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.     

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.     

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.     

The protective effects of high amylose maize (amylomaize) starch granules on the survival of Bifidobacterium spp. in the mouse intestinal tract

The possibility of using high amylose maize starch granules as a delivery system for probiotic bacteria has been investigated using Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B which were isolated from a healthy human. The Bifidobacterium cells were able to adhere to the amylomaize starch granules and were also able to hydrolyse the starch during growth. Initially, in vitro studies were carried out by studying the survival of strains Bifidobacterium LaftiTM 8B and LaftiTM 13B when exposed to pH 2.3, 3.5 and 6.5 as well as 0.03 and 0.05% w/v bile acids. Both strains were grown either in the absence or presence of high amylose maize starch granules, then mixed with the high amylose maize starch granules and exposed to acidic buffers or bile acid solutions. It was shown that growth in and the presence of high amylose maize starch granules led to enhanced survival of strains LaftiTM 8B and LaftiTM 13B. Subsequently, survival in vivo was monitored by measuring the faecal level of Bifidobacterium LaftiTM 8B after oral administration of the strain to mice. A sixfold better recovery of strain LaftiTM 8B from mice faeces after oral dosage was noted for cells grown in amylose-containing medium compared with controls. It was concluded that high amylose maize starch granules contributed to enhanced survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B.     

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.     

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.     

Damaged starch characterisation by ultracentrifugation

The relative molecular size distributions of a selection of starches (waxy maize, pea and maize) that had received differing amounts of damage from ball milling (as quantified by susceptibility to alpha-amylase) were compared using analytical ultracentrifugation. Starch samples were solubilised in 90% dimethyl sulfoxide, and relative size distributions were determined in terms of the apparent distribution of sedimentation coefficients g*(s) versus s(20,w). For comparison purposes, the sedimentation coefficients were normalised to standard conditions of density and viscosity of water at 20 degrees C, and measurements were made with a standard starch loading concentration of 8 mg/mL. The modal molecular size of the native unmilled alpha-glucans were found to be approximately 50S, 51S and 79S for the waxy maize, pea and maize amylopectin molecules, respectively, whilst the pea and maize amylose modal molecular sizes were approximately 14S and approximately 12S, respectively. As the amount of damaged starch increased, the amylopectin molecules were eventually fragmented, and several components appeared, with the smallest fractions approaching the sedimentation coefficient values of amylose. For the waxy maize starch, the 50S material (amylopectin) was gradually converted to 14S, and the degradation process included the appearance of 24S material. For the pea starch, the situation was more complicated than the waxy maize due to the presence of amylose. As the amylopectin molecules (51S) were depolymerised by damage within this starch, low-molecular-weight fragments added to the proportion of the amylose fraction (14S)--although tending towards the high-molecular-weight region of this fraction. As normal maize starch was progressively damaged, a greater number of fragments appeared to be generated compared to the other two starches. Here, the 79S amylopectin peak (native starch) was gradually converted into 61 and 46S material and eventually to 11S material with a molecular size comparable to amylose. Amylose did not appear to be degraded, implying that all the damage was focused on the amylopectin fraction in all three cases. Specific differences in the damage profiles for the pea and maize starches may reflect the effect of lipid-complexed amylose in the maize starch.     

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.     

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.     

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.     

Genetic evidence that outer membrane binding of starch is required for starch utilization by Bacteroides thetaiotaomicron

Mutagenesis of Bacteroides thetaiotaomicron with the transposon Tn4351 produced five classes of mutants that were not able to grow on amylose or amylopectin. These classes of mutants differed in their ability to grow on maltoheptaose (G7) and in the level of starch-degrading enzymes produced when bacteria were grown on maltose. All of the mutants were deficient in starch binding. Since one class of mutants retained normal levels of starch-degrading enzymes, this indicates that binding of the starch molecule by a cell surface receptor is necessary for starch utilization by B. thetaiotaomicron. Analysis of a starch-negative mutant that grew on G7 indicated that B. thetaiotaomicron possessed two starch-binding components or sites. One component (site A), apparently missing in this mutant, had an absolute preference for larger starch oligomers, whereas the other component (site M) also had a high affinity for maltodextrins (G4 through G7). Mutants not able to grow on maltodextrins (greater than G4) probably lacked both of these binding components. Only one class of mutants did not grow normally on maltose, but instead had a 4- to 5-h lag on maltose and a slower growth rate than the wild type. This class of mutants did not produce any of the starch-degrading enzymes or bind starch, even when growing on maltose. Such a phenotype probably resulted from transposon inactivation of a central regulatory gene or a gene encoding an enzyme that produces the inducer. The fact that both the degradative enzymes and the starch-binding activity were affected in this mutant indicates that genes encoding the cell surface starch-binding site are under the same regulatory control as genes encoding the enzymes.     

