Physical investigations of surface membrane-water relationship of intact and gelatinized wheat-starch systems

Water mobility in intact and dried gelatinized starch was investigated by gravimetric water sorption, scanning electron microscopy (SEM), and solid-state nuclear magnetic resonance (NMR). A multi-component exponential model quantitatively measured different spin-spin relaxation times of two water components, namely bound water (Tsi) at 3.16 ms and mobile or free water (Tsii) at 3.23 ms, as a function of water activity (aw). The starch samples were moistened to 30% moisture content. SEM confirmed the disrupted, absorbent microstructure in dried, gelatinized starch powder and revealed starch granules in an incomplete gelatinized state, as compared to the complete membrane surface of the intact starch granule. Starch granules sorbed significantly differently at low aw, but after aw=0.44, sorption leveled similarly with increasing aw. The presence and role of a surface membrane was concluded, in support of the hypothetical "water sink" properties of intact granules, and was considered to influence in part the sorption behavior of incompletely gelatinized starch granules.     

Total and structured water in cancer: an NMR experimental study of serum and tissues in DMBA-induced OF1 mice.

Total water and structured water (fraction of total water which remains unfrozen below the transition point from the semisolid to solid state) were characterized by 1H NMR relaxometry in the sera and tissues of 3 groups of 30 female mice (C, H and L) receiving a single administration of DMBA and different diets. Mice given the diet H, containing the highest proportion of saturated fatty acids and processed starch, and the lowest phytochemicals content, presented the highest tumor incidence (lymphoma). This allowed 3 subgroups to be defined: subnormal (SN), small (T+) and large tumor (T++). Spin-lattice relaxation times of total water (Tlobs) in the sera and tissues did not significantly differ between C, H and L groups, and SN, T+ and T++ subgroups. In T+ mice, a decrease in the relative amount of structured water was noticed in the serum, liver and heart, while changes in the temperature dependence of the Tl of structured water (Tlsw) were observed between -21 degrees C and -42 degrees C. These results suggest a moderate increase in the rotational mobility of structured water molecules in the serum and the heart, and a pronounced decrease in the liver. Likewise, the modification of the Tlsvv temperature dependence curve's shape tends to confirm the existence of important conformational changes in the macromolecular assemblies, which markedly affect the properties of structured water, especially in the earliest stage of cancer development.     

Fabrication and Characterization of Native and Oxidized Potato Starch Biodegradable Films

The need to replace conventional polymers due to environmental pollution caused by them has led to increased production of biodegradable polymers such as starch. Thus, the application possibilities of starch have increased. In this study, we produced and characterized biodegradable films derived from native and oxidized potato starch. The film-forming solution was prepared with different concentrations of extracted starch (native or oxidized) and a plasticizer (glycerol or sorbitol). Then, the mechanical, barrier, morphological, and structural properties of the films were characterized. The moisture content of the films varied from 15.35?±?1.31 to 21.78?±?0.49%. The elastic modulus of the films ranged from 219?±?14.97 to 2299?±?62.91 MPa. The film of oxidized starch plasticized with sorbitol in the lowest content was the most resistant and flexible; moreover, this film also presented lower water vapor permeability and low solubility in water. Fourier-transform infrared spectroscopic analysis of the biodegradable films indicated the presence of same functional groups as those of starch with bands in the same regions. The film thickness was lower for the films plasticized with glycerol whereas the color variation (Δ?) was lower for the ones plasticized with sorbitol. In case of both plasticizers, the increase in their content decreased the Δ? value. All the biodegradable films presented stability against water absorption owing to their low solubility in water. Morphological evaluation revealed the presence of partially gelatinized starch granules in the films. The roughness parameter (Rq) of the films varied from 3.39 to 10.9 nm, indicating that their surfaces are smooth. X-ray diffraction studies showed a B-type pattern for the starches, which is representative of tubers. Further, the films present higher relative crystallinity (RC) compared to the starches. The biodegradable starch films are uniform, transparent and with low solubility in water. The oxidation of starch and use of sorbitol as a plasticizer resulted in improved properties of the starch films, which is suitable for application.     

