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[Maize starch biosynthesis and its genetic manipulation].

Starch is the most important source of calories and a vital storage component in plants. The characterization and production of starch variants from mutation and with transgenic technology has improved our understanding of the synthesis of starch granule. In starch biosynthesis in plants, four enzymes, including ADP-glucose pyrophosphorylase, starch synthase, starch branching enzyme and starch debranching enzyme, are widely accepted from an enormous amount of research aimed primarily at enzyme characterization. As many genes encoding the enzymes and their multiple isoforms in starch biosynthesis pathway have been isolated, genetic manipulation of the starch biosynthesis pathway shows to be a practical way by which starch quantity is increased and starch with novel properties can be created.

English Abstract↗

Understanding and influencing starch biochemistry.

Starch is one of the most important products synthesized by plants that is used in industrial processes. If it were possible to increase production or modify starches in vivo, using combinations or either genetically altered or mutant plants, it may make them cheaper for use by industry, or open up new markets for the modified starches. The conversion of sucrose to starch in storage organs is, therefore, discussed. In particular the roles of the different enzymes directly involved in synthesizing the starch molecules on altering starch structure are reviewed, as well as the different models for the production of the fine structure of amylopectin. In addition, the process of starch phosphorylation, which is also important in determining the physical properties of starches, is reviewed. It is hoped that detailed knowledge of these processes will lead to the rational design of tailored starches. Starch degradation is also an important process, for example, in the cold-sweetening of potato tubers, but outside of cereal endosperm little is known about the processes involved. The enzymes thought to be involved and the evidence for this are discussed.

Glycogen Debranching Enzyme System↗

Starch powder contamination of surgical wounds.

OBJECTIVES: To determine if (1) lubricating starch glove powder contaminates surgical wounds even after powdered gloves have been washed and/or wiped: (2) starch powder can be eliminated from surgical wounds when the surgical team wears only powderless gloves; and (3) starch powder introduced into surgical wounds may increase scar formation. DESIGN AND OUTCOME MEASURES: Human surgical wounds were irrigated at the end of operations in which various combinations of powdered and powderless gloves were used. Team members who wore powdered gloves washed them in a saline solution and wiped them on surgical towels. The starch particles in the irrigant were counted. In addition, two series of breast biopsies were performed, one in which the surgeon wore powdered gloves and the other, powderless gloves. Pathologic specimens from reexcisions (for carcinoma) were examined for starch granules and inflammation. RESULTS: Starch granules were found in proportion to the number of surgical team members who wore powdered gloves and to the proximity of the wearer(s) to the operative site. Exclusive use of powderless gloves eliminated the presence of starch powder. Starch-containing phagocytes in tissue were surrounded by an inflammatory reaction, and in one patient the inflammation and scarring were severe. CONCLUSIONS: Starch powder is introduced into wounds by the use of powdered gloves despite glove washing and wiping. It can be eliminated by the exclusive use of powderless gloves. The inflammatory reaction to starch is variable and can be severe.

Equipment Contamination↗

Electron paramagnetic resonance study of water distribution in starch granules.

An electron paramagnetic resonance (EPR) study was performed for potato and wheat starch containing Cu2+ ions as a paramagnetic probe. Distribution of water in the starch granules as well as the interactions between the copper and starch matrix of different crystalline structures were determined. EPR spectra of the native starches consisted of two different centers of Cu2+. One of them, giving at 293 and 77 K an EPR signal of axial symmetry with a well-resolved hyperfine structure (HFS), was assigned to the Cu2+ -starch complex in which Cu2+ ions strongly interacted with oxygen atoms of the starch matrix. Another Cu2+ species, exhibiting an isotropic signal at 293 K and an axial signal with resolved HFS at 77 K, was attributed to a [Cu(H2O)6]2+ complex freely rotating at room temperature and immobilized at low temperatures. Interaction of Cu2+ with the starch matrix and the relative number of the particular copper species depended on the crystallographic type of starch. Dehydration at 393 K resulted in elimination of the rotating complex signal and decrease of the total intensity of the EPR spectrum caused by clustering of the Cu2+ ions. Freezing at 77 K and thawing led to restoring of the spectrum intensity and reappearing of the signal of the [Cu(H2O)6]2+ complex. This effect, related to liberation of water molecules from the granule semicrystalline growth rings on freezing/thawing, was especially visible for wheat starch, indicating differences in the water retention ability of starch granules of different crystallographic structure.

