In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria.

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.     

Spherulitic crystallization of gelatinized maize starch and its fractions

Binary systems of polymers often display spherulitic morphologies after cooling from the melt, but these phenomena have rarely been reported among food polymers of native-size. Here we report the observation of spherulitic and other morphologies in gelatinized maize starch. The morphology could be manipulated by choosing polymer compositions and kinetic regimes. Spherulites (10 μm diameter) formed from gelatinized high-amylose maize starches and purified amylose at cooling rates of order of magnitude 100 °C/min. They were more numerous and exhibited a higher melting point the greater the ratio of amylose to amylopectin. Rapid cooling rates (150–500 °C/min) resulted in a more even distribution of smaller spherulites. Very rapid (liquid nitrogen quench) or slow (0.1–1 °C/min) cooling rates resulted in mixed morphology, as did addition of 15 or 60% (w/w) sucrose to a 10% (w/w) dispersion of high-amylose starch (HAS). Spherulites were observed in aqueous suspensions of high-amylose maize starch between 5 and 30% (w/w). Lower starch concentrations resulted in a broader size distribution and spherulites of more distinct shape. WAXS patterns of B-type were observed. Negatively birefringent spherulites predominated, but positive spherulites were found. The spherulite melting range overlapped with that for amylose–lipid complex. Evidence indicated that micro-phase separation takes place when a holding period at 95 °C follows gelatinization at 180 °C. Despite the high maximum temperature of treatment (180 °C) there was evidence for a memory effect in samples of 30% HAS. Spherulite morphology closely resembled that of native starch granules in very early stages of development.     

Locations of hypochlorite oxidation in corn starches varying in amylose content

The general oxidation mechanism by hypochlorite on starch has been well studied, but the information on the distribution of the oxidation sites within starch granules is limited. This study investigated the locations where the oxidation occurred within corn starch granules varying in amylose content, including waxy corn starch (WC), common corn starch (CC), and 50% and 70% high-amylose corn starch (AMC). Oxidized corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The surface-gelatinized remaining granules were separated and studied for structural characteristics including carboxyl content, amylose content, amylopectin chain-length distribution, thermal properties, and swelling properties. Oxidation occurred mostly at the amorphous lamellae. More carboxyl groups were found at the periphery than at the core of starch granules, which was more pronounced in oxidized 70% AMC. More amylose depolymerization from oxidation occurred at the periphery of CC. For WC and CC, amylopectin long chains (>DP 36) were more prone to depolymerization by oxidation. The gelatinization properties as measured by differential scanning calorimetry also supported the changes in amylopectin fine structure from oxidation. Oxidized starches swelled to a greater extent than their unmodified counterparts at all levels of surface removal. This study demonstrates that the locations of oxidation and physicochemical properties of oxidized starches are affected by the molecular arrangement within starch granules.     

Carbon isotope ratios of amylose,amylopectin and mutant starches

Carbon isotope ratios (expressed as δ¹³C values) were determined for various sources of starch and the starch fractions amylose and amylopectin. The δ¹³C values of amylose were consistently less negative, 0.4–2.3 ‰, than those of amylopectin in kernal starch from maize (Zea mays) and barley (Hordeum vulgare) and in tuber starch from potato (Solanum tuberosum). Kernel starch isolated from the maize mutants wx1 and ae1, with known genetic lesions in the starch biosynthetic pathway, also showed significant differences in δ¹³C values. Collectively, these results suggest that variation in carbon isotope ratios in the amylose and amylopectin components of starch may be attributed to isotopic discrimination by the enzymes involved in starch biosynthesis.     

Sorption of Monoterpenoids and Sesquiterpenoids by Natural Polysaccharides

Sorption of terpenoids (essential oil components) from aqueous solutions by six types of native food starches was studied by capillary gas chromatography. Sorption of volatile substances did not depend on amylose content in starch and specific surface of its granules. The degree of sorption was maximum (86%) for corn starch containing 25–28% amylose and decreased in the following order: tapioca starch (77%) > potato starch (74%) > wheat starch (70%) > high-amylose corn starch (58%) > amylopectin corn starch (57%). Amylopectin corn starch differed from other starches in the mechanism of sorption and selectivity to compounds with various functional groups.     

Bacillus stearothermophilus Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin

The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10⁸ to 10⁷ Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.     

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.     