Formamide and 2-hydroxy-N-[2-(2-hydroxy-propionylamino)-ethyl] propionamide (HPEP) as a mixed plasticizer for thermoplastic starch

As a plasticizer of starch, 2-hydroxy-N-[2-(2-hydroxy-propionylamino)-ethyl] propionamide (HPEP) is synthesized by the reaction of lactic acid and ethylenediamine. Melt extrusion processing is used to prepare thermoplastic starch by the mixed plasticizers of formamide and HPEP (FHTPS). Fourier Transform Infrared proves that the mixed plasticizers can form the interaction with CO group of starch in FHTPS. Scanning Electron Microscope shows that formamide and original water are necessary to from the homogeneous FHTPS containing formamide/HPEP/original water (20/10/10). X-ray diffraction exhibits that the mixture of formamide and HPEP can effectively suppress starch re-crystallization, and the original water has no effect on starch re-crystallization when FHTPS are stored at RH 50% for 50 days. Water absorption testing shows that the more HPEP is, the better water resistance of FHTPS is. With the increasing of HPEP ratios, the tensile strength of FHTPS is increased while the elongation is decreased at the whole range of water contents.     

Influence of extrusion and digestion on the nanostructure of high-amylose maize starch

An in-depth characterization of the structural changes undergone by high-amylose starch after extrusion and digestion with a pancreatic alpha-amylase has been carried out. The combination of USAXS, SAXS, XRD, and SEM techniques has provided a wide "picture" of the morphological transformations of starch, covering a length scale from approximately 0.3 nm to approximately 230 microm. Depending on the extrusion conditions, either gelatinization was attained ("mild" conditions) or single-amylose helix formation was induced ("extreme" conditions). SAXS experiments demonstrated that upon contacting the extruded materials with water, retrogradation took place. A new type of molecular organization with a characteristic repeat length of 5 nm was observed in the dry resistant starch fractions from the extruded high-amylose starch. The crystalline morphology of the resistant starch fractions, as observed by XRD, varied from B-type crystallinity for the "mild" extruded starch to a mixture of C- and V-type crystallinity in the case of "extreme" extrusion.     

Atomic force microscopy of pea starch: origins of image contrast

Atomic force microscopy (AFM) has been used to image the internal structure of pea starch granules. Starch granules were encased in a nonpenetrating matrix of rapid-set Araldite. Images were obtained of the internal structure of starch exposed by cutting the face of the block and of starch in sections collected on water. These images have been obtained without staining, or either chemical or enzymatic treatment of the granule. It has been demonstrated that contrast in the AFM images is due to localized absorption of water within specific regions of the exposed fragments of the starch granules. These regions swell, becoming "softer" and higher than surrounding regions. The images obtained confirm the "blocklet model" of starch granule architecture. By using topographic, error signal and force modulation imaging modes on samples of the wild-type pea starch and the high amylose r near-isogenic mutant, it has been possible to demonstrate differing structures within granules of different origin. These architectural changes provide a basis for explaining the changed appearance and functionality of the r mutant. The growth-ring structure of the granule is suggested to arise from localized "defects" in blocklet distribution within the granule. It is proposed that these defects are partially crystalline regions devoid of amylose.     

EPR investigation of Cu(II)-complexes with nitrogen derivatives of dialdehyde starch

Dialdehyde starch obtained by periodate oxidation from potato starch was converted into its disemicarbazone (DSC), dithiosemicarbazone (DTSC), dihydrazone (DHZ) and dioxime (DOX). The Cu(II) complexes of these compounds were prepared and characterized by Raman and EPR spectra, as well as by the measurements of magnetic susceptibility. EPR investigations showed that two types of complexes with different surroundings of copper centres existed in each starch derivative. Besides nitrogen atoms of the CN moiety and sulphur atoms of the CS moiety, also oxygen atoms from starch hydroxyl groups and/or water molecule were proposed as the coordination sites for the central copper ions.     

