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Thermoplastic starch-waxy maize starch nanocrystals nanocomposites.

Waxy maize starch nanocrystals obtained by hydrolysis of native granules were used as a reinforcing agent in a thermoplastic waxy maize starch matrix plasticized with glycerol. Compared to our previous studies on starch nanocrystals reinforced natural rubber (NR) [Macromolecules 2005, 38, 3783; 2005, 38, 9161], the present system presents two particularities: (i) thermoplastic starch is a polar matrix, contrarily to NR, and (ii) the chemical structures of the matrix and the filler are similar. The influence of the glycerol content, filler content, and aging on the reinforcing properties of waxy maize starch nanocrystals (tensile tests, DMA) and crystalline structure (X-ray diffraction) of materials were studied. It was shown that the reinforcing effect of starch nanocrystals can be attributed to strong filler/filler and filler/matrix interactions due to the establishment of hydrogen bonding. The presence of starch nanocrystals leads to a slowing down of the recrystallization of the matrix during aging in humid atmosphere.

Crystallization↗

A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme.

The aim of this work was to identify enzymes that participate in the degradation of transitory starch in Arabidopsis. A mutant line was isolated by screening leaves at the end of the night for the presence of starch. The mutant had a higher starch content than the wild-type throughout the diurnal cycle. This accumulation was due to a reduction in starch breakdown, leading to an imbalance between the rates of synthesis and degradation. No reduction in the activity of endo-amylase (alpha-amylase), beta-amylase, starch phosphorylase, maltase, pullulanase or D-enzyme could be detected in crude extracts of leaves of the mutant. However, native PAGE in gels containing amylopectin revealed that a starch-hydrolysing activity, putatively identified as an endo-amylase and present in wild-type chloroplasts, was absent or appreciably reduced in the mutant. This is the first time that a specific enzyme required for starch degradation has been identified in leaves.

Amylases↗

Mutants of Arabidopsis lacking starch branching enzyme II substitute plastidial starch synthesis by cytoplasmic maltose accumulation.

Three genes, BE1, BE2, and BE3, which potentially encode isoforms of starch branching enzymes, have been found in the genome of Arabidopsis thaliana. Although no impact on starch structure was observed in null be1 mutants, modifications in amylopectin structure analogous to those of other branching enzyme II mutants were detected in be2 and be3. No impact on starch content was found in any of the single mutant lines. Moreover, three double mutant combinations were produced (be1 be2, be1 be3, and be2 be3), and the impact of the mutations on starch content and structure was analyzed. Our results suggest that BE1 has no apparent function for the synthesis of starch in the leaves, as both be1 be2 and be1 be3 double mutants display the same phenotype as be2 and be3 separately. However, starch synthesis was abolished in be2 be3, while high levels of alpha-maltose were assayed in the cytosol. This result indicates that the functions of both BE2 and BE3, which belong to class II starch branching enzymes, are largely redundant in Arabidopsis. Moreover, we demonstrate that maltose accumulation depends on the presence of an active ADP-glucose pyrophosphorylase and that the cytosolic transglucosidase DISPROPORTIONATING ENZYME2, required for maltose metabolization, is specific for beta-maltose.

1,4-alpha-Glucan Branching Enzyme↗

Acute metabolic response to high-carbohydrate, high-starch meals compared with moderate-carbohydrate, low-starch meals in subjects with type 2 diabetes.

OBJECTIVE: The monosaccharides resulting from the digestion of ingested carbohydrates are glucose, fructose, and galactose. Of these three monosaccharides, only ingested glucose resulted in a large increase in the plasma glucose concentration. Fructose (Metabolism 41:510-517, 1992) and galactose (Metabolism 42:1560-1567, 1993) had only a minor effect. Therefore, we were interested in determining whether we could design a mixed meal, using foods of known monosaccharide, disaccharide, and starch composition, the ingestion of which would result in only a small rise in plasma glucose concentration. RESEARCH DESIGN AND METHODS: The experimental meal was composed of very little readily digestible starch but rather large amounts of fruits and vegetables. It contained 43% carbohydrate, 22% protein, and 34% fat. The results were compared with a second type of meal that contained 55% carbohydrate, 15% protein, and 30% fat, with an emphasis on complex carbohydrates (starch). It also was compared with a third meal that contained 40% carbohydrate, 20% protein, and 40% fat, typical of that consumed by the average American. The test meals were ingested in random order by people with type 2 diabetes who were not treated with oral hypoglycemic agents or insulin. Each subject ingested each type of meal. The same identical meal was ingested at 0800, 1200, and 1700. RESULTS: The integrated 24-h plasma glucose area response was statistically significantly smaller (P < 0.05) after ingestion of the low-starch meals compared with the high-starch, high-carbohydrate meals or the typical American meals. The 24-h integrated serum insulin area response also was statistically significantly less (P < 0.05) after ingestion of the low-starch meals compared with the high-starch meals or the typical American meals. The serum triglyceride area response was similar after ingestion of all three test diets. CONCLUSIONS: A diet in which fruits, nonstarch vegetables, and dairy products are emphasized may be useful for people with type 2 diabetes.