Influence of amylose,amylopectin and pullulan on β-glucuronidase activity of starch-degrading Escherichia coli

Synthesis of -glucuronidase in starch-degrading Escherichia coli (S1) was induced by amylose, amylopectin and pullulan supplied in mineral medium as the sole carbon source (1%, w/v). The maximum activity occurred after 4 days when cultures reached the stationary phase of growth, but induction was also evident during log-phase. The effects obtained with amylose, amylopectin and pullulan were higher than that obtained with maize starch.     

Internal structure and physicochemical properties of corn starches as revealed by chemical surface gelatinization

The organization of amylose and amylopectin within starch granules is still not well elucidated. This study investigates the radial distribution of amylose and amylopectin in different corn starches varying in amylose content (waxy corn starch (WC), common corn starch (CC), and 50% and 70% amylose corn starches (AMC)). Corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The gelatinized surface starch and remaining granules were characterized for amylose content, amylopectin chain-length distribution, thermal properties, swelling power (SP), and water solubility index (WSI). Except for the outmost 10% layer, the amylose content in CC increased slightly with increasing surface removal. In contrast, amylose was more concentrated at the periphery than at the core for 50% and 70% AMC. The proportion of amylopectin A chains generally decreased while that of B1 chains generally increased with increasing surface removal for all corn starches. The gelatinization enthalpy usually decreased, except for 70% AMC, whereas the retrogradation enthalpy relatively remained unchanged for CC but increased for WC, 50% and 70% AMC with increasing surface removal. The SP and WSI increased with increasing surface removal for all corn starches, with WC showing a significant increase in SP after the removal of the outmost 10% layer. The results of this study indicated that there were similarities and differences in the distribution of amylose and amylopectin chains along the radial location of corn starch granules with varying amylose contents. More amylose-lipid complex and amylopectin long chains were present at the periphery than at the core for amylose-containing corn starches.     

Adhesion of Bifidobacteria to Granular Starch and Its Implications in Probiotic Technologies

Adhesion of 19 Bifidobacterium strains to native maize, potato, oat, and barley starch granules was examined to investigate links between adhesion and substrate utilization and to determine if adhesion to starch could be exploited in probiotic food technologies. Starch adhesion was not characteristic of all the bifidobacteria tested. Adherent bacteria bound similarly to the different types of starch, and the binding capacity of the starch (number of bacteria per gram) correlated to the surface area of the granules. Highly adherent strains were able to hydrolyze the granular starches, but not all amylolytic strains were adherent, indicating that starch adhesion is not a prerequisite for efficient substrate utilization for all bifidobacteria. Adhesion was mediated by a cell surface protein(s). For the model organisms tested (Bifidobacterium adolescentis VTT E-001561 and Bifidobacterium pseudolongum ATCC 25526), adhesion appeared to be specific for α-1,4-linked glucose sugars, since adhesion was inhibited by maltose, maltodextrin, amylose, and soluble starch but not by trehalose, cellobiose, or lactose. In an in vitro gastric model, adhesion was inhibited both by the action of protease and at pH values of ≤3. Adhesion was not affected by bile, but the binding capacity of the starch was reduced by exposure to pancreatin. It may be possible to exploit adhesion of probiotic bifidobacteria to starch granules in microencapsulation technology and for synbiotic food applications.     

The influence of amylose on starch granule structure

Starch granules are principally composed of the two glucose polymers amylose and amylopectin. Native starch granules typically contain around 20% amylose and 80% amylopectin. However, it is possible to breed plants that produce starch with very different amylose and amylopectin contents. At present, the precise structural roles played by these two polymers are incompletely understood. In this study, small-angle X-ray scattering techniques have been applied to investigate the effect of varying amylose content on the internal structure of maize, barley and pea starch species. The results suggest that amylose disrupts the structural order within the amylopectin crystallites.     