Response of Abies alba embryonal-suspensor mass to various carbohydrate treatments

Cultures of embryonal-suspensor mass of European silver fir (Abies alba) were screened for their response to various sucrose concentrations (29–175 mM) and to equimolar (100 or 200 mM) concentrations of glucose, fructose, galactose, sucrose, lactose and soluble starch. The higher concentrations were combined with 3.78 M abscisic acid. While fructose was ineffective in promoting any somatic embryo maturation glucose, sucrose and galactose promoted early maturation stages. Somatic embryo development up to the torpedo stage was observed only on media with soluble starch or lactose. Lactose was superior to other carbohydrate treatments and led to more synchronized maturation and more normally structured embryos. In comparison with the pronounced carbohydrate effect abscisic acid at this concentration proved to be of less importance. A sequence of culture steps was established in order to stimulate differentiation and maturation.Abbreviations ABA (±) 2-cis-4-trans-abscisic acid - BAP N6-benzylaminopurine - ESM embryonal-suspensor mass - L.S.D. Least Significant Difference - SE somatic embryo(s) - SH Schenk and Hildebrandt     

Characterization of a crosslinked high amylose starch excipient.

A controlled release excipient based on sodium trimetaphosphate (STMP) crosslinked high amylose starch has been examined by 13C CP/MAS NMR. The dry excipient powder is pressed to a hard tablet whose volume increase in H2O runs parallel to the STMP concentration used. The known polymorph resonances of single helix 'V' starch (hydrated) and double helix 'B' starch (hydrated) both contribute to the spectrum corresponding to the swollen tablet. The wet tablet when loaded with a pharmaceutical agent provides a near zero-order release profile for up to 20 h. The swelling and drug release behaviour is explained in terms of self-assembly of the STMP treated starch nanomolecular particles. These particles are drawn together by "self-assembly" due to formation of amylose double helices as water penetrates the tablet. An optimum level of chemical crosslinking ensures the integrity of the swollen tablet whose sponge-like structure enclosed by a membranous surface is responsible for sustained release.     

Modeling and experimental studies on intermittent starch feeding and citrate addition in simultaneous saccharification and fermentation of starch to flavor compounds

Simultaneous saccharification and fermentation (SSF) is a combined process of saccharification of a renewable bioresource and fermentation process to produce products, such as lactic acid and ethanol. Recently, SSF has been extensively used to convert various sources of cellulose and starch into fermentative products. Here, we present a study on production of buttery flavors, namely diacetyl and acetoin, by growing Lactobacillus rhamnosus on a starch medium containing the enzyme glucoamylase. We further develop a structured kinetics for the SSF process, which includes enzyme and growth kinetics. The model was used to simulate the effect of pH and temperature on the SSF process so as to obtain optimum operating conditions. The model was experimentally verified by conducting SSF using an initial starch concentration of 100 g/L. The study demonstrated that the developed kinetic was able to suggest strategies for improved productivities. The developed model was able to accurately predict the enhanced productivity of flavors in a three stage process with intermittent addition of starch. Experimental and simulations demonstrated that citrate addition can also lead to enhanced productivity of flavors. The developed optimal model for SSF was able to capture the dynamics of SSF in batch mode as well as in a three stage process. The structured kinetics was also able to quantify the effect of multiple substrates present in the medium. The study demonstrated that structured kinetic models can be used in the future for design and optimization of SSF as a batch or a fed-batch process. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Specific adsorption of starch oligosaccharides in the gel phase of starch granules