Biopolymers↗

Modeling the utilization of starch by activated sludge for simultaneous substrate storage and microbial growth.

This paper presents a mechanistic model incorporating microbial growth on external substrate with simultaneous formation of storage biopolymers (activated sludge model for growth and storage-ASMGS) for the utilization of starch by activated sludge. Model description and calibration utilized experimental data of an SBR fed with particulate native potato starch (NPS) and soluble starch (SolS) selected as model substrates. The fate of starch was monitored in a cycle together with glycogen and oxygen uptake rate (OUR) profiles. In the experiments, glycogen formation was significantly lower than predicted by total conversion of starch to glycogen, justifying the need to account for primary growth on starch. The proposed model basically modified Activated Sludge Model No.3 (ASM3), to include adsorption of starch, its hydrolysis and simultaneous growth and glycogen formation using the hydrolysis products, which was mainly maltose. Model simulations indicated hydrolysis of the adsorbed starch as the rate limiting process. The proposed model calibrated well the fate of all major model components, namely, starch, glycogen, and OUR. Particulate NPS and SolS were hydrolyzed with similar rates; however, primary and secondary growth processes on SolS were more efficient, with higher yields, due to the more easily utilizable products of SolS, both in terms of extracellular hydrolysis and of stored poly-glucose. Modeling with ASM3, assuming starch as either readily or slowly biodegradable, did not provide an equally acceptable fit for the glycogen and OUR curves; supporting the need to consider primary growth together with storage as defined in the proposed model.

Bioreactors↗

Water diffusivity in starch-based systems.

The objective of this study was to investigate the influence of structure, and component interactions, on the sorption and transport properties of water in starch-based systems. We compared the effective diffusivity (Deff) of water in two starches, with differing amylose-amylopectin ratios, using either kinetics of water adsorption or analysis of drying curves (water desorption) to estimate Deff. The effect of incorporating small sugar molecules into the granular or gelatinized starch matrices on Deff was measured by drying curve analysis. To investigate the possible mechanisms of water transport, the porosity and microscopic appearance of the samples at different stages of drying were determined. In a complementary study, sorption isotherms and the number of accessible "binding" sites in the starch and starch-sugar systems were determined using gravimetric analysis and inverse gas chromatography (IGC) 'probe analysis'. In the case of the starch-sugar systems, the measurements were made after the components had been 'mechanically mixed', or after more intimate mixing had been achieved by a co-freeze-drying process. The Deff of the starches was found to depend, in a complex way, on the moisture content of the samples. At relatively high moisture contents, the predominant mode of water transport was by liquid diffusion. As the samples became drier, their porosity increased, and the predominant mode of moisture transport was by vapor phase diffusion. As the samples became very dry (less than 10% water content), Deff fell significantly. Incorporation of sugars, in general, led to a reduction of Deff, which was correlated with a corresponding fall in porosity. In agreement with the findings of other workers, for the starches studied, the value of Deff determined from water adsorption measurements was significantly less than Deff determined from water desorption (drying curve analysis). The form of the Deff versus moisture content relationship was, however, independent of the method of measurement (adsorption or desorption). The water sorption and IGC probe analysis results indicated that some physicochemical interaction was expedited by the freeze-drying process. This interaction was manifested by a reduction in water sorption at a given relative vapor pressure, and by major changes in the accessibility of the co-freeze-dried samples to organic probe molecules. Taken together, the results indicate that water transport (diffusion) in starches and in starch-sugar mixtures is dependent significantly on gross structural features (development of porosity during drying), but that specific molecular, physico-chemical interactions must also be considered.

