Stabilisierung fettlöslicher Vitamine mit beta-Cyclodextrin

Stabilization of Fatsoluble Vitamins with ß-Cyclodextrin. Stability and dispersibility of the most important fatsoluble vitamins D₃, K₃, A and E can be improved by complexing with cyclodextrin. Two molecules of ß-cyclodextrin form a molecular “capsule” for one molecule cholecalciferol. The complexation enhances the solubility in water, the stability against heat, light, oxygen, cations, as well as biological activity of D₃-Vitamin. K₃-Vitamin forms a 1:1 complex with ß-cyclodextrin in aqueous solution, however, the isolated crystalline product contains 3 mol ß-cyclodextrin for one mol menadione. The complexed menadione is not sublimable, and retains its biological activity.     

Carboxymethyl Kefiran: Preparation and Viscometric Properties

Carboxymethyl (CM-) kefiran was prepared with monochloroacetic acid in isopropyl alcohol at 30, 40 and 50°C for 4 and 24 hr. Resulting CM-kefiran varied in degree of substitution (0.73-0.89), molecular weight (5.0-7.0 × 10⁵) and viscosity (4.5-14.5 cP in 0.5% aqueous solution) depending on the conditions. Carboxymethylation resulted in a maximum fourteenfold increase in viscosity. ¹³C-NMR spectra of CM-kefiran indicated C-6 of native kefiran was preferentially substituted with CM-groups.     

Studies on flavonol degradation by peroxidase (donor: H2O2-oxidoreductase,EC 1.11.1.7): Part 2—Quercetin

The peroxidatic transformation of quercetin was studied at pH 5·5 under UV-spectrophotometric control. The reaction products obtained after 3 minutes' incubation of quercetin (dissolved in ethylene glycol monomethyl ether) with peroxidase (Boehringer, Mannheim)/H₂O₂ were fractionated on preparative scale by Sephadex LH-20 liquid chromatography using methanol as solvent. More than twenty compounds could be detected by analytical HPLC, from which the following substances were characterized by their spectroscopic data (UV; IR; ¹H- and, in part, ¹³C-NMR; EI- and, in part, FAB- and FD-MS): 2,4,6-trihydroxybenzoic acid (Q1); 3,4-dihydroxybenzoic acid (Q2); methyl 2,4,6-trihydroxyphenyl-glyoxylate (Q3); 2-(3,4-dihydroxyphenyl)-2-hydro-3,5,7-trihydroxy-3-methoxy-4H-1-benzopyran-4-one (‘quercetinchinone’) (Q5); 2,3-epoxy-2-(3,4-dihydroxyphenyl)-3-[4O-[2-(3-hydroxyphenyl)-3,5,7-]trihydroxy-4H-1-benzopyran-4-onyl]-5,7-dihydroxy-4H-1-benzopyran-4-one (2 isomers) (Q7) and a trimer (Q8) structurally related to Q7.     

Stereoisomeric flavour compounds LXXX: alkyl-branched alkanals – stereodifferentiation, structure elucidation and structure – function relationship

The direct enantioseparation of 2-methylbutanal, 2-methylpentanal, 2-ethylhexanal, 3-methylpentanal and 4-methylhexanal was achieved using enantioselective gas chromatography and the chiral stationary phase heptakis-[2,3-di-O-acetyl-6-O-tert.butyldimethylsilyl]-β-cyclodextrin (DIAC-TBDMS-β-CD). Odour characteristics and threshold values of detection for compounds were evaluated by enantioselective gas chromatography/olfactometry. Enantiopure reference compounds with defined absolute configurations were synthesized starting from the corresponding alcohols or acids which were commercially available or prepared in the laboratory.     

Galactomannan esters—A simple, cost-effective method of preparation and characterization

Acetate, succinate and octenylsuccinate derivatives of galactomannans were prepared in anhydrous reaction conditions carried out at slightly elevated temperature (40–60 °C) using solid NaHCO₃ as a mild base catalyst. Prior surface wetting of the reactants with 5% absolute ethyl alcohol gave derivatives with a higher degree of substitution, although it decreased the slurry viscosity. Use of NaHCO₃ significantly minimized high pH-induced degradative reactions, and its quantitative removal later was easily accomplished by repeated washings with aqueous ethyl alcohol. FT-IR, ¹³C-NMR, HPSEC and SEM data provided additional structural information. These derivatives are useful as functional food ingredients.     