The gel phase of native starch-granules is penetrable by such low-molecular-weight solutes as oligosaccharides, amino acids, and salts [Lathe and Ruthven, Biochem. J., 62 (1956) 665]. Molecules larger than about 1000 daltons are effectively excluded. Starch oligosaccharides (maltotriose through maltoheptaose and perhaps higher) exhibit anomalous behavior in that they are taken up by the gel phase far in excess of the amount expected on the basis of their molecular size. Adsorption was measured by using radioactive starch oligosaccharides and counting weighed amounts of solution before and after equilibration with starch granules. The measurements were corrected for water sorption by the starch granules and for exclusion effects as ascertained by controls with nonstarch types of oligosaccharides. Maximum adsorption was observed with maltotetraose. The results indicate a specific binding between the starch oligosaccharides and molecular chains in the starch, presumably those chains in the gel phase. We suggest that these chains constitute interbranch regions of branched molecules, or segments of linear molecules in the gel or amorphous phase, the segments being of sufficient length to form a double helix or other association with the linear oligosaccharides.     

Transformation of water clusters in wet starch under changing environmental conditions

Transformation of the water cluster distribution in wet potato starch (with a water content of 27 to 45%) at temperatures that ranged from–50 to +80°C was studied by differential scanning calorimetry. A significant difference was observed between the transformations in the temperature ranges below and above 0°C. Both cooling and heating at T < 0°C enabled a reorganization of the initial size distribution of water clusters characteristic for room temperature. These changes could lead to an increase of the average cluster size during both crystallization and melting. The transformation intensity depended on the water content and scanning rate and differed between the native and amorphous states of starch. In this case, the cluster-size distribution remained unimodal. However, heating of wet native starch to temperatures close to the point of transition into the amorphous state (75–80°C) induced a bimodal distribution due to the emergence of large water clusters; thus, the heterogeneity of the water distribution within the native granules increased.     

Static water structure detected by heat capacity measurements on aqueous solutions of a triple-helical polysaccharide schizophyllan

Heat capacity measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan by precision adiabatic calorimetry over a wide range of concentrations 30.45-90.93 wt % at temperatures between 5 and 315 K. The heat capacity curves obtained were divided into four groups depending on the weight fraction of schizophyllan w regions I-IV. In region I, triple-helices with the sheath of bound water, structured water, and loosely structured water forming layers around the helix core are embedded in free water. In region II, there is no free water, and loosely structured water decreases until it vanishes, but structured water stays constant with increasing w. In region III, bound water remains unaffected, but structured water decreases with increasing w by overlapping each other. Finally, in region IV, only schizophyllan and bound water exist, the latter decreasing upon increasing w. The maximum thickness of each layer is 0.18(3) nm for bound water, 0.13(4) nm for structured water, and 0.23(6) nm for loosely structured water, and these layers of water are at the enthalpy levels of 53%, 93.7%, and nearly 100%, respectively, between ice (0%) and free water (100%).     

The retrogradation of waxy maize starch extrudates: effects of storage temperature and water content

The effects of water content and storage temperature on the kinetics of the retrogradation of nonexpanded waxy maize starch extrudates were studied using (1)H pulsed NMR and wide-angle X-ray diffraction. The increase in crystallinity observed by XRD was accompanied by a decrease in the relaxation times of the solid-like component of the NMR free induction and the spin-echo decays, and an increase in the contribution of the solid-like component to the total signal. The dependence of the rate of starch retrogradation on the storage temperature showed the typical "bell-shaped" behavior, which was successfully modeled using the Lauritzen-Hoffman theory of crystallization of chain-folded polymers. This theory was extended to model the effect of water content on the rate of isothermal crystallization by exploiting the ten-Brinke and Karasz, and the Flory equations to describe the dependence of the glass-transition and the melting temperatures on water content.     

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.     

