Michikatsu Sato

Isolation and characterization of branched cyclodextrins

Abstract Three branched cyclodextrins (CDs) were isolated by high-performance liquid chromatography (l.c.) from the mother liquors of a large-scale preparation of the unbranched CDs with Bacillus ohbensis cyclomaltodextrin glucanotransferase. Evidence from chromatographic behavior on three l.c. columns of different separation modes, fragmentation analysis, 13C-n.m.r. spectroscopy, methylation analysis, and fast-atom bombardment-mass spectrometry (f.a.b.—m.s.) indicated that these compounds were 6-O-α- d -glucopyranosylcyclomaltohexaose (1), 6-O-α- d -glucopyranosylcyclomaltohepataose (2), and 6,6‴-di-O-α- d -glucopyranosylcyclomaltoheptaose (3).

Purification and Characterization of an α- L -Rhamnosidase from Pichia angusta X349

An intracellular α-L-rhamnosidase from Pichia angusta X349 was purified to homogeneity through four chromatographic steps. The α-L-rhamnosidase appeared to be a monomeric protein with a molecular mass of 90 kDa. The enzyme had an isoelectric point at 4.9, and was optimally active at pH 6.0 and at around 40°C. The Ki for L-rhamnose inhibition was 25 mM. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The α-L-rhamnosidase was highly specific for α-L-rhamnopyranoside and liberated rhamnose from naringin, rutin, hesperidin, and 3-quercitrin. The α-L-rhamnosidase was active at the ethanol concentrations of wine. It efficiently released monoterpenols, such as linalool and geraniol, from an aroma precursor extracted from Muscat grape juice.

Colour and Sensory Characteristics of Merlot Red Wines Caused by Prolonged Pomace Contact

Pigment analytical parameters and sensory characteristics of Merlot red wines made with pomace contact during fermentation and subsequent maceration were investigated. The fermenting (at 25 ° C) musts were pressed at 0 (free-run juice), 1, 2, 4, 8, 16, 32 and 64 days after crushing and addition of the yeast. All wines were allowed to finish fermentation after pressing. High scores were given for wines made with 4, 8 and 16 days’ pomace contact. The sensory characteristics correlated with the wine component and pigment parameter results.

Prevention of Pathogenic Escherichia coli Infection in Mice and Stimulation of Macrophage Activation in Rats by an Oral Administration of Probiotic Lactobacillus casei I-5

Lactobacillus casei I-5 isolated from an alcohol fermentation broth enhanced immunity and prevented pathogenic infection as a probiotic. Mice fed with I-5 cells for 11 days prior to an intraperitoneal challenge with pathogenic Escherichia coli Juhl exhibited a high survival rate compared with the control group. Rats fed with I-5 cells for 10 days significantly increased the phagocytosis of peritoneal macrophages. In a cell culture system employing peritoneal macrophages from rats, the I-5 administration activated NF-κB stimulated by LPS. It also enhanced LPS-stimulated IL-12 and TNF-α production, but not IL-6 production. These results show that L. casei I-5 effectively prevented infection by pathogenic E. coli possibly through the activation of peritoneal macrophages. The strain would be useful to prevent pathogenic microbial infections in humans and farm animals.

Purification and Characterization of a Novel α- L -Arabinofuranosidase from Pichia capsulata X91

An intracellular α-L-arabinofuranosidase from Pichia capsulata X91 was purified and characterized. The enzyme was purified to homogeneity from a cell-free extract by ammonium sulfate treatment, Concanavalin A-Sepharose, ion-exchange chromatography with DEAE Bio-Gel A agarose, arabinose-Sepharose 6B affinity chromatography, and hydroxyapatite column chromatography. The apparent molecular mass of the enzyme was estimated to be 250 kDa by native-PAGE. The enzyme molecule was suggested to be a tetramer with a subunit molecular mass of 72 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.1, and was most active at pH 6.0 and at around 50°C. The α-L-arabinofuranosidase was active at ethanol concentrations of wine. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The enzyme hydrolyzed beet arabinan and arabinogalactan, and efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. A considerable amount of monoterpenols was produced in the Muscat wine coupled with the enzyme addition.

Prolyl Endopeptidase Inhibitory Peptides in Wine

Two peptides that inhibit prolyl endopeptidase were isolated from a red wine made from Cabernet Sauvignon grapes. Their amino acid sequences and IC50 values were Val-Glu-Ile-Pro-Glu (17.0 μM) and Tyr-Pro-Ile-Pro-Phe (87.8 μM). The peptides also suppressed the degradation levels of the bioactive peptides vasopressin, substance P, and neurotensin fragments 8-13, which are involved in memory and neural communication.