Improved Reaction Conditions for Preparation of β-Cyclodextrin (β-CD) from Cassava (Manihot esculenta Crantz) Starch

Experimental conditions for bioconversion of starch to get higher yield of ß-cyclodextrin using cyclodextrin glycosyl transferase (CGT-ase) (EC 2.4.1.19) were worked out. A 5% initial starch concentration gave ß-CD yield of 20-28% that could be separated as ß-CD bromobenzene complex from the reaction mixture. Addition of bromobenzene during the second phase of the reaction, i.e. CD formation phase, was found beneficial.     

An Improved Method for the Preparation of Schardinger β-Dextrin on a Industrial Scale by Cyclodextrin Glycosyl Transferase of an Alkalophilic Bacillus Sp. (ATCC 21783)

The formation of Schardinger ß-dextrin from starch by an alkalophilic Bacillus sp. (ATCC 21783) has been studied. Factors affecting the yield of cyclodextrins include the type, concentration, and dispersion of the starch; the time of the enzymolysis, the amount of enzyme used. A method for the preparation of Schardinger ß-dextrin on a industrial scale without using precipitants was devised. The final yield of ß-cyclodextrin was 3.6 kg from 15 kg of potato starch.     

Purification and application of α-galactosidase from germinating coffee beans (<Emphasis Type="Italic">Coffea arabica</Emphasis>)

The objective of this study was to prepare and purify α-galactosidase (α-Gal) from germinating coffee beans. The molecular weight of purified α-Gal from germinating coffee beans was determined to be 38.8 kDa by SDS-PAGE. Then the purified α-Gal was applied to synthesize galactosyl β-cyclodextrin using melibiose as donor and β-cyclodextrin as acceptor. Galactosyl β-cyclodextrin was isolated by preparative high-performance liquid chromatography (HPLC) and its structure was analyzed and elucidated by HPLC, fourier transform infrared spectrometer, electro spraying ionization-mass spectrometry, and ¹³C nuclear magnetic resonance. Our results strongly demonstrate that the synthesized product is a mono-6-O-α-d-galactopyranosyl-β-cyclodextrin and the purified α-galactosidase could transfer one galactosyl residue directly to the β-cyclodextrin ring and synthesize the mono-6-O-α-d-galactosyl β-cyclodextrin.     

Enhancement of Drug Bioavailability by Cyclodextrins

The reassuring results of toxicity of orally administered ß-cyclodextrin and the realization of its industrial production opens a new way in the formulation of non-parenteral drug preparations. It is assumed that the utilization of ß-cyclodextrin in oral drug preparations will be a common process within a decade. The molecular encapsulation of drugs – i.e. complexation with cyclodextrin – has numerous advantages out of which one of the most important is the improved bioavailability. By inclusion complexation of a drug with cyclodextrin the drug becomes molecularly dispersed in a hydrophylic matrix, therefore the poorly soluble drug will become more soluble and will dissolve with an increased dissolution rate in aqueous milieu. The improved solubility generally leads to higher blood-level of the drug, which is manifested also in the biological response. The same dose of the drug results in an higher biological effect when applied a cyclodextrin complex, or to achieve an unaltered effect the drug dose can be reduced.     

Production of Cyclodextrins by Simultaneous Actions of Two CGTases from Three Strains of Bacillus

