonds. Cultures were mutagenized with UV and ethyl methanesulfonate, and colony morphology mutants were selected on 10% sucrose plates. Colony morphology variants exhibited changes from parent cultures in the production of one or more glucosyltransferases (GTFs) and glucans. Mutants were characterized by measuring resistance of glucan products to dextranase digestion, by electrophoresis, and by high-pressure liquid chromatography of maltose acceptor products generated from sucrose-maltose mixtures. Some mutants produced almost pure fraction L dextran, and cultures exhibited a single principal GTF band on sodium dodecyl sulfate-acrylamide gels. Other mutants produced glucans enriched for alternan. Colony morphology characteristics (size, smoothness, and opacity) and liquid culture properties (clumpiness, color, and viscosity in 10% sucrose medium) were explained on the basis of GTF production. Three principal GTF bands were detected.     

Action of alpha-1,6-glucan glucohydrolase on oligosaccharides derived from dextran

The action of α-1,6-glucan glucohydrolase on α-(1→6)-D-glucosidic linkages in oligosaccharides that also contain an α-(1→2)-, α-(1→3)-, or α-(1→4)-D-glucosidic linkage has been investigated. The enzyme could hydrolyse α-(1→6)-D-glucosidic linkages from the non-reducing end, including those adjacent to an anomalous linkage. α-(1→6)-D-Glucosidic linkages at branch points were not hydrolysed, and the enzyme could neither hydrolyse nor by-pass the anomalous linkages. These properties of α-1,6-glucan glucohydrolase explain the limited hydrolysis of dextrans by the exo-enzyme. Hydrolysis of the main chain of α-(1→6)-D-glucans will always stop one D-glucose residue away from a branch point. The extent of hydrolysis by α-1,6-glucan glucohydrolase of some oligosaccharide products of the action on dextran of Penicillium funiculosum and P. lilacinum dextranase, respectively, has been compared. Differences in the specificity of the two endo-dextranases were revealed. The Penicillium enzymes may hydrolyse dextran B-512 to produce branched oligosaccharides that retain the same 1-unit and 2-unit side-chains that occur in dextran.     

One-step synthesis of isomalto-oligosaccharide syrups and dextrans of controlled size using engineered dextransucrase

Isomalto-oligosaccharides and dextrans of controlled molecular weight of about 10 and 40 kDa were produced using a simple one-step process using engineered L. mesenteroides NRRL B-512F dextransucrase variants. Isomalto-oligosaccharides were produced in a 58% yield by the acceptor reaction with glucose, and reached a degree of polymerization of at least 27 glucosyl units. Reaction conditions for optimal synthesis of dextrans of controlled molecular weight were defined, in respect of initial sucrose concentration and reaction temperature. Thus, we achieved synthesis with impressive yields of 69 and 75% for the 40 and 10 kDa dextran species, respectively. These two dextran sizes are particularly suitable for clinical applications, and are of great industrial demand. Compared with the traditional processes based on chemical hydrolysis and fractionation, which achieve only low yields, the new enzymatic methods offer improvement in quantity, quality and efficiency.     

The PGE2/IL-10 Axis Determines Susceptibility of B-1 Cell-Derived Phagocytes (B-1CDP) to Leishmania major Infection

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. A mononuclear phagocyte derived from B-1 cells (B-1CDP) has been described. As the B-1CDP cells migrate to inflammatory/infectious sites and exhibit phagocytic capacity, the microbicidal ability of these cells was investigated using the Leishmania major infection model in vitro. The data obtained in this study demonstrate that B-1CDP cells are more susceptible to infection than peritoneal macrophages, since B-1CDP cells have a higher number of intracellular amastigotes forms and consequently release a larger number of promastigotes. Exacerbated infection by L. major required lipid bodies/PGE₂ and IL-10 by B-1CDP cells. Both infection and the production of IL-10 were decreased when PGE₂ production was blocked by NSAIDs. The involvement of IL-10 in this mechanism was confirmed, since B-1CDP cells from IL-10 KO mice are more competent to control L. major infection than cells from wild type mice. These findings further characterize the B-1CDP cells as an important mononuclear phagocyte that plays a previously unrecognized role in host responses to L. major infection, most likely via PGE₂-driven production of IL-10.     

Production of glucosyltransferases by wild-type Leuconostoc mesenteroides in media containing sugars other than sucrose

Leuconostoc mesenteroides produces glucosyltransferases (GTFs) and fructosyltransferases (FTFs) which are inducible enzymes which respectively synthesize dextrans and levans from sucrose. Except for a few mutant strains which produce high activities in glucose medium, L. mesenteroides is thought not to produce GTFs and FTFs unless sucrose is present. We show here that cultures of eight strains produced low, but detectable GTF activity when glucose, maltose or melibiose replaced sucrose as the growth substrate. Four strains also produced FTFs of approximately 130 kDa in medium with or without sucrose. The GTFs and FTFs produced on sugars other than sucrose could be detected as bands on SDS gels even when not detected by other methods. Except for strain B-523, the number, sizes and relative intensities of the bands were independent of the sugar used for growing the cultures. Alternansucrase from strains B-1355 and B-1501 in glucose or maltose medium was almost entirely associated with the cell fraction, ruling out binding to glucans as the cause of the association. Received 06 October 1998/ Accepted in revised form 05 February 1999     

