Substitution patterns in methylated potato starch as revealed from the structure and composition of fragments in enzymatic digests

The effect of the granule structure on the methylation of starch was investigated by comparing the substitution patterns of potato starch methylated in granular suspension and in solution to DS 0.3. Substitution patterns were analyzed by successive digestion with alpha-amylase and amyloglucosidase, fractionation of the resulting malto-oligosaccharide mixture by GPC on a preparative scale, and characterization of the fractions by GLC and MALDI-MS. The mass composition of fractions with intermediate and higher degree of polymerization was indicative of enhanced clustering of substituents in granular methyl starch. On the contrary, the composition of the smaller saccharides was governed by enzyme specificity, which was also reflected in strong deviations in their monomer composition. A sequencing study on selected 'pure' small saccharides confirmed and complemented previous conclusions on enzyme specificity.     

Insights in starch acetylation in sub- and supercritical CO2

An in-depth study on the acetylation of starch with acetic anhydride (Ac₂O) and sodium acetate (NaOAc) as the catalyst in pressurized carbon dioxide (scCO₂) in a broad pressure range (8–25 MPa) and a temperature of 90 °C is provided. Highest degrees of substitution (DS) of 0.29 (1 h reaction time) and 0.62 (24 h reaction time) were found near the critical point of the mixture (15 MPa). The phase behavior of the system CO₂, starch and acetic anhydride (Ac₂O) was studied in a high pressure view cell. The critical points were a clear function of the temperature and increased from the range of 9.4–10 MPa to 14.5–14.8 MPa when going from 50 to 90 °C (Ac₂O mole fraction at the critical point in the range of 0.08–0.09). Acetylation experiments with a range of starch particles sizes showed a clear relation between the DS and the particle size.     

Gel formation in mixtures of high amylopectin potato starch and potato starch

The effects of starch granules on the rheological behaviour of gels of native potato and high amylopectin potato (HAPP) starches have been studied with small deformation oscillatory rheometry. The influence of granule remnants on the rheological properties of samples treated at 90 °C was evident when compared with samples treated at 140 °C, where no granule remnants were found. The presence of amylose in native potato starch gave to stronger network formation since potato starch gave higher moduli values than HAPP, after both 90 and 140 °C treatments. In addition, amylose may have strengthened the network of HAPP because higher moduli values were obtained when native potato starch was added to the system. The moduli values of the mixtures also increased with increasing polysaccharide concentration in the system, which is due to an increment in the polysaccharide chain contacts and entanglements. Finally, it was found that a mixture of commercial amylose from potato starch and HAPP resulted in lower values of G′ compared to native potato starch. This indicates that the source of amylose is important for the properties in a blend with native amylopectin.     

Mediator facilitated,laccase catalysed oxidation of granular potato starch and the physico-chemical characterisation of the oxidized products

The primary hydroxyl groups in potato starch were selectively oxidized to the corresponding aldehyde and carboxylic acid functionalities by mediators like TEMPO, using laccase from fungi as catalytic oxidant and oxygen as the primary oxidant. Oxidized starch products with degree of substitution (DS_CHO ranging from 0.16 to 16.4/100AGU and DS_COOH from 0.01 to 3.71 carboxyl groups/100AGU) were obtained with mediator facilitated enzymatic oxidation. Maximum conversion of the primary alcohol group was obtained at a pH of 5, with TEMPO as mediator, under oxygen bubbling and two step administration of Trametes versicolor laccase (200 + 200 nkat/g of starch). The oxidized products were characterised by IR spectroscopy, XRD and thermal studies. In the oxidized samples, the larger starch granules exhibited cracks and fractures in comparison to the smaller granules which were relatively unaffected, as observed from the microstructural studies.     

Controlled peeling of the surfaces of starch granules by gelatinization in aqueous dimethyl sulfoxide at selected temperatures

Microscopic examination of starch granules in 90:10 (v/v) Me(2)SO-H(2)O indicated that the granules were slowly being gelatinized from their surfaces. The rate of gelatinization was dependent on two variables: (1) the amount of water in Me(2)SO and (2) the temperature. An increase of water in Me(2)SO and/or an increase in temperature increased the rate of gelatinization and vice versa. Specific ratios of Me(2)SO and H(2)O (85:15-95:5) and temperatures (0-15 degrees C) were found to give controlled sequential peeling/gelatinization of eight kinds of starch granules in 1-12h, with amounts of 10-25% gelatinization per hour. It was observed that the percent of starch granule remaining versus time gave curves that were linear and others that had linear parts separated by one or more abrupt changes. No two starches had a similar gelatinization curve for the same two conditions of the amount of water and the temperature. It is hypothesized that these curves reflect different structural characteristics for the individual kinds of starch granules.     