A moderate thermophile, which produces a large amount of extracellular and thermostable cyclomaltodextrin glucanotransferase, was isolated from soil samples at hot spring areas in Japan. The isolate (A-147), that grew at 30–60°C with an optimum at 45–50°C, was identified as a strain of Bacillus coagulans. The enzyme, which was most active at pH 6.5 and 60°C, was stabilized by 2–3 mM Ca²⁺, and degraded about 40% (as dry basis) of 13% (w/w) potato starch solution into cyclodextrins composed of α- and β-cyclodextrins dominantly at 74°C and pH 6.5 in the presence of 3 mM Ca²⁺. The ratio of α-, β- and γ-cyclodextrins was almost constant (2:2.6:1) during all stages of the reaction. By simultaneous actions of two enzymes from the bacterium and other 2 kinds of Bacillus, starch hydrolysates containing various amounts and rations of cyclodextrins were produced from starch. Ein mäßiges Thermophiles, das eine große Menge an extracellulärer und thermostabiler Glucanotransferase produziert, wurde aus Bodenproben in Heißquellengebieten von Japan isoliert. Das Isolat (A-147), das bei 30–60°C mit einem Optimum von 45–50°C wuchs, wurde als ein Stamm von Bacillus coagulans identifiziert. Das bei pH 6,5 und 60°C aktivste Enzym wurde durch 2–3 mM Ca²⁺ stabilisiert und baute etwa 40% (TS) einer 30%igen (w/w) Kartoffelstärkelösung zu Cyclodextrin ab, bestehend aus α- und β-Cyclodextrin, vorwiegend bei 74°C und pH 6,5 in Gegenwart von 3 mM Ca²⁺. Das Verhältnis von α-, β- und γ-Cyclodextrinen war nahezu konstant (2:2,6:1) während sämtlicher Reaktionsschritte. Durch gleichzeitige Einwirkung von zwei Enzymen aus dem Bakterium und zwei anderen Arten von Bacteria wurden Stärkehydrolysate hergestellt, die verschiedene Mengen und Verhältnisse an Cyclodextrinen enthielten.     

Anwendung der13C-NMR-Spektroskopie in der Aminos?ureanalytik in Wein und Fruchts?ften

Zusammenfassung Mit Hilfe der¹³C-NMR-Spektroskopie können die Aminosäuren in Wein (Weinextrakt), Fruchtsäften und Fruchtsaftkonzentraten ohne Probenvorbehandlung und ohne vorherige Auftrennung strukturspezifisch nachgewiesen und quantitativ bestimmt werden. Bei der quantitativen Bestimmung werden weder die Signale der Carboxylgruppe noch die an Stickstoff gebundenen Kohlenstoffe berücksichtigt. Ein Vergleich der bei Wein und Orangensaftkonzentrat mit der¹³C-NMR-Methode erhaltenen quantitativen Meßwerten mit den Werten einer gebräuchlichen Aminosäureanalyse (Aminosäureanalysator) zeigt eine gute Übereinstimmung der Ergebnisse. Lediglich bei einigen Aminosäuren, die cyclische Säureamide bilden können (Glutaminsäure, 4-Aminobuttersäure) bzw. peptidisch oder glykosidisch gebunden vorliegen (Serin, Alanin) sind Abweichungen zwischen beiden Methoden vorhanden. In Orangensaftkonzentraten und Orangensaft konnte N,N-Dimethylprolin (in Orangensäften mit Gehalten von 200 bis 740 mg/L) identifiziert werden. Diese Komponente war bisher in Säften nicht bekannt.

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Application of¹³C-NMR spectroscopy for detection and quantitative determination of amino acids in wine and fruit juices

Summary With the help of¹³C-NMR spectroscopy, the amino acids in wine (wine extract), fruit juices and juice concentrates can be identified and quantified without sample preparation and prior separation in a structure-specific way. Neither the signals of carboxylic groups nor those of carbons bonded to nitrogen are used for the quantitative determination. Comparison of the results gained by the¹³C-NMR method to the results of the normal analysis with an amino acid analyser shows good accord. Only for some amino acids which form cyclic acid amides (glutamic acid, 4-amino butyric acid) or which have peptide or glycosidic bonds are deviations between the methods observed. In orange juice concentrates and juices,N,N-dimethylprolin (in orange juices 200–740 mg/L) was identified. This compound was not previously known in juices.

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Auszug aus der Dissertation von A. Markowetz, Universität Karlsruhe, Dezember 1989     

Synthesis of raffinose by fungal α-galacotosidase from Absidia corymbifera

In order to investigate the optimal conditions for raffinose synthesis, α-galactosidase was purified from Absidia corymbifera IFO8084 with a recovery yield of approximately 8.1% (8.36 mg). The molecular weight of the wild-type α-galactosidase was about 83 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native molecular mass of the enzyme was approximately 330 kDa by gel filtration chromatography, indicating that α-galactosidase from A. corymbifera IFO8084 is a homotetrameric enzyme. The purified enzyme displayed optimal enzyme activity at pH 4.5 and 60°C. When the purified α-galactosidase was incubated in a substrate solution of sucrose and D-galactose for 48 hr at 37°C, raffinose was synthesized and was confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and ¹³C-nuclear magnetic resonance (¹³C-NMR) spectrometer analysis. Maximum rates of conversion were observed with 1.67 M galactose, 2.04 M sucrose, and 100 U α-galactosidase at pH 6.0 and 70°C. Under the optimized conditions, the overall conversion ratio was 10%(w/v), representing 2.5 times the synthesis yield that would be possible without the optimized conditions.     