Aged↗

Measurement of resistant starch by enzymatic digestion in starch and selected plant materials: collaborative study.

Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSDr) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSDR) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSDr and RSDR values are unacceptably high.

Enzymes↗

Soluble starch synthases and starch branching enzymes from cotyledons of smooth- and wrinkled-seeded lines of Pisum sativum L.

Soluble starch synthase and branching enzyme were purified from 18-day-old cotyledons of the smooth-seeded pea cultivar Alaska (RR) and wrinkled-seeded pea cultivar Progress #9 (rr) by DEAE-cellulose chromatography. Two coeluting peaks of primed and citrate-stimulated starch synthase activity and a major and minor peak of branching enzyme activity were observed in Alaska. However, in Progress #9, only one peak of synthase activity was found. When crude extracts of Progress #9 were centrifuged, over 70% of the starch synthase activity was recovered in the pelleted fraction, and additional washings of the pellet released no further activity. The addition of purified starch granules to Alaska crude extracts also resulted in the recovery of a greater proportion of synthase activity in pelleted fractions. The two peaks of branching enzyme activity in Alaska differed in their stimulation of phosphorylase, amylose branching activity, and activity in various buffers. The DEAE-cellulose profile of Progress #9 showed no distinct peak of branching enzyme and less than 10% of the total activity found in Alaska. The association of one form of soluble starch synthase with the pelleted fraction and the greatly reduced levels of branching enzyme provide a partial explanation for the appearance of high-amylose starch in Progress #9 cotyledons.

1,4-alpha-Glucan Branching Enzyme↗

The structural organisation of the gene encoding class II starch synthase of wheat and barley and the evolution of the genes encoding starch synthases in plants.

Wheat and barley contain at least four classes of starch synthases in the endosperm, granule bound starch synthase I (GBSSI) and starch synthases I, II and III (SSI, SSII, SSIII). In this work, SSII in barley is shown to be associated with the starch granule by using antibodies. A cDNA from barley encoding SSII and the genes for SSII from barley and Aegilops tauschii ( A. tauschii, the D genome donor to wheat) are characterised. Fluorescent in situ hybridisation (FISH) and PCR were used to localise the wheat SSII gene to the short arm of chromosome 7, showing synteny with the location of the rice SSII gene to the short arm of chromosome 6. Comparison of the genes encoding SSII of A. tauschii, barley and Arabidopsis showed a conserved exon-intron structure although the size of the introns varied considerably. Extending such comparison between the genes encoding starch synthases (GBSSI, SSI, SSII and SSIII) from A. tauschii and Arabidopsis showed that the exon-intron structures are essentially conserved. Separate and distinct genes for the individual starch synthases therefore existed before the separation of monocotyledons and dicotyledons.

Base Sequence↗

The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase.

High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg x L(-1) and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x mL(-1) for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.

Aspergillus niger↗

Mechanisms by which resistant starches and non-starch polysaccharide sources affect the metabolism and disposition of the food carcinogen, 2-amino-3-methylimidazo[4,5-f]quinoline.

Although both non-starch polysaccharides (NSP) and resistant starches (RS) are included in current definitions of dietary fibre, our previous work has suggested fundamental differences in the way in which these two classes of material affect the disposition and absorption of a dietary carcinogen. The present studies explore whether different effects on carcinogen metabolism could play a role in the contrasting patterns seen previously. Groups of female Wistar rats were pre-fed for 4 weeks one of five types of defined diet (AIN-76). The control diet contained 35% maize starch and no dietary fibre. The RS-containing diets had all the maize starch substituted with either Hi-maize or potato starch. In the NSP-containing diets, 10% of the maize starch was substituted with dietary fibre in the form of either lignified plant cell walls (wheat straw) or soluble dietary fibre (apple pectin). Pre-fed rats were gavaged with the food carcinogen, [2-14C] 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and plasma and urinary metabolites characterized using HPLC at various time intervals after administration. After 4 h gavage, plasma from rats on both RS-containing diets contained significantly higher levels of intact IQ and lower levels of the major metabolites, IQ-5-O-glucuronide and IQ-5-sulfate, as compared with plasma from the negative control group at this time. In contrast, plasma from animals on the NSP-containing wheat straw diet (and to a lesser extent the apple pectin diet) showed significantly lower levels of intact IQ, and significantly higher levels of the two major metabolites, as compared with those from the control rats. These different metabolite profiles were also reflected in different urinary excretion profiles. Urine from rats pre-fed RS-containing diets revealed significantly slower metabolite excretion as compared with urine from rats that had been given the NSP-containing diets. Western blotting methodologies also profiled differences between the effects of these two types of dietary fibre in the expression of xenobiotic metabolizing enzymes. We conclude that changes in activity and expression of xenobiotic metabolising enzymes could play a role in the contrasting effects of these two types of dietary fibre on carcinogen uptake and disposition.