Adsorption↗

Deuterium nuclear magnetic resonance studies of potato starch hydration.

Deuterium Nuclear Magnetic Resonance (NMR) measurements at 4.7 Tesla were used to study the hydration properties of potato starch suspensions as a function of the starch-to-water ratio. The deuterium NMR spectrum of potato starch suspensions consisted of a relatively tall, single Lorentzian (D2O) peak and a resolved doublet ('powder' pattern) of about 1 kHz quadrupole splitting and low amplitude. The deuterium NMR transverse relaxation rate (R*2) was measured for the single Lorentzian, deuterium oxide peak; this rate increased with increasing starch-to-water ratio. Deviations of such R*2 dependences from linearity were observed only at high ratios of starch-to-water, above approximately 40% solids. In addition to the 'free' or bulk population (which has a very fast rotational correlation time of 5 ps), a second population of water weakly sorbed on the starch granule surface was monitored and found to be in fast exchange with the bulk water; this second water population has a rotational correlation time of 17 ps at 298 K, only about three times slower than that of bulk water. Additionally, a third population of slowly exchangeable water is present, which is "anisotropically bound" and has a highly restricted motion within the potato starch granule structure; well-defined quadrupole splittings are observed from this latter population of water in potato starch. Potato starch exhibits unique hydration properties that have not been found in cereal starches.

Chemical Phenomena↗

Native starch in tablet formulations: properties on compaction.

Maize, potato, rice and tapioca (cassava) starch were evaluated with respect to their properties on direct compression. Rice starch showed much better compactibility as compared to maize, potato and tapioca starch. Moreover, its binding capacity proved to be almost insensitive to mixing with magnesium stearate. This in contrast to the dramatic decrease in crushing strength of potato starch tablets containing the lubricant. The compactibility of the starches was found to be strongly affected by the equilibrium moisture content of the starches, which is dependent on the relative humidity of the atmosphere under which the powders were stored. All starches showed adequate capacity for water uptake to act as a disintegrant. Rice starch exhibited worst flowability, caused by its fine particle size as compared to the other starches. Granulation of rice starch changed it into a potential filler-binder in tablets prepared by direct compression.

Chemistry, Pharmaceutical↗

Effect of starch-based biomaterials on the in vitro proliferation and viability of osteoblast-like cells.

The cytotoxicity of starch-based polymers was investigated using different methodologies. Poly-L-lactic acid (PLLA) was used as a control for comparison purposes. Extracts of four different starch-based blends (corn starch and ethylene vinyl alcohol (SEVA-C), corn starch and cellulose acetate (SCA), corn starch and polycaprolactone (SPCL) and starch and poly-lactic acid (SPLA70) were prepared in culture medium and their toxicity was analysed. Osteoblast-like cells (SaOs-2) were incubated with the extracts and cell viability was assessed using the MTT test and a lactate dehydrogenase (LDH) assay. In addition DNA and total protein were quantified in order to evaluate cell proliferation. Cells were also cultured in direct contact with the polymers for 3 and 7 days and observed in light and scanning electron microscopy (SEM). LDH and DNA quantification revealed to be the most sensitive tests to assess respectively cell viability and cell proliferation after incubation with starch-based materials and PLLA. SCA was the starch blend with higher cytotoxicity index although similar to PLLA polymer. Cell adhesion tests confirmed the worst performance of the blend of starch with cellulose acetate but also showed that SPCL does not perform as well as it could be expected. All the other materials were shown to present a comparable behaviour in terms of cell adhesion showing slight differences in morphology that seem to disappear for longer culture times. The results of this study suggest that not only the extract of the materials but also their three-dimensional form has to be biologically tested in order to analyse material-associated parameters that are not possible to consider within the degradation extract. In this study, the majority of the starch-based biomaterials presented very promising results in terms of cytotoxicity, comparable to the currently used biodegradable PLLA which might lead the biocompatibility evaluation of those novel biomaterials to other studies.

