Kristine Koch

Quantitative analysis of amylopectin unit chains by means of high-performance anion-exchange chromatography with pulsed amperometric detection

Abstract High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) is a widely used technique to study the chain length distribution of amylopectin. The chromatograms, however, do not directly reflect this distribution, since the PAD response changes with the degree of polymerization. In this study, waxy maize starch was debranched and fractionated on a Bio-Gel P-6 column and the response factors for maltosaccharides with DP 3–65 were determined. The detector response per μg glucan chains was shown to decrease considerably for DP 3–7 while the curve leveled out for DP larger than 15.

Composition of chains in waxy-rice starch and its structural units

Abstract φ,β-Limit dextrins of fractions of intermediate products obtained by alpha-amylolysis of waxy-rice starch were analysed for their unit chain distributions by gel-permeation chromatography. The proportion of long chains decreased in fractions of low-molecular-weight dextrins, but the ratio of A:B-chains, A:Ba-chains, and Ba:Bb-chains remained almost constant. High-performance anion exchange chromatography was used for a detailed analysis of the composition of B-chains. Chains with a length intermediate to the groups of short and long B-chains increased in small dextrins. The shortest B-chain in the φ,β-limit dextrins was maltotriose and was preferentially produced during alpha-amylolysis. Models are proposed showing the fine structure of the clusters of the amylopectin, that probably originated from different structural domains within the starch granules.

Building block organisation of clusters in amylopectin from different structural types.

Clusters of chains consisting of tightly branched units of building blocks were isolated from 10 amylopectin samples possessing the 4 types of amylopectin with different internal unit chain profiles previously described. It was shown that clusters in types 1 and 2 amylopectins are larger than in types 3 and 4, but the average cluster size did not correspond to the ratio of short to long chains of the amylopectins. The size-distribution of the building blocks, having one or several branches, possessed generally only small differences between samples. However, the length of the interblock segments followed the type of amylopectin structure, so that type 1 amylopectins had shortest and type 4 the longest segments. The chains in the clusters were divided into characteristic groups probably being involved in the interconnection of two, three, and four - or more - building blocks. Long chains were typically found in high amounts in clusters from type 4 amylopectins, however, all cluster samples contained long chains. The results are discussed in terms of the building block structure of amylopectin, in which the blocks together with the interblock segments participate in a branched backbone building up the amorphous lamellae inside growth rings of the starch granules. In such a model, amylopectins with proportionally less long chains (types 1 and 2) possess a more extensively branched backbone compared to those with more long chains (types 3 and 4).

Three underutilised sources of starch from the Andean region in Ecuador Part I. Physico-chemical characterisation

The physico-chemical aspects of three native Andean starches from Arracacha xanthorriza, Canna edulis and Oxalis tuberosa were investigated. Scanning electron microscopy-investigations showed that granules of A. xanthorriza were the smallest with irregular shape and sizes between 7 and 23 pm, whereas granules of O. tuberosa and C. edulis were both ovally shaped with granular sizes between 20 and 55 mum and 35-101 mum, respectively. All three starches revealed a B-type X-ray diffraction pattern. The gelatinisation behaviour was investigated using differential scanning calorimetry (DSC). The gelatinisation enthalpy was 14.6 J/g of starch for O. tuberosa, 15.7 J/g for C. edulis and 17.6 J/g for A. xanthorriza, and the peak temperature of the endothermic DSC-transition was 55.9, 61.2 and 60.1 degreesC, respectively. The amylose content determined by gel permeation chromatography after debranching with isoamylase was 4% for A. xanthorriza, 18.4% for O. tuberosa. and 23.8% for C. edulis. The amylopectin showed different structures among the three starches with a beta-amylolysis limit of 67.6% for C edulis, 64.5% for O. tuberosa and 56.6% for A. xanthorriza. The average chain length of the amylopectin was highest for A. xanthorriza (22.6), followed by O. tuberosa with 22.4, and lowest for C. edulis (21.9). The complexation ability of the three starches was investigated by adding sodium dodecyl sulphate (SDS), and the amylose content was positively correlated with the enthalpy of the amylose-SDS complex

Molecular structure of citric acid cross-linked starch films

The effect of citric acid (CA) on starch films has been examined. A new method to detect cross-linking of starch by CA in solution-cast films by molecular weight measurements is described. Furthermore, we managed to distinguished between free, mono- and di-esterified CA and quantify di-ester content within starch films by using a modification in the method of complexometric titration with copper(II)-sulfate. Cross-linking of starch by CA occurred at low temperature, 70°C, which we assumed is so far the lowest temperature reported where cross-linking reaction occurred. This is essential for starch coating applications within paper industry since no high temperatures for curing will be required. However, curing at 150°C and high CA concentrations, 30 pph, increased cross-linking reaction. Furthermore, the physical properties like water solubility, gel content and glass transition temperature, were highly reflected by changes in the molecular structure i.e. cross-linking and hydrolysis, as well as CA content and curing temperature.