Functionality of Disintegrants and Their Mixtures in Enabling Fast Disintegration of Tablets by a Quality by Design Approach

Investigation of the effect of disintegrants on the disintegration time and hardness of rapidly disintegrating tablets (RDTs) was carried out using a quality by design (QbD) paradigm. Ascorbic acid, aspirin, and ibuprofen, which have different water solubilities, were chosen as the drug models. Disintegration time and hardness of RDTs were determined and modeled by executing combined optimal design. The generated models were validated and used for further analysis. Sodium starch glycolate, croscarmellose sodium, and crospovidone were found to lengthen disintegration time when utilized at high concentrations. Sodium starch glycolate and crospovidone worked synergistically in aspirin RDTs to decrease disintegration time. Sodium starch glycolate-crospovidone mixtures, as well as croscarmellose sodium-crospovidone mixtures, also decreased disintegration time in ibuprofen RDTs at high compression pressures as compared to the disintegrants used alone. The use of sodium starch glycolate in RDTs with highly water soluble active ingredients like ascorbic acid slowed disintegration, while microcrystalline cellulose and crospovidone drew water into the tablet rapidly and quickened disintegration. Graphical optimization analysis demonstrated that the RDTs with desired disintegration times and hardness can be formulated with a larger area of design space by combining disintegrants at difference compression pressures. QbD was an efficient and effective paradigm in understanding formulation and process parameters and building quality in to RDT formulated systems.KEY WORDS: disintegrants, quality by design, rapidly disintegrating tablets     

The Two Plastidial Starch-Related Dikinases Sequentially Phosphorylate Glucosyl Residues at the Surface of Both the A- and B-Type Allomorphs of Crystallized Maltodextrins But the Mode of Action Differs

