Anton Huber

Molecular background of technological properties of selected starches

Selected starches, i.e. waxy maize, amaranth, quinoa, wheat, millet and buckwheat starches, were investigated with respect to their technological properties such as gelatinization, stability to mechanical stress, resistance to conditions and stability in continuous freeze/thaw cycles. Technological properties are correlated with molecular features such as branching characteristics in terms of iodine-complexing potential, molar mass, occupied glucan-coil volume, packing density of glucan coils and rheological properties. Waxy maize and amaranth starches were found to be amylopectin-type short-chain branched (scb) glucans with weight average molar masses M w = 17 × 10 6 g/mol and 12 × 10 6 g/mol, respectively. Waxy maize starch had a high gelatinization potential, high viscosity at 95 °C (340 mPas) low stability at acidic conditions, average stability to shearing and good freeze/thaw stability. For amaranth starch a viscosity of 122 mPas at 95°C, low resistance to acid, but high stability to applied shearing and even high freeze/thaw stability was determined. Investigated quinoa starch was classified as scb-type glucan, however, the branches are significantly longer than those of waxy maize and amaranth. With a M w = 11 × 10 6 g/mol and a viscosity of 187 mPas at 95°C, this sample is comparably resistant to acidic conditions and to shearing, but instable in freeze/thaw experiments. Wheat, millet and buckwheat starches contain significant percentages of amylose-type long-chain branched (lcb) glucans (22.1, 32.1 and 24.3%, respectively) with M w values of 5 × 10 6 g/mol, 12 × 10 6 g/mol and 15 × 10 6 g/mol, respectively. Wheat starch, with a viscosity of 107 mPas at 95°C, shows low stability under acidic conditions, but high stability to shearing. Wheat and millet starches, but not buckwheat starch, form weak gels in the course of subsequent freeze/thaw cycles. Millet starch, with a viscosity of 101 mPas at 95 °C was found to be moderately stable under acidic conditions and to shearing. Buckwheat starch with a viscosity of 230 mPas at 95 °C shows no acid resistance and is instable upon shearing but performs very well in freeze/thaw experiments.

The anionic glycan from the cactus Cereus peruvianus : structural features and potential uses

The columnar cactusCereus peruvianus provides various compounds of interest that account for most of its 10% dry wt content. Included are acidic gum and cellulose as the highly polymerized carbohydrate components, and a complex waxy lipid fraction. The major gum fraction (1.5 g% of the fresh phytobiomass on single aqueous extraction) is an uronylated rhamnoarabinogalactan whose intrinsic viscosity may exceed 1000 mL/g. Its rheological behavior is, in part, influenced by the nativeo-acetyl and cation components, mainly Ca2+. A pigment-free powdered gum was obtained by precipitating and washing the fresh mucilage with 2–3 vol of ethanol. The almost protein-free polysaccharide forms viscous solution upon redissolution. The possible uses to be investigated for the pretreated cactus gum will be as an adjuvant in the flocculation of water impurities and in formulation of cosmetics.

Characterisation of the (1→4)-α-d-glucan-branching 6-glycosyltransferase by in vitro synthesis of branched starch polysaccharides

Abstract Starch branching enzyme (Q-enzyme; EC 2.4.1.18), isolated from young, mature potato tubers (Solanum tuberosum L.) and purified by ammonium sulfate precipitations, hydrophobic-interaction chromatography, and size-exclusion chromatography, was completely free of phosphorylase (EC 2.4.1.1) and alpha-amylase (EC 3.2.1.1) activity, had a molecular mass of 64 kDa, was homogeneous in SDS-PAGE, was inhibited by 4 × 10−5 m oxidised glutathione, and could be stored at −80° in the presence of SH-reducing agents. The actions of Q-enzyme alone and in combination with potato phosphorylase on amylose, pea starch, potato amylose, potato amylopectin, and waxy maize was investigated. The combination gave high molecular weight polysaccharides, debranching of which yielded patterns of short and long chains similar to those of debranched amylopectin. Treatment of amylose with Q-enzyme reseulted in a decrease in the molecular weight averages and broadening of the molecular weight distribution, and debranching of the product yielded a short-chain distribution pattern.