Animals↗

Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain.

Raw-starch-degrading glucoamylases have been known as multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain (SBD) by an O-glycosylated linker region. A molecular genetics approach has been chosen to find structural differences between two related glucoamylases, raw-starch-degrading Glm and nondegrading Glu, from the yeasts Saccharomycopsis fibuligera IFO 0111 and HUT 7212, respectively. We have found that Glm and Glu show a high primary (77%) and tertiary structure similarity. Glm, although possessing a good ability for raw starch degradation, did not show consensus amino acid residues to any SBD found in glucoamylases or other amylolytic enzymes. Raw starch binding and digestion by Glm must thus depend on the existence of a site(s) lying within the intact protein which lacks a separate SBD. The enzyme represents a structurally new type of raw-starch-degrading glucoamylase.

Amino Acid Sequence↗

Effect of gelatinization and starch-emulsifier interactions on aroma release from starch-rich model systems.

Release of selected volatile aldehyde compounds from starch-rich matrices was studied by headspace extraction, using solid-phase microextraction, and gas chromatography. Changes in the rheological properties of the starch-based matrices, due to starch concentration, gelatinization, and interactions with emulsifiers, were studied by steady shear and dynamic methods. The degree of volatile retention was found to depend on the compound properties, starch concentration, native structure of the granules, and presence of emulsifiers. The nongelatinized starch granules were more effective in lowering the volatile headspace quantities. Loss of the native structural integrity of the granules decreases the retention ability. For the nongelatinized starch dispersions, the more hydrophilic emulsifier showed a more pronounced effect on the matrix rheology and also on the aroma retention. A different behavior was observed for the gelatinized systems. Interpretation of the volatile release profiles was made on the basis of the matrix physical properties and interactions among components.

Chromatography, Gas↗

Regulatory effect of amino acids on the pasting behavior of potato starch is attributable to its binding to the starch chain.

The binding of an amino acid, glycine (Gly), alanine (Ala), epsilon-aminocaproic acid (-AC), monosodium glutamate (GluNa), or lysine (Lys), to starch was examined by a biomolecular interaction analyzer (IAsys). A starch sample (ATS) hydrolyzed to an extent of 1% hydrolysis rate with 15% sulfuric acid was used as a model starch for the binding examination. The reducing end of ATS was oxidized by the Somogyi reagent, and the conversion of the reducing end to the carboxyl group of ATS was confirmed by a carboxylic acid fluorescence labeling reagent. The oxidized ATS was immobilized to the amino group of a sensor cuvette by using water-soluble carbodiimide and N-hydroxysuccinimide through an amide bond. The IAsys examination showed that Gly, Ala, and epsilon-AC scarcely bound to the immobilized starch chains but that GluNa and Lys favorably bound with their increasing concentrations. The relative binding index (RBI) of each amino acid was defined by the ratio of the slope of the linear regression equation between the binding response and the concentration for each amino acid to that for Gly. Because the relationships between the RBI and the pasting characteristics (pasting temperature, peak viscosity, breakdown, and swelling index) could each be expressed by a linear regression equation with a high correlation coefficient, it is concluded that the regulation of the pasting behavior of starch with an amino acid is caused by binding of the amino acid to the starch chains.

Amino Acids↗

Influence of genotype and processing on the in vitro rate of starch hydrolysis and resistant starch formation in peas (Pisum sativum L.).

The formation of resistant starch (RS) and the rate of starch hydrolysis were evaluated in vitro in a wild type of green-seeded pea genotype RRRbRb BC3 (33-Am) with 32.7% amylose content and in two mutants RRrbrb BC3 (23-Am) and rrRbRb BC3 (65-Am) with amylose contents of 23.3 and 65.1%, respectively. Pea samples were intact or homogenized and subjected either to autoclaving or to boiling at atmospheric pressure. The amount of RS (total starch basis) varied from 6.2 to 12.9% in the 23-Am products and from 31.2 to 33.4% in the 65-Am products. The RS level of the 33-Am product with a regular amylose content was 11.0%. Both the 23-Am and the 65-Am products were abundant sources of dietary fiber (39 and 34%, dry matter basis, respectively) versus 23% in the regular pea product. The amylose/amylopectin ratio was an important determinant of the rate of starch hydrolysis. The hydrolysis indices (HI) and predicted glycemic indices were lowest in the 65-Am peas (HI range = 42-59) as compared to the 23-Am peas (HI range = 53-84). It is concluded that the pea genotypes covered a wide range in starch availability, which is likely to affect nutritional parameters such as glycemic responses and colonic delivery of starch.