In this study, two crystallized maltodextrins were generated that consist of the same oligoglucan pattern but differ strikingly in the physical order of double helices. As revealed by x-ray diffraction, they represent the highly ordered A- and B-type allomorphs. Both crystallized maltodextrins were similar in size distribution and birefringence. They were used as model substrates to study the consecutive action of the two starch-related dikinases, the glucan, water dikinase and the phosphoglucan, water dikinase. The glucan, water dikinase and the phosphoglucan, water dikinase selectively esterify glucosyl residues in the C6 and C3 positions, respectively. Recombinant glucan, water dikinase phosphorylated both allomorphs with similar rates and caused complete glucan solubilization. Soluble neutral maltodextrins inhibited the glucan, water dikinase-mediated phosphorylation of crystalline particles. Recombinant phosphoglucan, water dikinase phosphorylated both the A- and B-type allomorphs only following a prephosphorylation by the glucan, water dikinase, and the activity increased with the extent of prephosphorylation. The action of the phosphoglucan, water dikinase on the prephosphorylated A- and B-type allomorphs differed. When acting on the B-type allomorph, by far more phosphoglucans were solubilized as compared with the A type. However, with both allomorphs, the phosphoglucan, water dikinase formed significant amounts of monophosphorylated phosphoglucans. Thus, the enzyme is capable of acting on neutral maltodextrins. It is concluded that the actual carbohydrate substrate of the phosphoglucan, water dikinase is defined by physical rather than by chemical parameters. A model is proposed that explains, at the molecular level, the consecutive action of the two starch-related dikinases.In terms of quantity, starch is one of the most prominent photosynthesis-derived products. The global starch production by land plants has been estimated to be approximately 2,850 million tons per year (Burrell, 2003). Starch is highly relevant for nutrition in animals and humans, but it is also used for many industrial applications, such as additives in paper or textiles and in pharmacy products as well. In addition, starch appears to be increasingly important as a photosynthesis-based renewable energy source that can be converted into technologically relevant products such as bioethanol and hydrogen (Hannah and James, 2008; Zhang et al., 2008).Native starch is formed as a water-insoluble particle called a granule that is thought to comprise two types of polyglucans, amylopectin and amylose. The latter is an almost unbranched α-1,4-glucan and usually is the minor constituent of the starch particle, accounting for 10% to 35% of the total starch dry weight (Ball, 2000). However, in some mutants, the relative amylose content is strongly diminished, resulting in an essentially amylose-free starch (such as in the waxy mutant of maize [Zea mays]), or, alternatively, it is increased, forming up to 70% of the starch mass (e.g. in the amylose extender mutant from maize; Gérard et al., 2001). Nevertheless, in wild-type starches, amylopectin typically is the major constituent that also is essential for the molecular organization of the glucans within the entire starch granule (Ball and Morell, 2003). Like glycogen, amylopectin is a branched α-glucan with 4% to 6% of the inter-Glc linkages being α-1,6-bonds (Ball, 2000); however, as opposed to glycogen, the branching points occur as intramolecular clusters. Due to the length distribution of the side chains and the clustering of the branching points, neighboring glucan chains are capable of forming highly ordered double helices (Smith, 2001; Zeeman et al., 2002).As revealed by x-ray diffraction analysis, two major native starch structures are known that differ in the arrangement of the double helices. The A-type allomorph, which is typical of wild-type cereal starches but also occurs in lower plants, is more compact, as compared with the B type, and consists of flat layers of double helices. By contrast, in the B-type allomorph, six double helices are thought to surround a central cavity that is filled with water molecules. The B-type allomorph is found in starch synthesized by dicotyledonal storage organs, such as potato (Solanum tuberosum) tubers, in some high-amylose starches from cereal mutants (Gallant et al., 1997; Gérard et al., 2001), and in assimilatory starches from potato and Arabidopsis (Arabidopsis thaliana) as well (Hejazi et al., 2008). Legume starches are believed to represent another allomorph that is designated the C type. However, this allomorph is actually a mixture of both the A- and B-type crystallites within a single native starch particle rather than a third distinct type of the double helical arrangement (Imberty et al., 1991; Bogracheva et al., 2001).It should be noted that both the A- and B-type allomorphs of native starch granules often contain, as a minor constituent, an additional crystal structure designated the V type. Unlike the A- and B-type allomorphs, the V type is assumed to arise from single amylose helices, some of which are complexed with endogenous granular lipids. When estimated for the dry state, the V-type crystal structure accounts for only a small percentage of the total starch granule crystallinity (Lopez-Rubio et al., 2008).The physical structure of the native starch particle is likely to have important biochemical implications, as it affects the performance of carbohydrate-active enzymes and, thereby, the transition of carbohydrates from the solid phase to the soluble phase. This conclusion has been reached by in vitro experiments demonstrating that the pancreas α-amylase hydrolyzes A-type starch faster than the B-type counterpart (Gérard et al., 2001).Another metabolically important feature of amylopectin is the occurrence of covalent modification by phosphate esters that are found in a small proportion of the glucosyl residues. Most frequently phosphorylation occurs at the C6 position of the glucosyl residue, but C3 and, to a minor extent, C2 can also be esterified (Hizukuri et al., 1970). Recently, evidence has been presented that the esterification of the C6 and C3 positions of glucosyl residues differs in the structural effects on the neighboring inter-Glc bonds (Hansen et al., 2009). Phosphorylation at C6 is mediated by the recently identified α-glucan, water dikinase (GWD; EC 2.7.9.4), which utilizes ATP as dual phosphate donor and three distinct acceptors, two of which are sequentially used. The enzyme transfers the terminal phosphate group to water (thereby forming orthophosphate) and the β-phosphate group first to a conserved His residue within the catalytic domain of the monomeric GWD and, subsequently, to the C6 target of the glucosyl residue to be phosphorylated (Ritte et al., 2002, 