Microdialysis clean-up and sampling in enzyme-based methods for the characterisation of starch

Microdialysis was used for sampling enzyme hydrolysis products of starch hydrolysed with beta -amylase, pullulanase, and/or isoamylase, to obtain information about the molecular structure of starch. Starches from waxy, normal, and high amylose maize, and from normal and genetically modified potato (amylose deficient) were used, and also commercial potato amyloses. The hydrolysis products were analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Simultaneous sampling and sample clean-up were achieved with microdialysis, thus enabling on-line injection into the liquid chromatographic system. The molecular weight cut-off of the membrane allowed for diffusion of small molecules such as oligosaccharides through the membrane, but hindered large molecules, e.g. enzymes and large polysaccharides, from entering the chromatographic system. With microdialysis sampling, it was possible to investigate the short chain fractions of debranched starch in the presence of amylose without pre-fractionation. The microdialysis-HPAEC-PAD system was also used for determination of the A:B chain ratio and the P-limit value. After P-amylolysis, only liberated maltose diffused through the dialysis membrane, which resulted in on-line sample clean-up from branched P-limit dextrin as well as from the enzyme. The proposed method is fast and easy to handle since clean-up of the hydrolysate is achieved on-line with the chromatographic system

Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var. azul

Fructo-oligosaccharides (FOSs) of a six year old agave plant variety, Agave tequilana, were isolated and fractionated by 2D preparative chromatography (SEC and rpHPLC). Structural analyses of different FOS-fractions were performed by reductive methylation analysis connected to GC/FID identification and NMR-analysis. FOSs from leaves (d.p. 3-8) contain single α-d-Glcp residues as well in terminal as internal position, however (2→1)-linked β-d-Fruf residues only. FOSs from stem, however, contain as well (2→1)- and (2→6)-linked β-d-Fruf residues with branched oligomeric repeating units. These characteristics indicate an enzymatically catalyzed metabolic regulation for the biosynthesis and transformation of fructans in A. tequilana which strongly depends on location and transport activities.

The cuticle of the cactusCereus peruvianus as a source of a homo-α-d-galacturonan

The waxy pecto-cellulosic cuticle of cladodes of the columnar cactusCereus peruvianus (19% of the whole phytobiomass; dry wt) is a source of an α-d-polygalacturonic or pectic acid (35–40% yield, on a dry wt based on the wax-free pectocellulose layer). Warm EDTA/oxalate or room temperature strong acid/alkali cycles are efficient for pectic acid extraction, since divalent cation (mainly Ca2+) is a barrier to be removed within the native and compact architecture of the cuticle. Despite some molecular dispersion arising from the application of strong mineral acid in the first extraction step, the pectic material appears to be quite homogeneous and, on acid or enzymatic analyses, was shown to contain onlyd-galacturonic acid as its monomer.Cereus cuticle pectate (sodium salt) tends to gel above a concentration of 1%, a useful property that can be more easily obtained by the inclusion of sucrose, light addition of calcium salt, and/or mild acidification.

Effect of varying flow regimes upon elution behaviour, apparent molecular characteristics and hydrodynamic properties of amylopectin isolated from normal corn starch using asymmetrical flow field-flow fractionation

A detailed study of the elution behaviour, apparent molecular characteristics and hydrodynamic properties of amylopectin-type fraction (isolated from normal corn starch) in aqueous media employing asymmetrical flow field-flow fractionation (AF4) was undertaken by systematically varying the channel flow (F(ch)), cross flow (F(cr)) and F(cr)/F(ch) ratios. Distributions of apparent molar masses and radii of gyration, mass recoveries and hydrodynamic radii decreased as a function of increasing F(cr) at a fixed F(ch), due to the increase in the retention of amylopectin-type fraction in the AF4 channel. Increased retention of the amylopectin-type fraction in the AF4 channel was also observed at low F(ch) and high F(cr)/F(ch) ratios. Large amylopectin-type molecules/particles (possibly aggregates) eluted at high F(ch), low F(cr) and low F(cr)/F(ch) ratios.