The priming of amylose synthesis in Arabidopsis leaves

We investigated the mechanism of amylose synthesis in Arabidopsis leaves using (14)C-labeling techniques. First, we tested the hypothesis that short malto-oligosaccharides (MOS) may act as primers for granule-bound starch synthase I. We found increased amylose synthesis in isolated starch granules supplied with ADP[(14)C]glucose (ADP[(14)C]Glc) and MOS compared with granules supplied with ADP[(14)C]Glc but no MOS. Furthermore, using a MOS-accumulating mutant (dpe1), we found that more amylose was synthesized than in the wild type, correlating with the amount of MOS in vivo. When wild-type and mutant plants were tested in conditions where both lines had similar MOS contents, no difference in amylose synthesis was observed. We also tested the hypothesis that branches of amylopectin might serve as the primers for granule-bound starch synthase I. In this model, elongated branches of amylopectin are subsequently cleaved to form amylose. We conducted pulse-chase experiments, supplying a pulse of ADP[(14)C]Glc to isolated starch granules or (14)CO(2) to intact plants, followed by a chase period in unlabeled substrate. We detected no transfer of label from the amylopectin fraction to the amylose fraction of starch either in isolated starch granules or in intact leaves, despite varying the time course of the experiments and using a mutant line (sex4) in which high-amylose starch is synthesized. We therefore find no evidence for amylopectin-primed amylose synthesis in Arabidopsis. We propose that MOS are the primers for amylose synthesis in Arabidopsis leaves.     

Scanning Electron Microscopy and Fermentation Studies on Selected Known Maize Starch Mutants Using STARGEN? Enzyme Blends

The conversion of maize (corn) kernels to bio-ethanol is an energy-intensive process involving many stages. One step typically required is the liquefaction of the ground kernel to enable enzyme hydrolysation of the starch to glucose. The enzyme blends STARGEN? (Genencor) are capable of hydrolysing starch granules without liquefaction, reducing energy inputs and increasing efficiency. Studies were conducted on maize starch mutants amylose extender 1 (ae1), dull 1 (du1) and waxy 1 (wx1) in the inbred line Oh43 to determine whether different maize starches affected hydrolysation rates by STARGEN? 001 and STARGEN? 002. All mutants contained similar proportions of starch in the kernel but varied in the amylose to amylopectin ratio. Ground maize kernels were incubated with STARGEN? 001 and viewed using scanning electron microscopy to examine the hydrolysis action of STARGEN? 001 on the starch granules. The ae1 mutant exhibited noticeably less enzymic hydrolysis action, on the granules visualised, than wx1 and background line Oh43. Kernels were batch-fermented with STARGEN? 001 and STARGEN? 002. The ae1 mutant exhibited a 50% lower ethanol yield compared to the wx1 mutant and background line. A final study compared hydrolysation rates of STARGEN? 001 and STARGEN? 002 on purified maize starch, amylopectin and amylose. Though almost twice the amylopectin was hydrolysed using STARGEN? 002 than STARGEN? 001 in this trial, fermentations using STARGEN? 002 resulted in lower ethanol yields than fermentations using STARGEN? 001. Both STARGEN? enzyme blends were more suitable for the fermentation of high amylopectin maize starches than high amylose starches.     

Starch hydrolysis modeling: application to fuel ethanol production

Efficiency of the starch hydrolysis in the dry grind corn process is a determining factor for overall conversion of starch to ethanol. A model, based on a molecular approach, was developed to simulate structure and hydrolysis of starch. Starch structure was modeled based on a cluster model of amylopectin. Enzymatic hydrolysis of amylose and amylopectin was modeled using a Monte Carlo simulation method. The model included the effects of process variables such as temperature, pH, enzyme activity and enzyme dose. Pure starches from wet milled waxy and high-amylose corn hybrids and ground yellow dent corn were hydrolyzed to validate the model. Standard deviations in the model predictions for glucose concentration and DE values after saccharification were less than ±0.15% (w/v) and ±0.35%, respectively. Correlation coefficients for model predictions and experimental values were 0.60 and 0.91 for liquefaction and 0.84 and 0.71 for saccharification of amylose and amylopectin, respectively. Model predictions for glucose (R ² = 0.69–0.79) and DP4⁺ (R ² = 0.8–0.68) were more accurate than the maltotriose and maltose for hydrolysis of high-amylose and waxy corn starch. For yellow dent corn, simulation predictions for glucose were accurate (R ² > 0.73) indicating that the model can be used to predict the glucose concentrations during starch hydrolysis.     