2006). Phosphorylation at C3 is catalyzed by a second dikinase, designated phosphoglucan, water dikinase (PWD; EC 2.7.9.5; Ritte et al., 2006). The amino acid sequence of the catalytic (C-terminal) domain of PWD shares similarity with that of GWD, and in principle, the PWD-mediated catalysis follows the same mode of action as GWD, including the transient autophosphorylation at a conserved His residue (Baunsgaard et al., 2005; Kötting et al., 2005). However, PWD deviates from GWD in the amino acid sequence of the N-terminal domain, especially in the carbohydrate-binding region. PWD possesses a single carbohydrate-binding module that has been grouped into the family CBM20 (Machovič and Janaček, 2006a, 2006b). By contrast, the N-terminal domain of GWD contains two putative carbohydrate-binding motifs similar to those of an α-amylase that presumably is located in the chloroplasts (Yu et al., 2005). However, the structure of these motifs is still not known; therefore, a sequence-based prediction of the actual carbohydrate target is not yet possible.GWD- and PWD-deficient Arabidopsis mutants possess to some extent similar but not equal phenotypes. Leaves of GWD-deficient lines (which contain essentially unchanged levels of functional PWD) have starch levels that are at least five times higher than those of the wild type and remain high even after prolonged darkness. Growth of the entire plant is strongly compromised. The phenotype of PWD-deficient mutants (which express functional GWD) is less severe, as growth is only slightly diminished and transitory starch levels are elevated but not as strongly as in the GWD-deficient lines. Mutants lacking functional PWD can degrade transitory starch, but net degradation occurs at a lower rate as compared with wild-type plants (Kötting et al., 2005). These data clearly indicate that, in vivo, PWD cannot substitute for GWD and that glucosyl 6-phosphate residues are involved in a more strict control of the starch turnover as compared with the C3 phosphate esters.When considering the metabolic function(s) of starch phosphorylation, it should be noted that phosphorylation occurs during both net starch synthesis and degradation, although the rates of phosphorylation are likely to be different (Nielsen et al., 1994; Ritte et al., 2004). It is reasonable, therefore, to assume that starch phosphorylation exerts an important role in the entire transitory starch metabolism, rather than being functional only during the degrading process (and, consequently, the starch-related dikinases cannot, in a strict sense, be considered as “starch-degrading enzymes”).Depending on the botanical source, the degree of starch phosphorylation varies strongly. In potato tuber starch, approximately 0.1% to 0.5% of the glucosyl residues are phosphorylated (Ritte et al., 2002), and this value is considered to be indicative of a high level of phosphorylation. By contrast, cereal starches contain a far lower relative phosphate content that often is close to the limit of detection (approximately 0.002%; Glaring et al., 2006). In principle, these differences could be due to different rates of phosphorylation, as catalyzed by the two starch-related dikinases, and this assumption seems to be supported by the observation that, in general, starches of the B-type allomorph appear to have a higher degree of phosphorylation as compared with those of the A-type allomorph. If so, the dikinases may preferentially act on the B-type allomorph. Alternatively, the phosphorylation catalyzed by the two dikinases could be balanced by counteracting phosphatases, such as SEX4. This plastidial enzyme has been shown to act as a (phospho)glucan phosphatase that is involved in leaf starch metabolism (Kötting et al., 2009). If antagonistic enzyme activities are taken into consideration, the actual level of starch phosphorylation is determined by the rate of both phosphorylation and the subsequent hydrolysis of phosphate esters and, consequently, does not necessarily reflect the action of the starch-related dikinases.Recently, crystallized maltodextrins (MD_cryst) have been prepared that, by using x-ray diffraction, were identified as being the B-type allomorph and to possess a highly ordered structure (which exceeds that of native starch granules). MD_cryst have been applied as a substrate for a recombinant GWD from potato. Using a carefully optimized assay, the rate of phosphorylation was by far higher than that observed with any other carbohydrate substrate, such as native starch granules or starch-derived polysaccharides. By contrast, solubilization by heat treatment of the MD_cryst almost completely abolished the activity of GWD. Phosphorylation resulted in the formation of singly, doubly, and triply phosphorylated glucans and favored the solubilization of both neutral glucans and phosphoglucans (Hejazi et al., 2008). Recombinant PWD also phosphorylated MD_cryst, provided the MD_cryst had been prephosphorylated by GWD and were not solubilized by heat treatment (Hejazi et al., 2008).Because of the high phosphorylation rates and the phosphorylation pattern obtained, MD_cryst are a suitable model carbohydrate that mimics phosphorylation-relevant features of highly ordered regions within the native starch granule. It allows study of the action of the two starch-related dikinases and the transition of carbohydrates from the solid to the soluble state without any other starch-related enzyme being required.Until now, only the B-type allomorph of the MD_cryst has been applied as substrate of the two dikinases. Using native starch granules as a target, the rates of phosphorylation as obtained with recombinant GWD varied largely within the B-type allomorph (Hejazi et al., 2008); therefore, it is reasonable to assume that additional but largely unknown features of the native starch granule also strongly affect the action of GWD. This implies that any preference or specificity of the starch-related dikinases for a given allomorph can be analyzed most convincingly if MD_cryst preparations representing both the B- and A-type allomorphs are available.In this study, we used two MD_cryst preparations that are indistinguishable in their oligoglucan patterns but differ in the physical arrangement of the double helices and represent the highly ordered A- and B-type allomorphs. Using these two MD_cryst preparations, we analyzed the action of the two starch-related dikinases. The size distribution of the MD_cryst particles has been determined using the Coulter counter, and surface properties of both allomorphs were monitored by scanning electron microscopy. Thermal stability of the two allomorphs was analyzed by measuring the temperature dependence of light scattering. Finally, the phosphorylation-dependent solubilization of both allomorphs and the transition of (phospho)glucans into the soluble state have been studied.     