Biocompatible Materials↗

Impact of beta-cyclodextrin and resistant starch on bile acid metabolism and fecal steroid excretion in regard to their hypolipidemic action in hamsters.

To examine the impact on bile acid metabolism and fecal steroid excretion as a mechanism involved in the lipid-lowering action of beta-cyclodextrin and resistant starch in comparison to cholestyramine, male golden Syrian hamsters were fed 0% (control), 8% or 12% of beta-cyclodextrin or resistant starch or 1% cholestyramine. Resistant starch, beta-cyclodextrin and cholestyramine significantly lowered plasma total cholesterol and triacylglycerol concentrations compared to control. Distinct changes in the bile acid profile of gallbladder bile were caused by resistant starch, beta-cyclodextrin and cholestyramine. While cholestyramine significantly reduced chenodeoxycholate independently of its taurine-glycine conjugation, beta-cyclodextrin and resistant starch decreased especially the percentage of taurochenodeoxycholate by -75% and -44%, respectively. As a result, the cholate:chenodeoxycholate ratio was significantly increased by 100% with beta-cyclodextrin and by 550% with cholestyramine while resistant starch revealed no effect on this ratio. beta-Cyclodextrin and resistant starch, not cholestyramine, significantly increased the glycine:taurine conjugation ratio demonstrating the predominance of glycine conjugated bile acids. Daily fecal excretion of bile acids was 4-times higher with 8% beta-cyclodextrin and 19-times with 1% cholestyramine compared to control. beta-Cyclodextrin and cholestyramine also induced a 2-fold increase in fecal neutral sterol excretion, demonstrating the sterol binding capacity of these two compounds. Resistant starch had only a modest effect on fecal bile acid excretion (80% increase) and no effect on excretion of neutral sterols, suggesting a weak interaction with intestinal steroid absorption. These data demonstrate the lipid-lowering potential of beta-cyclodextrin and resistant starch. An impaired reabsorption of circulating bile acids and intestinal cholesterol absorption leading to an increase in fecal bile acid and neutral sterol excretion is most likely the primary mechanism responsible for the lipid-lowering action of beta-cyclodextrin. In contrast, other mechanisms involving the alterations in the biliary bile acid profile or repressed hepatic lipogenesis, e.g., VLDL production, appear to be involved in the hypolipidemic effect of resistant starch.

Animals↗

Organisation of the external region of the starch granule as determined by infrared spectroscopy.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) was used to study the external regions of starch granules. Native starches (wheat, potato, maize, waxy maize and amylomaize) were analysed and compared to gelatinised and acid-hydrolysed starches. The IR spectra of potato and amylomaize starches were closer to that of highly ordered acid-hydrolysed starch than the other starches. FTIR was not able to differentiate between A- and B-type crystallinity so the difference observed between starches was not related to this factor. The variation between starch varieties was interpreted in terms of the level of ordered structure present on the edge of starch granules with potato and amylomaize being more ordered on their outer regions. This could explain the high resistance of both these starches to enzyme hydrolysis.

Acids↗

Ex vivo bioadhesion and in vivo testosterone bioavailability study of different bioadhesive formulations based on starch-g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures.