Investigation of the Branching Characteristic of Glycogen by Means of Two-Dimensional1H and 13C NMR Spectroscopy

Summary. The assignment of 13C and 1H NMR signals of three highly short-chain branched α-D-glucan samples from animal tissue (glycogen) of different provenience, dissolved in dimethylsulfoxide, was achieved using two-dimensional H-H and C-H correlated spectroscopy. The results were comparable to those recently obtained for plant derived short-chain branched α-D-glucans (amylopectin) and non-branched/long-chain branched α-D-glucans (amylose). From these assignments and the integration of pertinent proton signals, the branching degree of the glycogen samples was derived to amount 13.5%. These samples were also analyzed with respect to their degree of polymerization distribution by means of the SEC-DRI/LALLS method. These data and the line broadening of the 1H and 13C NMR spectra of the glycogen samples compared to those of amylopectin and amylose supported the assumption of a broad (homogeneous) distribution of glucose residues in glycogen.Zusammenfassung. Eine Zuordnung von 13C- und 1H-NMR-Signalen von drei in Dimethylsulfoxid gelösten kurzkettenverzweigten α-D-Glucanen aus tierischen Geweben (Glycogen) unterschiedlicher Provenienz konnte mit Hilfe zweidimensionaler H-H- und C-H-Korrelationsspektroskopie erzielt werden. Die Ergebnisse waren vergleichbar zu den kürzlich an pflanzlichen kurzkettenverzweigten α-D-Glucanen (Amylopektin) und linearen bzw. langkettenverzweigten α-D-Glucanen erhaltenen Zuordnungen. Aus diesen Zuordnungen und der Integration entsprechender Protonensignale konnte ein Verzweigungsgrad dieser Glycogenproben von 13.5% abgeleitet werden. Diese Proben wurden auch mit Hilfe der SEC-DRI/LALLS-Methodik in Hinblick auf deren Polymerisationsgradverteilung untersucht. Diese Daten und die Signalverbreiterung in den 1H- und 13C-NMR Spektren der Glycogenproben verglichen mit jenen des Amylopektins und der Amylose erhärten die Vorstellung einer breiten (homogenen) Verteilung der Glucose-Reste in Glycogen.

Characterization of branching-characteristics of starch-glucans by means of combined application of complexation, enzymatically catalyzed modification, and liquid-chromatography

Abstract Molecular characteristics, especially the branching-structure, of glucans from starch granules isolated from tubers of potato species Ukomo are determined. Granule-fraction with different mean-diameters are provided by sedimentation as inital separation step for further investigations. Then aqueous/DMSO dissolved starch glucans were separated into a non-branched/long-chain-branched and a short-chain-branched fraction by precipitation with n-butanol and methanol, respectively. By means of preparative SEC glucan pools of these fractions were collected and as well analyzed by means of methylation/GC-MS as checked for their individual potential to complex with polyiodide anions. Additionally equivaalents of these fractions were debranched by the catalytic action of pullulanase to determine the constituting glucan chain distribution by means of analytical SEC. Results of branching characteristics in terms of average number of monomers between two adjacent branching positions (bp→bp), average percentag...

Light Scattering Experiments to Follow the Degradation Process of Hydroxethyl-Cellulose Due to Trichoderma Reesei Cellulase

Abstract The process of enzymatic degradation of the high-molecular hydroxyethyl-cellulose was followed by means of static low angle laser light scattering (static LALLS) which proved to be an excellent analytic method to obtain continuously an information about the decrease of the weight average molecular weight Mw without any manipulation of the substrate and enzyme, respectively. Moreover, due to the high sensitivity of LALLS the required substrate as well as enzyme concentration can be made much lower than needed for other physico-chemical techniques. For a kinetic treatment the experimentally obtained Mw-data must be converted to Mn-values in order to get the number of bonds broken as a function of time. Exploitation of a formal concept based on the assumption of random scission of bonds of an initially mono-disperse substrate would be one way to gain the number average molecular weight.