Permeation of volatile compounds through starch films

The aim of this study was to gain insight into the factors that affect the permeation of volatiles through starch films. These films were obtained by casting gelatinized starch/water/glycerol mixtures. The films were dried and conditioned under different conditions (temperature and relative humidity) resulting in films that vary in the degree of starch crystallinity and glycerol and water content. The permeation of two model volatiles (carvone and diacetyl) at 20 degrees C and at 30, 60, or 90% relative humidity (RH) was analyzed gravimetrically. Further, the solubility of the two model compounds (under conditions where the permeation experiments were carried out) was determined. From the obtained permeation and solubility data, the diffusion coefficients of these compounds in the different starch films were calculated. The crystallinity in the starch films increased with increasing water content of the films during preparation. The water content of the resulting films in turn increased with increasing glycerol and when the films were exposed to a higher RH during drying or conditioning. For films with the same composition, the flux for diacetyl was greater than for carvone. The solubilities of diacetyl and carvone were slightly dependent on the properties of the films. It was found that with increasing starch crystallinity the diffusion coefficient for both compounds decreases, which is probably due to the impermeability of starch crystallites. Interestingly, in films with about the same extent of crystallinity, the diffusion can be described with the free volume model, with water and glycerol determining the amount of free volume.     

芸香地上器官的生物学研究

对芸香的茎、叶及花等器官的形态结构进行了较为系统的观察研究,对花芽形成及开花过程进行了客观描述.芸香花有顶生和侧生两种,柱头为"湿柱头",叶片具有典型旱生型植物特点,茎叶中均含有簇晶和油腔,这些特征可作为分类学上鉴别该物种的性状.茎的淀粉积累过程:花前期淀粉积累很少(6月下旬),10月下旬达高峰,之后略有减少并保持稳定直至被冻死.对此结合当地温度及水分条件进行了抗寒、抗旱的讨论.     

State of water in starch-water systems in the gelatinization temperature range as investigated using dielectric relaxation spectroscopy

The dielectric response of native wheat starch-water slurries containing 5-60% starch (w/w) was measured in the frequency range of 0.2-20 GHz after heating the slurries to 7 different temperatures between 25 and 90 °C for 30 min. Three relaxations, with relaxation time range of 4-9 ps, 20-25 ps and 230-620 ps at 25 °C, were identified from the dielectric spectra of starch slurries. The fastest relaxation process (4-9 ps) was attributed to bulk water while the two slower relaxations were attributed to the confined water molecules present in the starch-water system. The amount of water exhibiting the slowest relaxation (230-620 ps) was calculated to be 0.08-0.16 g water/g starch, which was close to the monolayer water associated with wheat starch. Mobility of bulk water was significantly reduced (P < 0.001) upon gelatinization at low starch concentration (10% starch), but remained unaffected at higher starch concentrations. The mobility of two slower relaxing water species was not significantly influenced (P > 0.19) by gelatinization at all starch concentrations.