On the presence of starch bound phosphate in potato leaf starch

The presence of starch-bound phosphate in potato leaves collected late afternoon in the middle of July when the starch content is high (12.9% dry matter basis) was studied. Starch was extracted from the leaves with dimethylsulphoxide and fragments of starch were purified by ultrafiltration in two steps in combination with an -amylase hydrolysis. The fragments were analysed with ³¹P-NMR and no signals corresponding to phosphate monoesters linked to glucose at the C₃ and/or C₆ positions were detected. The results show that starch in potato leaves does not contain any detectable amounts of phosphate monoesters.     

Gelatinization and freeze-concentration effects on recrystallization in corn and potato starch gels

Freeze-concentration of starch gels was controlled by temperature and gelatinization with glucose and lactose. The aim of the study was to evaluate the effects of freezing temperature and gel composition on starch recrystallization behaviour of corn and potato starch gels (water content 70%, w/w) in water or glucose or lactose (10%, w/w) solutions. Starch gels were obtained by heating in differential scanning calorimetry (DSC). Samples of starch gels were frozen at -10 degrees C, -20 degrees C and -30 degrees C for 24h and, after thawing, stored at +2 degrees C for 0, 1, 2, 4 and 8 days. The extent of starch recrystallization was taken from the enthalpy of melting of the recrystallized starch by DSC. Freezing temperatures, glucose, lactose and the origin of the starch affected the recrystallization behaviour greatly. The recrystallization of amorphous starch during storage was enhanced by freeze-concentration of gels at temperatures above T'(m). Molecular mobility was enhanced by unfrozen water and consequently molecular rearrangements for nucleation could take place. Further storage at a higher temperature enhanced the growth and the maturation of crystals. In particular, glucose decreased the T'(m) of the gels and consequently lower freezing temperatures were needed to reduce enhanced recrystallization during storage. Freeze-concentration temperatures also showed a significant effect on the size and the perfection of crystals formed in starch recrystallization.     

Nucleosome acetylation sequencing to study the establishment of chromatin acetylation

The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.     

On the effect of surface active agents and their structure on the temperature-induced changes of normal and waxy wheat starch in aqueous suspension. Part I. Pasting and calorimetric studies

Pasting and calorimetric studies of normal and waxy wheat starch were performed in the presence of a series of ionic (sulphates, trimethyl ammonium bromides) and non-ionic (monoglycerides, maltosides) short (12 carbon atoms) and long (16 carbon atoms) n-alkyl chain surfactants. With the exception of the alkyl ammonium bromides, all of the short chain surfactants lower the pasting temperature (PT) in normal wheat starch, while the long chain surfactants have the opposite effect. Contrary, regardless of their chain length, all ionic surfactants lower the PT in waxy wheat starch while the non-ionic surfactants induce small, sometimes almost negligible changes in the PT. Calorimetric studies revealed the absence of a direct connection between the effect of surfactants on the onset of the starch gelatinization transition and the PT. However, in the presence of all surfactants, except the alkyl ammonium bromides, the PT of normal wheat starch was found to lie within or very close the temperature range within which the dissociation of the amylose–surfactant complexes takes place. Waxy wheat starch, in contrast, pasted at temperatures that fell within the temperature range of the starch gelatinization transition. This is taken as evidence of the existence of a correlation between the PT and the dissociation of the amylose–surfactant complexes.     

Extrusion processing of high amylose potato starch materials

Thermoplastic starch was prepared by mixing native high amylose potato starch and normal potato starch in a Buss co-kneading extruder at starch to glycerol ratios of 100:45 and 100:30. The materials were also conditioned to different moisture contents at different relative humidities at 23 °C. After the mixing, the compounds were extruded into sheets with a Brabender laboratory extruder. The thermoplastic high amylose materials exhibited a higher melt viscosity than the normal potato starch materials when conditioned at 53% relative humidity. Increasing the moisture content in HAP from 27% to 30% (by weight) lowered the melt viscosity to the same level as that of normal potato starch with a moisture content of 28%. In general, the high amylose materials were more difficult to extrude than the thermoplastic material based on normal starch. The main extrusion problems encountered with the high amylose starch were unstable flow, insufficient melt tenacity and clogging of the die. By increasing the moisture content, increasing the compression ratio of the screw and increasing the rotation rate of the screw, the problems were reduced or eliminated. However, only with a starch to glycerol ratio of 100:45 was an acceptable extrusion result obtained. Extruded sheets of such high amylose materials had a stress at break of about 5 MPa at room temperature and 53% relative humidity, whereas the corresponding value for normal potato (thermoplastic) starch was 3 MPa. The elongation at break was also higher in the case of the high amylose material. The results are discussed in terms of residual crystallinity of the starch materials.     