Effect of Ultra-Violet B on Release of Volatiles in Tea Leaf

Many volatiles in fresh tea leaf are in the forms bound with glycosides and they are released by action of ß–primeverosidase and ß-glucosidase during tea processing. Effect of ultra-violet B on release of volatiles in fresh tea leaves of two tea cultivars, “Maoxie” and “Anhui-9,” were investigated. Types of volatile and their concentration increased when ultra-violet B was applied at 3.0 μmol m^?2 s^?1 for 2 h and then decreased as the irradiation was extended to 6 h. The relation of expression of ß–primeverosidase and ß-glucosidase genes to the release of volatiles were also discussed.     

Darstellung von Methyl-beta-D-fructopyranosid,Methyl-alpha-D-fructofuranosid und Methyl-beta-D-fructofuranosid

Preparation of Methyl-ß-D-fructopyranoside, Methyl-α-D-fructofuranoside and Methyl-ß-D-fructopyranoside. A crude mixture of methyl-D-fructosides obtained by the reaction of D-fructose in dry protonated methanol (Fischer-reaction) easily can be separated on a column with Dowex 1 × 2 (OH⁻). Thus pure methyl-ß-D-fructofuranoside, methyl-ß-D-fructofuranoside and methyl-ß-D-fructopyranoside can be isolated. The Fischer-reaction can be watched by means of gas-liquid-chromatography after trimethylsilylation of reactionmixtures. In the first time of reaction the furanoside fructosides are formed in a kinetic controlled reaction; after equilibration the methyl-ß-D-fructopyranoside is the thermodynamic more stable main component.     

IONIZATION BEHAVIOUR OF HOP COMPOUNDS AND HOP-DERIVED COMPOUNDS

The ionization behaviour of colupulone, (?)-humulone, trans-isohumulone and trans-humulinic acid was investigated using a number of techniques. When a potentiometric technique was employed, precise estimates of pKa could not be obtained for any of the compounds, since a drift in the observed pKa values occurred during the titrations. Approximate, or equilibrium pKa values for the most acidic function of each of the compounds were: colupulone, 6.1; (?)-humulone, 5.0; trans-isohumulone, 3.1; trans-humulinic acid, 2.7. Evidence from spectroscopic measurements (UV, ¹H-NMR, ¹³C-NMR), conductiometric experiments and measurements of the pH dependency of solvent partition behaviour and aqueous solubility of the compounds is consistent with the possibility that, in aqueous solution, compounds such as trans-isohumulone are present as covalent hydrates. It is likely that reversible hydration occurs at one or more carbonyl groups. As a consequence of this behaviour, no single pKa value is sufficient to describe the behaviour of any one hop compound or hop-derived compound. The potential significance of this phenomenon is discussed.     

Comparison of HPLC Separation of Phenylalanine Enantiomers on Different Types of Chiral Stationary Phases

HPLC separation of phenylalanine by using chiral stationary phases (CSPs) with chiral selectors β-cyclodextrin, isopropylcarbamate cyclofructan 6, teicoplanin, and ristocetin were studied. The effects of mobile phase composition and column temperature from 0 to 30 °C on the retention and resolution of enantiomers were investigated. β-cyclodextrin and isopropylcarbamate cyclofructan 6 CSPs showed chiral recognition in normal phase separation mode. The sufficient enantioseparations (resolution values 1.59 and 2.75) were reached using teicoplanin and ristocetin-based stationary phases at 23 °C in reversed-phase mode with mobile phases consist of acetonitrile and water at ratios 75/25 and 60/40 (v/v), respectively. Chiral HPLC system coupled with circular dichroism and polarimetry detector could be used to determine enantiomeric elution order. The optimized and validated HPLC-UV method with teicoplanin-based chiral stationary phase was applied for determination of phenylalanine in real samples. Both of the enantiomeric forms were detected in samples of dietary supplements and L-phenylalanine in samples of energy drinks. Obtained recoveries were higher than 82% with an RSD less than 10%. The HPLC method showed good linearity (correlation coefficients >?0.998) in concentration range 0.1–500 μg mL^?1. The limit of detection was 0.1 μg mL^?1 for both enantiomers.     

