Novel Wx alleles generated by base editing for improvement of rice grain quality.

Amylose content (AC), which is regulated by the Waxy (Wx) gene, is a major indicator of eating and cooking quality (ECQ) in rice (Oryza sativa). Thus far, only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein. Here, nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors. The nucleotide substitutions led to changes in 15 amino acid residues of Wx, and a series of novel Wx alleles with ACs of 0.3%-29.43% (wild type with AC of 19.87%) were obtained. Importantly, the waxyabe2 allele showed a "soft rice" AC, improved ECQ, favorable appearance, and no undesirable agronomic traits. The transgenes were removed from the waxyabe2 progeny, generating a promising breeding material for improving rice grain quality.

Rheological Properties and Electrospinnability of High-Amylose Starch in Formic Acid.

Starch derivatives, such as starch-esters, are commonly used as alternatives to pure starch due to their enhanced mechanical properties. However, simple and efficient processing routes are still being sought out. In the present article, we report on a straightforward method for electrospinning high-amylose starch-formate nanofibers from 17 wt % aqueous formic acid (FA) dispersions. The diameter of the electrospun starch-formate fibers ranged from 80 to 300 nm. The electrospinnability window between starch gelatinization and phase separation was determined using optical microscopy and rheological studies. This window was shown to strongly depend on the water content in the FA dispersions. While pure FA rapidly gelatinized starch, yielding solutions suitable for electrospinning within a few hours at room temperature, the presence of water (80 and 90 vol % FA) significantly delayed gelatinization and dissolution, which deteriorated fiber quality. A complete destabilization of the electrospinning process was observed in 70 vol % FA dispersions. Optical micrographs showed that FA induced a disruption of starch granule with a loss of crystallinity confirmed by X-ray diffraction. As a result, starch fiber mats exhibited a higher elongation at break when compared to brittle starch films.

Role of molecular entanglements in starch fiber formation by electrospinning.

We have demonstrated a method of fabricating pure starch fibers with an average diameter in the order of micrometers. In the present study, correlation between the rheological properties of starch dispersions and the electrospinnability was attempted via the extrapolation of the critical entanglement concentration, which is the boundary between the semidilute unentangled regime and the semidilute entangled regime. Dispersions of high amylose starch containing nominally 80% amylose (Gelose 80) required 1.2-2.7 times the entanglement concentration for effective electrospinning. Besides starch concentration, molecular conformation, and shear viscosity were also of importance in determining the electrospinnability. The rheological properties and electrospinnability of different starches were studied. Hylon VII and Hylon V starches, containing nominally 70 and 50% amylose, respectively, required concentrations of 1.9 and 3.7 times their entanglement concentrations for electrospinning. Only poor fibers were obtained from mung bean starch, which contains about 35% amylose, while starches with even lower amylose contents could not be electrospun.

Effects on the structure and properties of native corn starch modified by enzymatic debranching (ED), microwave assisted esterification with citric acid (MCAE) and by the dual ED/MCAE treatment.

Native corn starch was modified by enzymatic debranching (ED), microwave assisted citric acid esterification (MCAE), and by dual ED/MCAE. The structure and properties of native starch (NS), and the resulting debranched starch (DS), microwave assisted citric acid esterified starch (MCS), and microwave assisted citric acid debranched starch (MCDS) were determined and compared. Both the morphology and crystalline regions of the modified starches were changed by ED and MCAE. ED increased significantly the amylose content and transparency, but decreased the in vitro enzymatic digestibility, freeze thaw stability and relative crystallinity of DS compared to those of NS. MCAE produced a decrease in amylose content, transparency, in vitro enzymatic digestibility, and relative crystallinity, but increased the freeze-thaw stability of MCS compared to NS, and of MCDS compared to DS. The A-type crystalline structure of NS and DS was changed to B-type crystalline structure after MCAE treatment, and a new FTIR characteristic band at 1735 cm-1 was observed for MCS and MCDS. This work provides insights for producing esterified corn starches by a combined enzyme, microwave and organic acid novel technology.

B-->A Allomorphic transition in native starch and amylose spherocrystals monitored by in situ synchrotron X-ray diffraction.