The granular structure of C-type pea starch and its role in gelatinization

We have used a combination of techniques to study the structure and properties of C-type starch from pea seeds. It was found that all C-type starch granules contain both types of polymorph; the B polymorphs are in the center of the granule and are surrounded by the A polymorphs. During heating in excess salt solution the A and B polymorphs within C-type granules melt independently, giving a double transition in heat capacity and a two-step swelling, compared with single transitions for A- and B-type starches. It was shown that B polymorphs gave a transition with a lower peak temperature than A. The disruption of crystallinity during gelatinization began from the hilum area and was propagated along the granule, accompanied by swelling of disrupted areas. It is proposed that the swelling of disrupted parts of the granule decreases the melting temperature of the neighboring crystallites resulting in the progressive disruption of crystalline areas. The gelatinization process is dependent on the arrangement of A and B polymorphs within the granule. © 1998 John Wiley & Sons, Inc. Biopoly 45: 323–332, 1998     

Physical, chemical and microscopic characterization of a new starch from chayote (Sechium edule) tuber and its comparison with potato and maize starches

In this work, the chayote tuber starch (CHS) was isolated and its chemical composition and its physical and microscopic characteristics were determined, and compared with potato (PS) and maize (MS) starches. The starch content in chayote tubers (728 g kg⁻¹ dry weight) was similar to potato tubers (700 g kg⁻¹ dry weight), with a high level of purity (>98%), while its phosphorous content was higher (0.15%) than PS (0.08%) and MS (0.01%). Starch granules were oval, irregular, truncated and rounded with sizes between 7 and 50 μm with smooth surfaces. CHS dispersions (1% and 4%, w/w) showed higher viscosity (75 and 1715 mPa s), than PS (350% and 50% lower) and MS (715% and 600% lower). The gelatinization temperature (65 to 74 °C) was similar in CHS and PS. The pasting properties (RVA) of the starches suggest that CHS showed better characteristics than the commercial potato and maize starches. Therefore, CHS could be used as a thickening agent and a substitute to PS in food dispersions where a high viscosity is needed.     

Ionic liquids as solvents for biopolymers: Acylation of starch and zein protein

Biopolymers such as starch and zein protein were found to be soluble at 80 °C in ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIMCl) and 1-butyl-3-methylimidazolium dicyanamide (BMIMdca) in concentrations up to 10% (w/w). Higher concentrations of biopolymers in these novel solvents resulted in solutions with too high viscosity to stir. Solutions of both starch and zein in BMIMCl were acylated with anhydrides in presence of pyridine to give acetyl starch and benzoyl zein with various degrees of substitution. Without pyridine the acylation reaction did not proceed. ¹H NMR and IR spectroscopies were used to determine the degree of substitution of starch. Viscosity studies indicated that the starch underwent slight reduction in molecular weight during the course of acylation. Starch was also soluble in other non-conventional solvents such as choline chloride/oxalic acid and choline chloride/ZnCl₂. However, zein was insoluble in these solvents.     

On the effect of surface active agents and their structure on the temperature-induced changes of normal and waxy wheat starch in aqueous suspension. Part II: A confocal laser scanning microscopy study

The location and penetration patterns of two fluorescently labelled, surface active molecules into normal and waxy wheat starch granules prior, during and after the temperature-induced gelatinization were studied by means of confocal laser scanning microscopy (CLSM). Amphiphilic dyes were found to have a tendency to penetrate wheat starch granules in aqueous suspension. The penetration patterns were however found to be dependent on the contact time, type of starch and the chain length (C₁₂ vs. C₁₆) of the amphiphilic dye. The penetration of amphiphilic dyes through the starch granule matrix proved to be less restricted in waxy than in normal wheat starch. For a given type of starch, the penetration of the longer chain dye was more constrained than that of the shorter chain one. The extent to which the dye diffuses into the granule matrix as it gelatinizes is also affected by the chain length of the dye, diffusion of the shorter chain dye occurring more profusely and at lower temperatures than for the longer chain one. These differences are suggested to be related to the dissociation temperature of the AM-amphiphilic dye complexes.     

Behaviour of granular starches in low-pressure glow plasma

Granular starches of nine botanical origins were exposed to low-pressure glow plasma generated in the air from either a high voltage DC supply or an AC inductor. Experiments with low energy air-plasma (10⁻² Torr) and starches provided insight into some details of their surface structure. Starches were partly oxidized to carboxylic starches and partly depolymerized. Their affinity to plasma depended on their botanical origin. The structure of starch granules seemed to be a crucial factor, which determined the behaviour of starches in plasma. Lipids and/or proteins stabilized polysaccharides to plasma being preferentially decomposed.     