Determination of Iron in Edible Oil by FAAS After Extraction with a Schiff Base

The synthesis of a new Schiff base has been achieved by condensation reaction of 1,3-diamino-2-propanol and 2-hydroxy-5-methoxy-benzaldehyde in alcoholic media. The Schiff base was characterized by elemental analysis, IR, UV–Visible, ¹H-NMR, and ¹³C-NMR spectroscopy and used for the extraction of iron in liquid edible oils. Iron complex with the Schiff base was investigated in order to determine experimental conditions of the complexation. The extraction of iron from oils to aqueous phase was succeeded by the complexation with the Schiff base. A central composite design was employed in order to optimize the extraction conditions of iron. Optimum conditions for the iron extraction with N,N′-bis(5-methoxy-salicylidene)-2-hydroxy-1,3-propanediamine were found to be 19.3 °C, 2.1 mL g^?1, 10.0 min for the temperature, the ratio of the volume of used Schiff base solution to the amount of oil, and the stirring time, respectively. Method validation was performed with recovery experiments in the analysis of the oil-based metal standard by flame atomic absorption spectrometry, limit of detection, and the relative standard deviation of the proposed method were found to be 0.09 μg g^?1 and 1.04 %, respectively. Additionally, an alternative determination procedure (inductively coupled plasma optical emission spectrometry) was applied for the comparison. This paper describes a simple, cheap, rapid, efficient, sensitive, and accurate alternative analytical method for the iron determination in oils. The proposed method was applied on six different oil samples and the iron concentration was found in the range of 0.38–0.70 μg g^?1.     

Advances in the instrumental analysis of food flavours

Zusammenfassung Es wird eine Übersicht über die Entwicklung der instrumentellen Meßtechniken gegeben, die bei der Untersuchung von Lebensmittelaromen eingesetzt werden: Probenaufarbeitung, Trenn-(Injektionstechniken; multidimensionale Gaschromatographie; Säulentechnologie; Enantiomerentrennung) und Identifizierungstechniken (Kapillargaschromatographie-Massenspektrometrie; TandemMassenspektrometrie; Kapillargaschromatographie-FTIR-Spektroskopie; Hochdruckflüssigkeitschroma-tographie-Massenspektrometrie;¹H- und¹³C-NMR-Spektroskopie).

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Advances in the instrumental analysis of food flavours

Summary A review is provided on recent developments in instrumental measurement techniques used in food flavour analysis comprising sample preparation, separation methods (injection techniques; multidimensional gas chromatography; column technology; enantiomer separation) and identification techniques (capillary gas chromatography-mass spectrometry; tandem mass spectrometry; capillary gas chromatography-FTIR spectroscopy; high-performance liquid chromatography-mass spectrometry;¹H- and¹³C-NMR spectroscopy).

     

AN APPROACH TO THE IDENTIFICATION OF A ß-GLUCAN SOLUBILASE FROM BARLEY1

A ß-glucan solubilase activity was demonstrated in barley extract. This enzyme catalyzed the dissolution of barley ß-glucan by releasing a product having a narrow molecular weight distribution and a molecular weight of about 20,000. The enzyme was partially purified by ion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Bio-Gel P-100. Although carboxypeptidase activity was present in the crude extract of barley flour the partially purified ß-glucan solubilase did not hydrolyse N-CBZ-Phe-ala. Examination of extracts from different barley tissues indicated that the ß-glucan solubilase activity was associated with the husks only; a large portion of the activity was extractable from whole barley kernels. About 85% of the enzyme activity in crude extracts from barley flour was retained after 40 min at 62°C. However, the enzyme was much more heat-labile in extracts of whole barley kernels. The pH of maximal activity was found to be about pH 5.7 and results from column chromatography suggested that the enzyme had a low pl value and a MW between 5 × 10⁴ and 6 × 10⁴.     

Study on Specific Modification of Glucosyl Cyclodextrins

The time courses of the periodate oxidation of glucosyl cyclodextrins complexed with organic solvents indicated the preferential oxidation of glucosyl residues on side chains, and the modified cyclodextrins could be purified by using a reversed phase column. HPLC and ¹³C-NMR analyses ensured that the modified cyclodextrins were cyclodextrins having aldehyde residues on their side chains (aldehyde cyclodextrins).