Differences of Starch Granule Distribution in Grains from Different Spikelet Positions in Winter Wheat

Wheat starch development is a complex process and is markedly difference by changes in spikelet spatial position. The present study deals with endosperm starch granule distribution and spatial position during filling development. The study was conducted with pure starch isolated from wheat (Triticum aestivum L.), Jimai20 and Shannong1391, at 7–35 days after anthesis (DAA). The results showed that grain number, spikelet weight and grain weight per spikelet in different spatial position showed parabolic changes. Upper spikelets had highest starch and amylose content followed by basal spikelets, then middle spikelets. The paper also suggested the volume percents of B-type and A-type granule in grain of middle spikelets were remarkably higher and lower than those of basal and upper spikelets, respectively. However, no significant difference occurred in the number percents of the two type granule. The ratio of amylase to amylopectin was positively correlated with the volume proportion of 22.8–42.8 µm, but was negatively related to the volume proportion of <9.9 µm. The results indicated that the formation and distribution of starch granules were affected significantly by spikelet position, and grains at upper and basal spikelet had the potential of increasing grain weight through increasing the volume of B-type granules.     

Starch Granule Biosynthesis in Arabidopsis Is Abolished by Removal of All Debranching Enzymes but Restored by the Subsequent Removal of an Endoamylase

Several studies have suggested that debranching enzymes (DBEs) are involved in the biosynthesis of amylopectin, the major constituent of starch granules. Our systematic analysis of all DBE mutants of Arabidopsis thaliana demonstrates that when any DBE activity remains, starch granules are still synthesized, albeit with altered amylopectin structure. Quadruple mutants lacking all four DBE proteins (Isoamylase1 [ISA1], ISA2, and ISA3, and Limit-Dextrinase) are devoid of starch granules and instead accumulate highly branched glucans, distinct from amylopectin and from previously described phytoglycogen. A fraction of these glucans are present as discrete, insoluble, nanometer-scale particles, but the structure and properties of this material are radically altered compared with wild-type amylopectin. Superficially, these data support the hypothesis that debranching is required for amylopectin synthesis. However, our analyses show that soluble glucans in the quadruple DBE mutant are degraded by α- and β-amylases during periods of net accumulation, giving rise to maltose and branched malto-oligosaccharides. The additional loss of the chloroplastic α-amylase AMY3 partially reverts the phenotype of the quadruple DBE mutant, restoring starch granule biosynthesis. We propose that DBEs function in normal amylopectin synthesis by promoting amylopectin crystallization but conclude that they are not mandatory for starch granule synthesis.     

Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping

Background

Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis.

Methods

Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme.

Results

Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5 μm in diameter.

Conclusions

Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before.

General significance

μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.     

Sorption of mono- and sesquiterpenes by natural polysaccharides

Sorption of terpenoids (essential oil components) from aqueous solutions by six types of native food starches was studied by capillary gas chromatography. Sorption of volatile substances did not depend on amylose content in starch and specific surface of its granules. The degree of sorption was maximum (86%) for corn starch containing 25-28% amylose and decreased in the following order: tapioca starch (77%) > potato starch (74%) > wheat starch (70%) > high-amylose corn starch (58%) > amylopectin corn starch (57%). Amylopectin corn starch differed from other starches in the mechanism of sorption and selectivity to compounds with various functional groups.     

Partial purification and characterization of granule-bound starch synthases from normal and waxy maize

The granule-bound starch-synthases from normal and waxy maize kernels have been solubilized, partially purified, and characterized. Two broad categories of starch synthases were revealed with representatives in the soluble phase and also on granules from both normal and waxy maize though the activity associated with granules from waxy was small. Data for native molecular weights, kinetic parameters, and immunological relatedness are used to demonstrate that the granule-bound isozymes from normal maize are different from the soluble enzymes. These distinct granule-bound enzymes are missing in waxy maize granules and a further novel form of starch synthase is revealed. These findings are discussed in relation to the type of starch produced in tissue affected by the waxy mutation.     

Production of very-high-amylose potato starch by inhibition of SBE A and B

High-amylose starch is in great demand by the starch industry for its unique functional properties. However, very few high-amylose crop varieties are commercially available. In this paper we describe the generation of very-high-amylose potato starch by genetic modification. We achieved this by simultaneously inhibiting two isoforms of starch branching enzyme to below 1% of the wild-type activities. Starch granule morphology and composition were noticeably altered. Normal, high-molecular-weight amylopectin was absent, whereas the amylose content was increased to levels comparable to the highest commercially available maize starches. In addition, the phosphorus content of the starch was increased more than fivefold. This unique starch, with its high amylose, low amylopectin, and high phosphorus levels, offers novel properties for food and industrial applications.