The B-->A phase transition in native starch granules and spherocrystals prepared from DP 20-40 synthetic amylose chains was investigated in situ at intermediate moisture content (20-30%) by wide-angle synchrotron X-ray scattering, using a temperature-controlled pressure cell. The transition in native starch was monitored at hydrostatic pressures of 1.6-11.0 MPa and occurred in a temperature range of 90-110 degrees C. The transition temperature increased with increasing amylose content and the transition was incomplete in amylose-rich starch. The B-->A transition in highly crystalline amylose spherocrystals was monitored at pressures between 2.0 and 28.5 MPa. The transition temperature was higher than in native starch, ranging from 125 to 135 degrees C. At 2.0 MPa, after conversion, the hydrated spherocrystals melted at 185 degrees C. Surprisingly, at the same pressure, in excess water, the spherocrystals did not solubilize but converted to allomorph A at 100 degrees C and melted at 160 degrees C. For all samples, the transition occurred in a matter of minutes and a higher pressure decreased the transition temperature. For the first time, thermal expansion coefficients were estimated for A- and B-amylose at intermediate moisture. A strong thermal anisotropy was observed for A-amylose, the expansion being higher along the b-axis than along the a-axis of the monoclinic unit cell. This anisotropy was attributed to the fact that, in the b-direction, amylose double helices lie at the same height along the chain axis while, in the a-direction, they are more closely packed in a zigzag fashion.

Understanding the multi-scale structure and digestibility of different waxy maize starches.

This work concerns different cultivars of waxy maize starch (WMS), from which a significant correlation between the multi-scale structure and the digestibility has been identified. WMSs show a typical A-type crystalline polymorph. The surface porosity of WMS granules facilitates their digestibility. In contrast, the in vitro digestion results indicate that the resistant starch (RS) content increased with higher contents of amylose, single helices, and surface short-range ordered structures. Resistant starch (RS) was found to be made up of single helices and perfect crystallites formed by the fraction of chains with a degree of polymerization (DP) between 13 and 24. Slowly digestible starch (SDS) consists of single helices. Rapidly digestible starch (RDS) is mainly composed of disordered molecular chains in the amorphous regions of starch. This work reveals the relationship between the multiscale structure and digestibility of different WMSs and can provide guidance for the application of WMSs in food or non-food fields.

Influence of germination time on the morphological, morphometric, structural, and physicochemical characteristics of Esmeralda and Perla barley starch.

The aim of this study was to analyzing the impact of germination time on the morphology, crystallinity, gelatinization and viscosity properties on the starch of Esmeralda and Perla barley variety. The two barley were germinated for 1 to 8 days, at 26 °C and 65% relative humidity. Micrographs showed the presence of pinholes and eroded surfaces. Starch in Esmeralda was hydrolyzed completely at 8 days of germination. Birefringence was reduced from day 4, losing molecular structuring of the crystalline area. Morphometric data: fractal dimension, area, perimeter, circularity, and roundness decreased significantly along germination time in both varieties. The entropy increased significantly, from 0.79 to 10.09 in Esmeralda and from 0.46 to 7.57 in Perla. Relative crystallinity decreased significantly in the Perla from 24.7% to 23.6%. Viscosity peaks were also significantly reduced, pasting temperature was constant in Esmeralda but in Perla was significantly reduced from 95.43 to 95.19 °C with germination, the gelatinization temperature increased significantly in the Esmeralda while in Perla it remained constant. Enthalpy decreased significantly to 75.8% and 37% in Esmeralda and Perla respectively. The study of germination impact on structural and physicochemical properties is important to identify the use of hydrolyzed starches in the food industry or others.

Effect of chemical composition, granule structure and crystalline form of pigmented rice starches on their functional characteristics.

The present research was carried out to analyze the effect of chemical composition, granule morphology and crystalline structure of pigmented rice starches on their functional characteristics. The starches of these rice cultivars were observed to possess novel characteristic as compared to hybrids or non-pigmented cultivars in terms of physicochemical, pasting and thermal characteristics. The diameter of the analyzed starch particles depicted a considerable range varying from 5.139 µm to 8.453 µm as their median particle size visualized at ×50. The color values of the analyzed starch samples indicated a high degree of whiteness and purity. The compact nature of starch granules in Kaw quder and Kaw kareed rice cultivars accounted for their higher transition temperatures as compared to other cultivars. Crystalline pattern by X-Ray diffractometer showed an A-type pattern for analyzing starches. The starch granules showed the irregular polyhedral morphology with spherical granules having a polyhedral angular shaped morphology packed tightly with a relatively smooth surface.

Characterization of the surface morphology of durum wheat starch granules using atomic force microscopy.

Knowledge of the structure and properties of microscopic surfaces of durum wheat starch granules is essential for understanding the functional and physico-chemical properties. The nanoscale surface undulations on the starch granules inside durum wheat macroscopically influence the milling properties. The objective of this study was to visualize the surface morphology and the size of starch grains of vitreous and nonvitreous durum wheat kernels using atomic force microscopy. The distribution of starch granules in the vitreous and nonvitreous durum wheat starch samples were examined and compared. The results of our study confirm the 'blocklet' model of the ultrastructure of the starch granule surface. Image contrast enhancement using UV/ozone treatment of microtomed starch samples improved the imaging of growth rings on the starch samples. The observation of growth rings in the nonvitreous starch granule surfaces indicates that amylopectin is more common than amylose in nonvitreous starch when compared with vitreous starch.