Preparation of clear noodles with mixtures of tapioca and high-amylose starches

Ways to simulate the making of clear noodles from mung bran starch were investigated by studying the molecular structures of mung bean and tapioca starches. Scanning electron micrographs showed that tapioca starch granules were smaller than those of mung bean starch. X-ray diffraction patterns of mung bean and tapioca starch were A- and C_A-patterns, respectively. Iodine affinity studies indicated that mung bean starch contained 37% of apparent amylose and tapioca starch contained 24%. Gel permeation chromatograms showed that mung bean amylopectin had longer peak chain-length of long-branch chains (DP 40) than that of tapioca starch (DP 35) but shorter peak chain-length of short-branch chains (DP 16) than that of tapioca starch (DP 21). P-31 n.m.r. spectroscopy showed that both starches contained phosphate monoesters, but only mung bean starch contained phospholipids. Physical properties, including pasting viscosity, gel strength, and thermal properties (gelatinization), were determined. The results of the molecular structure study and physical properties were used to develop acceptable products using mixtures of cross-linked tapioca and high-amylose maize starches. Tapioca starch was cross-linked by sodium trimetaphosphate (STMP) with various reaction times, pH values, and temperatures. The correlation between those parameters and the pasting viscosity were studied using a visco/amylograph. Starches, cross-linked with 0.1% STMP, pH 11.0, 3.5 h reaction time at 25, 35, and 45°C (reaction temperature), were used for making noodles. High-amylose maize starch (70% amylose) was mixed at varying ratios (9, 13, 17, 28, 37, and 44%) with the cross-linked tapioca starches. Analysis of the noodles included: tensile strength, water absorption, and soluble loss. Noodle sensory properties were evaluated using trained panelists. Noodles made from a mixture of cross-linked tapioca starch and 17% of a high-amylose starch were comparable to the clear noodles made from mung bean starch.     

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.     

Structural features of non-granular spherulitic maize starch

Complementary analyses of the internal structure of spherulites crystallized from high-amylose maize starch were obtained using light, electron and atomic force microscopy. Radially oriented crystalline lamellae were observed in transmission and scanning electron microscopy, as well as AFM. Internal structures consistent with the central hilum region of starch granules were observed. Spherulites were composed largely of linear or lightly branched starch polymers. Degradation of amylopectin at gelatinization temperatures of 180 degrees C was evident, but iodine binding suggested a high molecular weight (>100 DP) for the spherulitic polymers.     

Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an alpha-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and alpha-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the alpha-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the alpha-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual alpha-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch-degrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.     

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.     

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.     

The effects of amylose content on the molecular size of amylose, and on the distribution of amylopectin chain length in maize starches

The amylose to amylopectin ratios in six maize starch samples of differing amylose contents were measured by enzymatic debranching, followed by high performance size exclusion chromatography (HPSEC). The molecular size of amyloses, estimated by -log K_wav, shows progressive decrease with the increase in amylose content in maize starches. The gel permeation chromatographs of the corresponding amylopectins, debranched with isoamylase, showed bimodal distributions containing long and short chains. The average chain length of amylopectin has a correlation with amylose content. The correlation coefficients between amylose content and average chain length, long chain length, weight ratio and the mole ratio of long and short chain length, were 0.97, 0.92, 0.96, 0.94 respectively. The maize starch with the highest amylose content has the lowest amylose molecular size and the longest chains, with a high ratio of long to short chains in its amylopectin fraction. Comparing the values of amylose content determined by HPSEC of starch or debranched starch with those of the iodinecomplex method, we conclude that long chains of amylopectin in high amylose starches contribute significantly to apparent amylose content.     

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.     

Preparation and characterization of gelatinized granular starches from aqueous ethanol treatments

In order to modify the properties of native starch granules, the formation of gelatinized granular forms (GGS) from normal, waxy, and high amylose maize, as well as potato and tapioca starches was investigated by treating granules with aqueous ethanol at varying starch:water:ethanol ratios and then heating in a rotary evaporator to remove ethanol. The modified starches were characterized using bright field, polarized and electron microscopy. Short/long range molecular order and enthalpic transitions on heating were also studied using infrared spectroscopy, X-ray diffractometry and differential scanning calorimetry respectively. A diffuse birefringence pattern without Maltese cross was observed for most GGS samples. Treatment with aqueous ethanol resulted in starch-specific changes in the surface of granules, most noticeably swelling and disintegration in waxy maize, surface wrinkling in normal maize and tapioca, swelling and opening-up in potato starches, and swelling and bursting in high amylose maize. The ratio of ethanol to water at which original granular order was disrupted also varied with starch type. GGS had less short range molecular order than native granules as inferred by comparing 1047/1022 wave number ratio from infrared spectroscopy. Similarly, A- and B-type diffraction reflections were either reduced or completely lost with evolution of V-type patterns in GGS.