On the architecture of starch granules revealed by iodine vapor binding and lintnerization. Part 1: Microscopic examinations.

Structural nature of glucan chains in the amorphous part of granular starch was examined by iodine vapor treatment and lintnerization. Four iodine-stained amylose-containing normal starches and their waxy counterparts were examined under a microscope before, during, and after lintnerization. The presence of amylose retarded the lintnerization rate. The degree of retardation correlated with the structural type of the amylopectin component, suggesting that potato amylopectin (type 4 structure) interacts with amylose in the granules, whereas in barley granules (type 1 structure) the interaction is very weak. The inclusion complexes with iodine were not degraded by the acid treatment. Therefore, the iodine-glucan chain complex formation could be used to study the structural nature of the flexible, amorphous parts of the starch granules. Indeed, at the end of lintnerization, when 20%-30% of the granules remained, substantial amounts of blue-stained complexes were washed out from the granules especially from amylose-containing barley and maize starch, but also from both normal and waxy cassava and potato starch. The complexation with iodine did not affect the rate of lintnerization. This suggested that single helical structures were present during lintnerization also in the absence of iodine and this conformation was the reason for the acid resistance.

Temperature of plasma-activated water and its effect on the thermal and chemical surface properties of cereal and tuber starches.

High amylose and waxy starches from maize and potato were incubated with plasma-activated water (PAW) at 25 °C, 60 °C, and 80 °C temperatures to investigate PAW treatment effects on the starches' properties. At 60 °C incubation temperature, the starches were basically annealed with PAW. Annealing starches with PAW significantly increased (p < 0.05) the gelatinization parameters except for the enthalpy of gelatinization of waxy potato starch. Furthermore, starch swelling power significantly decreased while the water absorption capacity and solubility increased significantly when incubated at 80 °C. X-ray photoelectron spectroscopy (XPS) analysis showed the oxidation of C-C/C-H and C-O into carboxyl groups in waxy and high amylose maize starches incubated with PAW at 60 °C and 80 °C, respectively. In addition, cross-linking was observed in waxy maize and high amylose potato incubated with PAW at 80 °C and 25 °C, respectively. Overall, the results indicated PAW temperature is an important factor in modifying cereals and tuber starches with PAW.

Different genetic strategies to generate high amylose starch mutants by engineering the starch biosynthetic pathways.

This review systematically documents the major different strategies of generating high-amylose (HAS) starch mutants aiming at providing high resistant starch, by engineering the starch biosynthesis metabolic pathways. We identify three main strategies based on a new representation of the starch structure: 'the building block backbone model': i) suppression of starch synthases for reduction of amylopectin (AP) side-chains; ii) suppression of starch branching enzymes (SBEs) for production of AM-like materials; and iii) suppression of debranching enzymes to restrain the transformation from over-branched pre-AP to more ordered AP. From a biosynthetic perspective, AM generated through the second strategy can be classified into two types: i) normal AM synthesized mainly by regular expression of granule-bound starch synthases, and ii) modified linear AP chains (AM-like material) synthesized by starch synthases due to the suppression of starch branching enzymes. The application of new breeding technologies, especially CRISPR, in the breeding of HAS crops is also reviewed.

Structural characterization and enzymatic hydrolysis of radio frequency cold plasma treated starches.