Synthesis of novel sugars, oligoglucosyl-inositols, and their growth stimulating effect forBifidobacterium

SummaryNovel sugars, oligoglucosyl-inositols, which were synthesized using CGTase fromBacillusohbensis, stimulated the growth ofBifidobacterium. The enzyme catalyzed transglucosylation from α-1,4-maltodextrin (donor) tomyo-inositol (acceptor). Of donors examined, β-cyclodextrin gave superior oligoglucosyl-inositol yield of 56.6% (w/w) based on the conversion ratio of incubated inositol. Maltosyl-inositol stimulated growth ofB.adolescentis by 194% when compared with glucose.

Synthesis of glucosyl-inositol using a CGTase, isolation and characterization of the positional isomers, and assimilation profiles for intestinal bacteria

A novel disaccharide, glucosyl-inositol, was obtained by glucoamylase digestion of the oligoglucosylinositols synthesized frommyo-inositol as an acceptor and β-cyclodextrin as a donor by transglucosylation of CGT ase fromBacillus ohbensis. The glucosyl-inositol fraction was separated by ion-exchange column chromatography and two positional isomers contained in the fraction were isolated by crystallization and HPLC on a graphitized carbon column. The structure of one of the two isomers isolated was fully determined as 1L(D)-5-O-α-D-glucopyranosyl-myo-inositol and another one was presumed as 1D-4-O-α-D-glucopyranosyl-myo-inositol from physicochemical data, H-H and C-H COSY NMR analyses and FAB-MS spectra. The disaccharide was assimilated byBifidobacterium adolescentis, B. breve, B. infantis andB. longum. On the other hand, it was not utilized byE. coli, Clostridium butyricum, C. clostridiiforme andKlebsiella pneumoniae.

Thermodynamics of the Hydrolysis and Cyclization Reactions of α-, β-, and γ-cyclodextrin

A thermodynamic investigation of the hydrolysis and cyclization reactions of cyclomaltohexa-, hepta-, and octa-ose (alpha-, beta-, and gamma-cyclodextrins) has been performed using microcalorimetry and high-performance liquid-chromatography. The calorimetric measurements lead to standard molar enthalpy changes delta rHm0 (T = 298.15 K, KH2PO4 buffer (m = 0.10 mol kg-1), pH = 4.58 to 5.15) for the following reactions: alpha-cyclodextrin(aq) + 6H2O(l) = 6 D-glucose(aq), beta-cyclodextrin(aq) + 7H2O(l) = 7 D-glucose(aq), gamma-cyclodextrin(aq) + 8H2O(l) = 8 D-glucose(aq). Equilibrium constants were determined for the following generalized cyclization reactions (T = 329.6 K, 0.005 mol kg-1 K2HPO4 buffer adjusted to pH = 5.55 with H3PO4) catalyzed by cyclomaltodextrin glucanotransferase: Gu(aq) = alpha-cyclodextrin(aq) + G(u-6)(aq), Gv(aq) = beta-cyclodextrin(aq) + G(v-7)(aq), Gw(aq) = gamma-cyclodextrin(aq) + G(w-8)(aq). Here, G1 is D-glucose and the Gns (n is a positive integer) are linear maltodextrins; u, v, and w are, respectively, integers > or = 7, > or = 8, and > or = 9. Values of the equilibrium constants, standard molar Gibbs energy change delta rGm0, standard molar enthalpy change delta rHm0, standard molar entropy change delta rSm0, and standard molar heat-capacity change delta rCp,m0 are tabulated for the above reactions at T = 298.15 K. The values of delta rGm0 and delta rSm0 for the first three above-mentioned reactions rely upon an estimated value of delta rSm0 for the hydrolysis reaction of maltose to D-glucose. The thermodynamics of the disproportionation reaction Gm(aq) + Gn(aq) = Gm-1(aq) + Gn+1(aq) is also discussed. Values of the quantities delta rHm0/N, delta rGm0/N, delta rSm0/N, and delta rCp,m0/N for the three above-mentioned hydrolysis reactions where N is the number of (1-->4)-alpha-D-glucosidic bonds broken in each of these reactions, have been calculated and compared with thermodynamic quantities for the similar hydrolysis reaction of a linear oligosaccharide.

Changes in Radical Scavenging Activity of Japanese Cabernet Sauvignon Red Wines during Ageing

We investigated the relationship between the concentration of phenols in Cabernet Sauvignon red wines produced over 21 years from 1987 to 1998 and their radical scavenging activities [RSA]. The correlation between the concentrations of total phenols, or between those of either of the phenolic fractions and RSA was close. In contrast, the correlation between RSA and non-flavonoid phenols was poor. Total and flavonoid phenol concentrations and the total RSA of wines decreased during a period of about 10 years of storage, but their relative RSA (RSA mg -1 total phenols or flavonoids) gradually increased.