Influence of amylose content and oxidation level of potato starch on acetylation, granule structure and radicals' formation.

This study was aimed at determining the effect of the amylose content of starch and oxidation level of potato starch on the structure of starch granules, and susceptibility to chemical modification (acetylation) and subsequent generation of radicals. Potato starch and waxy potato starch were oxidised with sodium hypochlorite applied in doses corresponding to 10, 20, and 30gCl/kg starch, and then acetylated with acetic acid anhydride. The carboxyl, carbonyl, acetyl groups were determined in modified starches. Structural properties of starch granules were evaluated based on gelatinisation, crystallinity, specific surface, intrinsic viscosity, and microphotographs by SEM microscope. The electron paramagnetic resonance (EPR) measurements were carried out to establish starch susceptibility to radical creation upon chemical modification and UV radiation. The amount of formed radicals was treated as a measure of the starch structure stability. The higher amount of amylose and the highest level of oxidation led to strong starch structure destruction and consequently facilitated radical generation. Study results showed also that amylose content as well as the degree of starch oxidation modified consecutive acetylation process. The different effectiveness of the acetylation processes influenced the morphology and structure of starch granules.

Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose.

The interaction of the two binding sites of the starch-binding domain (SBD) of Aspergillus niger glucoamylase 1 (GA-I) with substrate has been investigated by using atomic force microscopy (AFM) and UV difference spectroscopy in combination with site-specific mutants of both SBD and GA-I. The SBD possesses two binding sites with distinct affinities towards the soluble linear substrate maltoheptaose; dissociation constants (K(d)) of 17 and 0.95 microM were obtained for W563 K (binding site 2 mutant) and W590 K (binding site 1 mutant), respectively, compared to an apparent K(d) of 23 microM for the wild-type SBD. Further, the two sites are almost but not totally independent of each other for binding, since abolishing one site does not prevent the amylose chain binding to the other site. Using AFM, we show that the amylose chains undergo a conformational change to form loops upon binding to the SBD, using either the recombinant wild-type SBD or a catalytically inactive mutant of GA-I. This characteristic conformation of amylose is lost when one of the SBD binding sites is eliminated by site-directed mutagenesis, as seen with the mutants W563 K or W590 K. Therefore, although each binding site is capable of simple binding to a ligand, both sites must be functional in order to induce a gross conformational change of the amylose molecules. Taken together these data suggest that for the complex with soluble amylose, SBD binds to a single amylose chain, site 1 being responsible for the initial recognition of the chain and site 2 being involved in tighter binding, leading to the circularisation of the amylose chain observed by AFM. Binding of the SBD to the amylose chain results in a novel two-turn helical amylose complex structure. The binding of parallel amylosic chains to the SBD may provide a basis for understanding the role of the SBD in facilitating enzymatic degradation of crystalline starches by glucoamylase 1.

Imaging of carrageenan macrocycles and amylose using noncontact atomic force microscopy.

Samples of kappa-carrageenan, iota-carrageenan, and synthetic amylose have been examined by atomic force microscopy (AFM). All samples were spray deposited from aqueous solutions onto freshly cleaved mica, air dried, and imaged in air using noncontact atomic force microscopy (NCAFM). Images of single stranded amylose and carrageenan are presented. At relatively low polymer concentrations in the presence of NaCl iota-carrageenan formed circles that appear to be predominantly head-to-tail associated unimeric duplex (double stranded) structures. At higher iota-carrageenan concentrations the polymer forms circles and aggregates that appear to involve dimeric duplex structure. Direct comparison of synthetic amylose molecular weights determined from NCAFM images with results from solution measurements showed that NCAFM provides an excellent way to measure amylose molecular weight and molecular weight distribution. It is shown that synthetic amylose is single stranded in aqueous solution and that the chain length distribution is broader than the Poisson distribution anticipated from polymerization theory.

Two-dimensional macromolecular distributions reveal detailed architectural features in high-amylose starches.

Two-dimensional (2D) structural distributions based on macromolecular size and branch chain-length are obtained for three maize starches with different amylose contents (one normal and two high-amylose varieties). Data were obtained using an analytical methodology combining chemical fractionation, enzymatic debranching, and offline 2D size-exclusion chromatography with multiple detection. The 2D distributions reveal novel features in the branching structure of high-amylose maize starches. Normal maize starch shows well-resolved structural topologies, corresponding to the amylopectin and amylose macromolecular populations. However, high-amylose maize starches exhibit very complex topologies with significant features between those of amylose and amylopectin, showing the presence of distinct intermediate components. These have the macromolecular size of amylose but similar branching structure to amylopectin, except for a higher proportion of longer branches. These structural features of the intermediate components can be related to a less tightly controlled biosynthesis of the branching structures in high-amylose maize starch mutants, which may prevent these molecules from maturing into full-size amylopectin. This altered macromolecular branched architecture of high-amylose starches probably contribute to their better nutritional properties.