Starch-g-poly(acrylic acid) copolymers or grafted starches synthesized by 60Co irradiation or chemical modification and co-freeze-dried starch/poly(acrylic acid) mixtures were evaluated on their ex vivo bioadhesion capacity. The buccal absorption of testosterone from a bioadhesive tablet formulated with the grafted starches or starch/poly(acrylic acid) mixtures was investigated. The results were compared to a reference formulation (physical mixture of 5% Carbopol 974P and 95% Drum Dried Waxy Maize). Rice starch-based irradiated grafted starches showed the best bioadhesion results. Partial neutralization of the acrylic acid with Ca(2+) ions resulted in significantly higher bioadhesion values compared to the reference. Ca(2+) and Mg(2+) partially neutralized maltodextrin-based irradiated grafted starches showed significantly higher bioadhesion values compared to the reference formulation. The chemically modified grafted starches showed significantly higher adhesion force values than for the reference tablet. None of the co-freeze-dried starch/poly(acrylic acid) mixtures showed significantly higher bioadhesion results than the reference (Bonferroni test, P<0.05). A chemically modified grafted starch could sustain the 3 ng/ml plasma testosterone target concentration during +/- 8 h (T(>3 ng/ml)). By lyophilization of a partially neutralized irradiated grafted starch, the in vivo adhesion time (22.0 +/- 7.2 h) and the T(>3 ng/ml) (13.5 +/- 1.3 h) could be increased. The absolute bioavailability of the lyophilized formulation approached the reference formulation. Some of the grafted starches showed to be promising buccal bioadhesive drug carriers for systemic delivery.

Adhesives↗

Composition and physical properties of starch in microgravity-grown plants.

The effect of spaceflight on starch development in soybean (Glycine max L., BRIC-03) and potato (Solanum tuberosum, Astroculture-05) was compared with ground controls by biophysical and biochemical measurements. Starch grains from plants from both flights were on average 20-50% smaller in diameter than ground controls. The ratio delta X/delta rho (delta X --difference of magnetic susceptibilities, delta rho--difference of densities between starch and water) of starch grains was ca. 15% and 4% higher for space-grown soybean cotyledons and potato tubers, respectively, than in corresponding ground controls. Since the densities of particles were similar for all samples (1.36 to 1.38 g/cm3), the observed difference in delta X/delta rho was due to different magnetic susceptibilities and indicates modified composition of starch grains. In starch preparations from soybean cotyledons (BRIC-03) subjected to controlled enzymatic degradation with alpha-amylase for 24 hours, 77 +/- 6% of the starch from the flight cotyledons was degraded compared to 58 +/- 12% in ground controls. The amylose content in starch was also higher in space-grown tissues. The good correlation between the amylose content and delta X/delta rho suggests, that the magnetic susceptibility of starch grains is related to their amylose content. Since the seedlings from the BRIC-03 experiment showed elevated post-flight ethylene levels, material from another flight experiment (GENEX) which had normal levels of ethylene was examined and showed no difference to ground controls in size distribution, density, delta X/delta rho and amylose content. Therefore the role of ethylene appears to be more important for changes in starch metabolism than microgravity.

Amylopectin↗

Effect of resistant starch from corn or rice on glucose control, colonic events, and blood lipid concentrations in streptozotocin-induced diabetic rats.

To examine the effect of two types of resistant starch on blood glucose and insulin levels, colonic events, hypolipidemic actions and humoral immune responses, Sprague-Dawley streptozotocin-induced diabetic rats were fed diet containing resistant starch from corn or rice. The marked body weight loss by inducing diabetes was not recovered by feeding resistant starch, even though there are no differences in food intakes compared to the non-diabetic control rats. No significant effect of resistant starch feeding on blood glucose and insulin was found. Even though the length of small intestines, and cecum, colon and rectum together with the tissue weight of cecum were not affected by feeding resistant starch, the intestinal transit time was markedly shortened by both types of resistant starch and resistant starch from corn had a more pronounced effect. The short chain fatty acids in the intestinal contents did not appear to be different among the groups. Nonetheless, both of resistant starch from corn and rice significantly lowered plasma total lipid and cholesterol concentrations compared to the diabetic control. The total liver cholesterol lowering effect was observed with resistant starch from rice. Neither immunoglobulin G nor C(3) were influenced by resistant starch.

Adipose Tissue↗

Amylopectin molecular structure reflected in macromolecular organization of granular starch.