Amylose↗

Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening.

We have cloned a gene involved in starch metabolism that was identified by the ability of its product to bind to potato starch granules. Reduction in the protein level of transgenic potatoes leads to a reduction in the phosphate content of the starch. The complementary result is obtained when the protein is expressed in Escherichia coli, as this leads to an increased phosphate content of the glycogen. It is possible that this protein is responsible for the incorporation of phosphate into starch-like glucans, a process that is not understood at the biochemical level. The reduced phosphate content in potato starch has some secondary effects on its degradability, as the respective plants show a starch excess phenotype in leaves and a reduction in cold-sweetening in tubers.

Amino Acid Sequence↗

An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilisation is abolished.

The Arabidopsis thaliana tpt-1 mutant which is defective in the chloroplast triose phosphate/phosphate translocator (TPT) was isolated by reverse genetics. It contains a T-DNA insertion 24 bp upstream of the start ATG of the TPT gene. The mutant lacks TPT transcripts and triose phosphate (TP)-specific transport activities are reduced to below 5% of the wild type. Analyses of diurnal variations in the contents of starch, soluble sugars and phosphorylated intermediates combined with 14CO2 labelling studies showed, that the lack of TP export for cytosolic sucrose biosynthesis was almost fully compensated by both continuous accelerated starch turnover and export of neutral sugars from the stroma throughout the day. The utilisation of glucose 6-phosphate (generated from exported glucose) rather than TP for sucrose biosynthesis in the light bypasses the key regulatory step catalysed by cytosolic fructose 1,6-bisphosphatase. Despite its regulatory role in the feed-forward control of sucrose biosynthesis, variations in the fructose 2,6-bisphosphate content upon illumination were similar in the mutant and the wild type. Crosses of tpt-1 with mutants unable to mobilise starch (sex1) or to synthesise starch (adg1-1) revealed that growth and photosynthesis of the double mutants was severely impaired only when starch biosynthesis, but not its mobilisation, was affected. For tpt-1/sex1 combining a lack in the TPT with a deficiency in starch mobilisation, an additional compensatory mechanism emerged, i.e. the formation and (most likely) fast turnover of high molecular weight polysaccharides. Steady-state RNA levels and transport activities of other phosphate translocators capable of transporting TP remained unaffected in the mutants.

Arabidopsis↗

Granule-bound starch synthase I. A major enzyme involved in the biogenesis of B-crystallites in starch granules.

Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A- and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and in vitro. We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B-type crystals either in close association with pre-existing amorphous amylopectin or by crystallization of entirely de novo synthesized material.

Amino Acid Sequence↗

Processing effects on susceptibility of starch to digestion in some dietary starch sources.

Maize flour, potato flour, cocoyam flour, plantain flour, yam flour, and rice flour were assayed for starch digestibility by an established enzymatic procedure. These were either autoclaved, microwaved, or parboiled and then freeze-dried. Freeze-dried samples were stored for 10 days either below freezing or at ambient temperature. Parameters assessed were readily digestible starch (RDS), slowly digestible starch (SDS), and total starch (TS). Data was analyzed by t-test (P < or = 0.05). RDS levels among raw flours ranged from 1.01 g/100g in rice flour to 8.16 g/100 g in cocoyam flour. Autoclaving and parboiling increased RDS levels in most flours, while microwaving significantly reduced RDS compared with raw flour. Ambient temperature storage reduced the RDS content. SDS levels ranged from 4.95 g/100 g in yam flour to 22.2 g/100 g in maize flour. SDS levels were increased by autoclaving and parboling, but significantly reduced by microwaving, compared with the raw flour. Storage at ambient temperature resulted in lower SDS. The TS content in raw flour ranged from 28.0 g/100 g in plantain flour to 68.4 g/100 g in rice flour. Autoclaving resulted in reduced TS levels insome flours. Moist heat processing and the post-process storage temperature therefore result in significant changes in starch susceptibility to enzymic digestion. This information will be useful in developing food processing and storage procedures that modify starch resistance to digestion in order to optimize its nutritional quality and to enhance the physiological benefits.

Dietary Carbohydrates↗

Cell structure and starch nature as key determinants of the digestion rate of starch in legume.

The factors responsible for the slow digestibility of starch in leguminous seeds have been studied by examining microscopically the cooked seeds after various treatments and by measuring starch digestion in vitro. Starch in leguminous seeds is entrapped in parenchyma cells and swells only partially during cooking. The alpha-amylase cannot easily penetrate within the gelatinized starch granules due to steric hindrance and the physical nature of the leguminous starch. Disruption of the cells, especially before cooking increases the susceptibility of starch to alpha-amylase digestion.

Cooking↗