The cluster structure of barley amylopectins of different genetic backgrounds.

The unit chains of amylopectin are organized into clusters. In this study, the cluster structure was analysed in detail in four different genotypes of barley, of which two possessed the amo1 genetic background. Amylose content of the barley starches differed from 0 to 32.6%. Isolated amylopectin was hydrolysed with α-amylase from Bacillus subtilis into domains, defined as groups of clusters, which were size-fractionated by methanol. The domain fractions were further treated with α-amylase to release single clusters. Amylopectin, domains and clusters were subsequently treated with phosphorylase and β-amylase to produce φ,β-limit dextrins and the detailed internal structures of these different structure levels were investigated. Analysis was performed with gel-permeation and anion-exchange chromatography. Equal amount of A-chains were detected in all barleys, but the distribution of B-chains differed. At least two types of domain structures were identified in all four barley varieties. Large domains were built up by large clusters and small domains by small clusters. In all four barley samples the number of long chains was small suggesting that shorter chains with a degree of polymerization of 25-35 also are involved in the interconnection of clusters. The cluster structure of the amylopectin correlated with the genetic background. The two barley samples with amo1 genetic background possessed a more dense structure. Internal chain lengths in these two barleys were shorter resulting in larger domains built up by larger clusters.

The effect of pH on hydrolysis, cross-linking and barrier properties of starch barriers containing citric acid.

Citric acid cross-linking of starch for e.g. food packaging applications has been intensely studied during the last decade as a method of producing water-insensitive renewable barrier coatings. We managed to improve a starch formulation containing citric acid as cross-linking agent for industrial paper coating applications by adjusting the pH of the starch solution. The described starch formulations exhibited both cross-linking of starch by citric acid as well as satisfactory barrier properties, e.g. fairly low OTR values at 50% RH that are comparable with EVOH. Furthermore, it has been shown that barrier properties of coated papers with different solution pH were correlated to molecular changes in starch showing both hydrolysis and cross-linking of starch molecules in the presence of citric acid. Hydrolysis was shown to be almost completely hindered at solution pH≥4 at curing temperatures≤105 °C and at pH≥5 at curing temperatures≤150 °C, whereas cross-linking still occurred to some extent at pH≤6.5 and drying temperatures as low as 70 °C. Coated papers showed a minimum in water vapor transmission rate at pH 4 of the starch coating solution, corresponding to the point where hydrolysis was effectively hindered but where a significant degree of cross-linking still occurred.

Structure of building blocks in amylopectins

Building blocks represent the smallest, branched units found inside clusters of amylopectins. The building blocks from clusters of 10 different amylopectins, representing a wide variety of plants, were isolated and size-fractionated. The unit chain compositions were then analysed. It was found that the number of chains in building blocks increased in proportion to their size similarly in all samples regardless plant source. The smallest blocks (DP 5-9) consisted of 2 chains and the largest blocks (DP ≥45), of which generally only little existed, possessed ≥10 chains. Generally, the degree of branching increased with building block size, but the organisation of chains inside the blocks was unique for each sample. Nevertheless, compared to other plants, amylopectins from cereals (represented by rye, oats, rice and waxy maize) possessing elevated number of the shortest internal B-chains (DP 3-7), tended to have blocks with a lower ratio of A:B-chains, indicative of a preferred Haworth type of structure as opposed to the Staudinger configuration.

Calibration of a size-exclusion chromatography system using fractions with defined amylopectin unit chains

A high-performance size-exclusion chromatography (HPSEC) system was successfully calibrated using fractions of debranched amylopectin unit chains, obtained by gel filtration, and with the average chain length of the fractions determined by high-performance anion-exchange chromatography. As a comparison, calibration of the HPSEC system was also performed using maltoheptaose and pullulan standards as reference substances. The relationships between the degree of polymerization and the retention time for the defined amylopectin unit chain fractions and the commercial standards, i.e. maltoheptaose and pullulans, were very similar, indicating similar elution behaviour.

The building block structure of barley amylopectin

Amylopectin branchpoints are present in amorphous lamellae of starch granules and organised into densely branched areas, referred to as building blocks. One single amylopectin cluster contains several building blocks. This study investigated the building block structure of domains (groups of clusters) and clusters in four different barley genotypes. Two of the barleys possessed the amo1 mutation, Glacier Ac38 and the double recessive SW 49427 with both wax and amo1 mutations, and were compared with the two waxy type barleys Cinnamon and Cindy. A previous detailed study on these four barley genotypes showed that the amo1 mutation affected the internal structure of amylopectin as manifested in the composition of clusters. In this work the building blocks were isolated from domains and clusters by extensive treatment with liquefying α-amylase of Bacillus subtilis and structurally characterised with enzymatic and chromatographic techniques. The proportion of large building blocks with a high number of chains was increased in the amo1 barleys, and the chain length between the blocks was short, which explained the previous findings of large clusters with more dense structure in the amo1 amylopectins.