Alkynyl polysaccharides: synthesis of propargyl potato starch followed by subsequent derivatizations

Potato starch propargyl ethers (PgS) with degrees of substitution (DS) from 0.1 to 2.2 have been prepared by etherification of starch with sodium hydroxide or Li dimsyl in Me(2)SO and propargyl bromide. DS values and substituent distribution were determined after hydrolysis and acetylation by GC-MS. The order of reactivity was 2>6>3, with O-3 substitution being preferably observed in the trisubstituted units. Repeated analysis of the starch derivatives revealed that propargyl residues were lost during storage, a phenomenon that was not fully understood until now. Selected PgS were further functionalized: (a) O- and C-methylated to O-(2-butynyl)-O-methyl starch (BMS), (b) in a Mannich type reaction with diethylamine and formaldehyde to yield O-(4-diethylamino)-2-butinyl starch (DEABiS), (c) in a 1,3-dipolar cycloaddition with benzyl azide ('click-chemistry') to a N-benzyltriazole derivatized starch (BTrS), and (d) with carbon dioxide to O-(3-carboxy)-2-butinyl starch (CBiS). While the yield of carboxylation was only poor, conversion was high or nearly quantitative for reactions a-c. Thus, it is demonstrated that starch propargyl ethers are valuable intermediates for the preparation of functional polysaccharides.     

Molecular and granular characteristics of corn starch modified by HCl-methanol at different temperatures

Native corn starch was hydrolyzed with 0.36% HCl in methanol at 25 and 45 °C for periods of time up to 240 h. The action of acid penetration and hydrolysis was investigated by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), high-performance anion-exchange chromatography (HPAEC) and high-performance size-exclusion chromatography (HPSEC) equipped with viscometry, right-angle laser light scattering (RALLS) and refractive index (RI) detectors. Corn starch hydrolyzed at 45 °C for 240 h showed strong intensity of APTS (8-amino-1,3,6-pyrenetrisulfonic acid) fluorescence and sharp growth ring structure. Exocorrosion over the surface of corn starch was only observed on the corn starch hydrolyzed at 25 °C for 240 h and observed on all corn starch hydrolyzed at 45 °C. The M_w and R_h of acid-hydrolyzed corn starch decreased with increasing the degree of hydrolysis. The acid hydrolysis rate in methanol of corn starch was mainly dependent on the temperature, which dominated the penetration efficiency of acid.     

The influence of alterations in ADP-glucose pyrophosphorylase activities on starch structure and composition in potato tubers

In order to examine whether alterations in the supply of precursor molecules into the starch biosynthetic pathway affected various characteristics of the starch, starch was isolated from potato (Solanum tuberosum L.) tubers containing reduced amounts of the enzyme ADP-glucose pyrophosphorylase (AGPase). It was found that although the type of crystalline polymorph in the starch was not altered, the amylose content was severely reduced. In addition, amylopectin from the transgenic plants accumulated more relatively short chains than that from control plants and the sizes of starch granules were reduced. The starch granules from the transgenic plants contained a greater amount of granule-bound starch synthase enzyme, which led to an increase in the maximum activity of the enzyme per unit starch tested. The K _m for ADP-glucose was, at most, only slightly altered in the transgenic lines. Potato plants containing reduced AGPase activity were also transformed with a bacterial gene coding for AGPase to test whether this enzyme can incorporate phosphate monoesters into amylopectin. A slight increase in phosphate contents in the starch in comparison with the untransformed control was found, but not in comparison with starch from the line with reduced AGPase activity into which the bacterial gene was transformed. Received: 2 February 1999 / Accepted: 25 March 1999     

Shear-thickening and shear-induced pattern formation in starch solutions

Since Dintzis et al. reported shear-thickening behavior and shear-induced pattern formation in semidilute starch solutions for the first time in 1995, considerable efforts have been made to understand the science behind these observations. Despite these efforts, however, many questions regarding this behavior of starch solutions remain. Using a Brookfield programmable rheometer and a custom-built shear microscope, starch solutions in alkaline solution medium were investigated. In this report, we present data leading to the following conclusions: (1) gently prepared starch solutions are macroscopically heterogeneous with regions of highly concentrated gel-like structures dispersed in dilute starch solution; (2) shear breaks up these heterogeneous regions, increasing in viscosity (shear-thickening) which is thus seen to be a result of an increase in the concentration of dissolved starch; (3) pattern formation, observed when the solution is exposed to higher shear rate, is the result of a separate shear-induced aggregation process; and (4) aggregations are not induced below a certain critical threshold shear rate and time is also a factor in the behavior of the aggregate.     

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters. H.M. Chen and C.T. Lin contributed equally to this work.