The effect of carbon dioxide-argon radio frequency cold plasma treatment on the in vitro digestion and structural characteristics of granular and non-granular waxy maize, potato, and rice starches was investigated in this study. The effect on the fine structure of waxy potato was very minimal after plasma treatment irrespective of their granular or non-granular form. The short chain length (SCL) of waxy maize and rice (granular and non-granular) starches was reduced leading to subsequent increases in the long chain length (LCL). In vitro digestibility studies showed that cold plasma treatment enhanced (p < 0.05) the amount of slowly digestible starches (5.62%; 10.24%) and resistant starches (0.28%; 85.66%) in non-granular waxy maize (WMS NG) and granular waxy potato starches (WPS G), respectively. The amount of rapidly digestible starches increased in granular waxy maize starch (WMS G) (85.08%) but was unaffected in non-granular waxy rice (WRS NG), WPS G, and non-granular waxy potato starches after plasma treatment. FTIR-ATR data confirmed the ability of cold plasma to induce cross-linking in waxy starches specifically in WMS NG, WRS G, WRS NG, and WPS G. Overall, the unit and internal chain structure of the waxy starches were mostly unaffected by radio frequency plasma treatment. Cross-linking served as the dominant mechanism by which plasma altered the structure and digestibility of these starches. PRACTICAL APPLICATION: Cold plasma technology has been suggested as a green technique for starch modification. More research is, however, needed to facilitate the industrial scale up of this technology. In this study, we utilized a carbon dioxide-argon radio frequency cold plasma to modify waxy maize, rice and potato starches. Cold plasma treatment resulted in starches that were resistant to digestion and were highly cross-linked. The cross-linking would give the starches the ability to possibly withstand the high temperatures and shear that can be applied during industrial processing.

Relationship between molecular structure and lamellar and crystalline structure of rice starch.

The relationship between molecular structure and crystalline and lamellar structures of fifteen types of rice starches was studied. GPC and HPAEC were used for the molecular chain analysis and WAXS, SAXS, and CP/MAS 13C NMR were employed for aggregation structural analysis. The amylopectin content and the average lengths of fb1-chains (the degree of polymerization (DP) 13-24) were positively correlated with the amount of double helices (r2 = 0.92 and 0.57, respectively). In contrast, amylose content was positively correlated with the amounts of amorphous materials in starch (r2 = 0.77). The amount of double helices, which constitute a major part of the crystalline matrix, was positively correlated with the lamellar ordering (r2 = 0.81), and negatively correlated with the thickness of crystalline lamellae (r2 = 0.90) and lamellar repeat distance (r2 = 0.84). Conversely, the amount of the amorphous matrix was correlated with these parameters in the opposite way (r2 = 0.50, 0.75, and 0.75, respectively).

A Review of Starch Biosynthesis in Relation to the Building Block-Backbone Model.

Starch is a water-insoluble polymer of glucose synthesized as discrete granules inside the stroma of plastids in plant cells. Starch reserves provide a source of carbohydrate for immediate growth and development, and act as long term carbon stores in endosperms and seed tissues for growth of the next generation, making starch of huge agricultural importance. The starch granule has a highly complex hierarchical structure arising from the combined actions of a large array of enzymes as well as physicochemical self-assembly mechanisms. Understanding the precise nature of granule architecture, and how both biological and abiotic factors determine this structure is of both fundamental and practical importance. This review outlines current knowledge of granule architecture and the starch biosynthesis pathway in relation to the building block-backbone model of starch structure. We highlight the gaps in our knowledge in relation to our understanding of the structure and synthesis of starch, and argue that the building block-backbone model takes accurate account of both structural and biochemical data.

On the role of the internal chain length distribution of amylopectins during retrogradation: Double helix lateral aggregation and slow digestibility.

A structure-digestion model is proposed to explain the formation of α-amylase-slowly digestible structures during amylopectin retrogradation. Maize and potato (normal and waxy) and banana starch (normal and purified amylopectin through alcohol precipitation), were analyzed for amylose ratio and size (HPSEC) and amylopectin unit- and internal-chain length distribution (HPAEC). Banana amylopectin (BA), like waxy potato (WP), exhibited a larger number of B3-chains, fewer BS- and Bfp-chains and lower S:L and BS:BL ratios than maize, categorizing BA structurally as type-4. WP exhibited a significantly greater tendency to form double helices (DSC and 13C-NMR) than BA, which was attributed to its higher internal chain length (ICL) and fewer DP6-12-chains. However, retrograded BA was remarkably more resistant to digestion than WP. Lower number of phosphorylated B-chains, more S- and Bfp-chains and shorter ICL, were suggested to result in α-amylase-slowly digestible structures through further lateral packing of double helices (suggested by thermo-rheology) in type-4 amylopectins.