For lintners with negligible amylose retrogradation, crystallinity related inversely to starch amylose content and, irrespective of starch source, incomplete removal of amorphous material was shown. The latter was more pronounced for B-type than for A-type starches. The two predominant lintner populations, with modal degrees of polymerization (DP) of 13-15 and 23-27, were best resolved for amylose-deficient and A-type starches. Results indicate a more specific hydrolysis of amorphous lamellae in such starches. Small-angle X-ray scattering showed a more intense 9-nm scattering peak for native amylose-deficient A-type starches than for their regular or B-type analogues. The experimental evidence indicates a lower contrasting density within the "crystalline" shells of the latter starches. A higher density in the amorphous lamellae, envisaged by the lamellar helical model, explains the relative acid resistance of linear amylopectin chains with DP > 20, observed in lintners of B-type starches. Because amylopectin chain length distributions were similar for regular and amylose-deficient starches of the same crystal type, we deduce that the more dense (and ordered) packing of double helices into lamellar structures in amylose-deficient starches is due to a different amylopectin branching pattern.

Amylopectin↗

Thermal properties of partially hydrolyzed starch-glycerophosphatidylcholine complexes with various acyl chains.

Complexes of starch and monoacyl-sn-glycerophosphatidylcholine (GPC) containing various acyl (myristoyl, palmitoyl, and stearoyl) chains were subjected to hydrolysis with glucoamylase (EC 3.2.1.3). The enzyme hydrolyzed approximately 40% of starch control and 20-28% of starch-GPC complexes. Among the GPCs examined, 1- and 2-monomyristoyl-sn-GPC showed the highest resistance to enzyme hydrolysis, and the hydrolysis rate of starch-GPCs was greater with longer chains. Enzymatic hydrolysis strongly affected the thermal properties of the starch. After enzymatic hydrolysis of starch-GPC complexes for 24 h, their thermograms had broader peaks with lower enthalpies than the corresponding starch without enzyme; however, the starch-GPC complexes showed little change. The surface of starch-GPC granules was less eroded. These results showed that the increasing amount of starch-GPC complexes could be more resistant to hydrolysis.

Acylation↗

Comparison of MALDI-TOF mass spectrometric to enzyme colorimetric quantification of glucose from enzyme-hydrolyzed starch.

Successful quantification of the glucose produced by enzyme hydrolysis of starch was achieved by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protocol, using sorbitol as an internal standard. The starch contents measured by MALDI-TOF MS of corn starch, fiber-enriched oat flour derivatives, oat and barley flours, and barley flour/corn starch composites were evaluated in comparison to a widely accepted and validated method of starch determination, which relies on enzyme colorimetry (EC). The average starch content measured in a series of corn starch samples of different masses was 93 and 101% for EC and MALDI-TOF MS, respectively, values that represent the estimated purity of the sample. There was an agreement of 99% between the starch contents determined by the two analytical methods for complex flour-derived samples. Starch values estimated by MALDI-TOF MS consistently showed a greater degree of variability than those determined by EC, but this limitation was readily compensated by rapid acquisition of multiple mass spectra. This study is the first to report the quantification of glucose by MALDI-TOF MS, and it offers new perspectives into the potential utility of MALDI-TOF MS as a definitive tool for monosaccharide analysis and rapid starch determination in complex samples.

Avena↗

Relationships among the composition and physicochemical properties of starches with the characteristics of their films.

The physical, molecular, and functional properties of corn, cassava, and yam starches were related to the film properties of these starches. Corn, cassava, and yam starches contained 25%, 19%, and 30% amylose, respectively. Amylose from yam starch showed the smallest molecular weight among the starches and amylopectin from corn starch the smallest molecular weight. Cassava starch presented a higher amylopectin content, and its gels and films were less strong, more transparent, and more flexible than corn and yam films. Plasticized films of the three starches were more flexible, with a higher strain and lower stress at break when the glycerol content increased. Unplasticized films were brittle and had water vapor permeability values ranging from 6.75 x 10(-10) to 8.33 x 10(-10) g m(-1) s(-1) Pa(-1). These values decreased when the glycerol content reached 20 g/100 g of starch because a more compact structure was formed. Then, at a glycerol content of 40 g/100 g of starch, the WVP increased because the film matrixes became less dense.

Amylopectin↗