Influence of the Ionic Composition of Fluid Medium on Red Cell Aggregation

The effects of ionic strength and cationic valency of the fluid medium on the surface potential and dextran-induced aggregation of red blood cells (RBC's) were investigated. The zeta potential was calculated from cell mobility in a microelectrophoresis apparatus; the degree of aggregation of normal and neuraminidase-treated RBC's in dextrans (Dx 40 and Dx 80) was quantified by microscopic observation, measurement of erythrocyte sedimentation rate, and determination of low-shear viscosity. A decrease in ionic strength caused a reduction in aggregation of normal RBC's in dextrans, but had no effect on the aggregation of neuraminidase-treated RBC's. These findings reflect an increase in electrostatic repulsive force between normal RBC's by the reduction in ionic strength due to (a) a decrease in the screening of surface charge by counter-ions and (b) an increase in the thickness of the electric double layer. Divalent cations (Ca⁺⁺, Mg⁺⁺, and Ba⁺⁺) increased aggregation of normal RBC's in dextrans, but had no effect on the aggregation of neuraminidase-treated RBC's. These effects of the divalent cations are attributable to a decrease in surface potential of normal RBC's and a shrinkage of the electric double layer. It is concluded that the surface charge of RBC's plays a significant role in cell-to-cell interactions.     

Differentiation of Dextran-Producing Leuconostoc Strains by a Modified Randomly Amplified Polymorphic DNA Protocol

Seven dextran-producing Leuconostoc strains were differentiated by using a modified randomly amplified polymorphic DNA (RAPD) protocol that incorporated specific primers designed from conserved regions of dextransucrase genes. RAPD profiles showed intraspecies differences among the Leuconostoc mesenteroides strains tested. This modified RAPD protocol will aid in the differentiation of polymer-producing leuconostocs, which are currently distinguished by time-consuming analyses of the dextrans they synthesize.     

Halogranum salarium sp. nov., a halophilic archaeon isolated from sea salt

Three halophilic archaea, strains B-1^T, B-3 and B-4, were isolated from evaporitic salt crystals from Namhae, Korea. Cells of the strains were Gram-stain-negative, motile and pleomorphic, and colonies were red-pigmented. The three isolates had identical 16S rRNA gene sequences and formed a tight phylogenetic clade with Halogranum rubrum RO2-11^T in the genus Halogranum, showing 99.5% sequence similarity. The next most closely related species were Halogranum amylolyticum and Halogranum gelatinilyticum (97.4 and 96.3% similarity to the respective type strains). The phylogeny based on the full-length RNA polymerase subunit B′ gene (rpoB′) was in agreement with the 16S rRNA gene sequence analysis, but allowed better discrimination. DNA-DNA hybridization between a representative strain (B-1^T) and the type strains of Hgn. rubrum, Hgn. amylolyticum and Hgn. gelatinilyticum revealed less than 40% relatedness. Polar lipid analysis showed that the three isolates contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and three glycolipids. Combined genotypic and phenotypic data supported the conclusion that strains B-1^T, B-3 and B-4 represent a novel species of the genus Halogranum, for which the name Halogranum salarium sp. nov. is proposed. The type strain is B-1^T (=KCTC 4066^T = DSM 23171^T).     

Enzymatic synthesis of l-DOPA α-glycosides by reaction with sucrose catalyzed by four different glucansucrases from four strains of Leuconostoc mesenteroides

l-DOPA α-glycosides were synthesized by reaction of l-DOPA with sucrose, catalyzed by four different glucansucrases from Leuconostoc mesenteroides B-512FMC, B-742CB, B-1299A, and B-1355C. The glucansucrases catalyzed the transfer of d-glucose from sucrose to the phenolic hydroxyl position-3 and -4 of l-DOPA. The glycosides were fractionated and purified by Bio-Gel P-2 column chromatography, and the structures were determined by ¹H NMR spectroscopy. The major glycoside was 4-O-α-d-glucopyranosyl l-DOPA, and the minor glycoside was 3-O-α-d-glucopyranosyl l-DOPA. The two glycosides were formed by all four of the glucansucrases. The ratio of the 4-O-α-glycoside to the 3-O-α-glycoside produced by the B-512FMC dextransucrase was higher than that for the other three glucansucrases. The glycosylation of l-DOPA significantly reduced the oxidation of the phenolic hydroxyl groups, which prevents their methylation, potentially increasing the use of l-DOPA in the treatment of Parkinson’s disease. The use of one enzyme, glucansucrase, and sucrose as the d-glucosyl donor makes the synthesis considerably simpler and cheaper than the formerly published procedure using cyclomaltodextrin and cyclomaltodextrin glucanyltransferase, followed by glucoamylase, and β-amylase hydrolysis.