Solvent-responsive behavior of inclusion complexes between amylose and polytetrahydrofuran.

Highly crystalline amylose-polytetrahydrofuran (PTHF) complexes can be obtained by employing organic solvents as washing agents after complex formation. The X-ray diffraction (XRD) of the washed complexes appear sharp at 12.9°-13.2° and 19.6°-20.1°, clear signs of the presence of V6I -amylose. Other diffraction peaks correlate with V6II -amylose, which indicates that the complexed amylose helices are in the form of an intermediate or a mixture of V6I - and V6II -amylose. SEM imaging reveals that the amylose-PTHF complexes crystallize in the form of lamellae, which aggregate in a round shape on top of one another with a diameter around 4-8 µm. Some lamellas aggregate as flower-like or flat-surface spherulitic crystals. There is a visible matrix in between the aggregated lamellas which shows that a part of the amylose-PTHF complexes is amorphous.

Interaction between amylose and tea polyphenols modulates the postprandial glycemic response to high-amylose maize starch.

High-amylose maize starch (HAM) is a common source material to make resistant starch with its high content of amylose (>70%). In the current investigation, the self-assembly of amylose in the presence of bioactive tea polyphenols (TPLs) and resulting slow digestion property of starch were explored. The experimental results using a mouse model showed a slow digestion property can be achieved with an extended and moderate glycemic response to HAM starch cocooked with TPLs. Further studies using a dilute aqueous amylose solution (0.1%, w/v) revealed an increased hydrodynamic radius of amylose molecules, indicating that TPLs could bridge them together, leading to increased molecular sizes. On the other hand, the bound TPLs interrupted the normal process of amylose recrystallizaiton evidenced by a decreased viscosity and storage modulus (G') of HAM (5%) gel, a rough surface of the cross-section of HAM film, and decreased short-range orders examined by Fourier transform infrared spectral analysis. Single-step degradation curves in the thermal gravimetric profile demonstrated the existence of a self-assembled amylose-TPL complex, which is mainly formed through hydrogen bonding interaction according to the results of iodine binding and X-ray powder diffraction analysis. Collectively, the amylose-TPL complexation influences the normal self-assembling process of amylose, leading to a low-ordered crystalline structure, which is the basis for TPLs' function in modulating the digestion property of HAM starch to produce a slowly digestible starch material that is beneficial to postprandial glycemic control and related health effects.

Accumulation of multiple-repeat starch-binding domains (SBD2-SBD5) does not reduce amylose content of potato starch granules.

This study investigates whether it is possible to produce an amylose-free potato starch by displacing the amylose enzyme, granule-bound starch synthase I (GBSSI), from the starch granule by engineered, high-affinity, multiple-repeat family 20 starch-binding domains (SBD2, SBD3, SBD4, and SBD5). The constructs were introduced in the amylose-containing potato cultivar (cv. Kardal), and the starches of the resulting transformants were compared with those of SBD2-expressing amylose-free (amf) potato clones. It is shown that a correctly sized protein accumulated in the starch granules of the various transformants. The amount of SBD accumulated in starch increased progressively from SBD to SBD3; however, it seemed as if less SBD4 and SBD5 was accumulated. A reduction in amylose content was not achieved in any of the transformants. However, it is shown that SBDn expression can affect physical processes underlying granule assembly, in both genetic potato backgrounds, without altering the primary structure of the constituent starch polymers and the granule melting temperature. Granule size distribution of the starches obtained from transgenic Kardal plants were similar to those from untransformed controls, irrespective of the amount of SBDn accumulated. In the amf background, granule size is severely affected. In both the Kardal and amf background, apparently normal oval-shaped starch granules were composed of multiple smaller ones, as evidenced from the many "Maltese crosses" within these granules. The results are discussed in terms of different binding modes of SBD.

Corn starches as film formers in aqueous-based film coating.

The purpose of this study was to evaluate the film formation ability and mechanical stress-strain properties of aqueous native corn starches, using free films and film coatings applied to tablets. Free films were prepared from high-amylose corn (Hylon VII), corn and waxy corn starches, using sorbitol and glycerol as plasticizers. The tablets and pellets were film-coated using an air-suspension coater, and characterized with respect to the film coating surface topography, cross-sectional structure and thickness (SEM), and dissolution in vitro. The amylose content of the starch film formers affected both the tensile strength and the elongation. The elongations were under 5% for even the plasticized starches, and in most cases, no plasticization effect was seen by either of the plasticizers. Dissolution of native corn starch film-coated tablets (weight gain 1%) did not differ from uncoated ones. A notable delay in dissolution of the drug was found by increasing Hylon VII film coating thickness, suggesting controlled-release characteristics.