Modification of cereal and tuber waxy starches with radio frequency cold plasma and its effects on waxy starch properties.

The use of carbon dioxide-argon gas radio frequency cold plasma in modifying waxy rice, maize and potato was explored in this paper. Treatment with plasma at 120 W or 0 W (carbon dioxide-argon gas mixture only) resulted in significant increases in the enthalpy of gelatinization of all three waxy starches. Treatment with plasma or gas resulted in a significant increase in the resistant starch content of maize and potato with rice increasing only after gas treatment. Significant decreases were observed in the setback and final viscosities after 120 W treatment in all starches. Plasma and gas treatment resulted in a 5.5% and 2.8% decrease in crystallinity of potato but not rice and maize starch. NMR results showed the presence of V-type single helices in mostly maize and rice starches. Carbon dioxide-argon radio frequency cold plasma served as a useful tool in modifying the properties of all three waxy starches.

Effect of diurnal photosynthetic activity on the fine structure of amylopectin from normal and waxy barley starch.

The impact of diurnal photosynthetic activity on the fine structure of the amylopectin fraction of starch synthesized by normal barley (NBS) and waxy barley (WBS), the latter completely devoid of amylose biosynthesis, was determined following the cultivation under normal diurnal or constant light growing conditions. The amylopectin fine structures were analysed by characterizing its unit chain length profiles after enzymatic debranching as well as its φ,ß-limit dextrins and its clusters and building blocks after their partial and complete hydrolysis with α-amylase from Bacillus amyloliquefaciens, respectively. Regardless of lighting conditions, no structural effects were found when comparing both the amylopectin side-chain distribution and the internal chain fragments of these amylopectins. However, the diurnally grown NBS and WBS both showed larger amylopectin clusters and these had lower branching density and longer average chain lengths than clusters derived from plants grown under constant light conditions. Amylopectin clusters from diurnally grown plants also consisted of a greater number of building blocks, and shorter inter-block chain lengths compared to clusters derived from plants grown under constant light. Our data demonstrate that the diurnal light regime influences the fine structure of the amylopectin component both in amylose and non-amylose starch granules.

Influence of diurnal photosynthetic activity on the morphology, structure, and thermal properties of normal and waxy barley starch.

This study investigated the influence of diurnal photosynthetic activity on the morphology, molecular composition, crystallinity, and gelatinization properties of normal barley starch (NBS) and waxy barley starch (WBS) granules from plants cultivated in a greenhouse under normal diurnal (16h light) or constant light photosynthetic conditions. Growth rings were observed in all starch samples regardless of lighting conditions. The size distribution of whole and debranched WBS analyzed by gel-permeation chromatography did not appear to be influenced by the different lighting regimes, however, a greater relative crystallinity measured by wide-angle X-ray scattering and greater crystalline quality as judged by differential scanning calorimetry was observed under the diurnal lighting regime. NBS cultivated under the diurnal photosynthetic lighting regime displayed lower amylose content (18.7%), and shorter amylose chains than its counterpart grown under constant light. Although the relative crystallinity of NBS was not influenced by lighting conditions, lower onset, peak, and completion gelatinization temperatures were observed in diurnally grown NBS compared to constant light conditions. It is concluded that normal barley starch is less influenced by the diurnal photosynthetic lighting regime than amylose-free barley starch suggesting a role of amylose to prevent structural disorder and increase starch granule robustness against environmental cues.

Morphological, Thermal, and Rheological Properties of Starches from Maize Mutants Deficient in Starch Synthase III.

Morphological, thermal, and rheological properties of starches from maize mutants deficient in starch synthase III (SSIII) with a common genetic background (W64A) were studied and compared with the wild type. SSIII deficiency reduced granule size of the starches from 16.7 to ∼11 µm (volume-weighted mean). Thermal analysis showed that SSIII deficiency decreased the enthalpy change of starch during gelatinization. Steady shear analysis showed that SSIII deficiency decreased the consistency coefficient and yield stress during steady shearing, whereas additional deficiency in granule-bound starch synthase (GBSS) increased these values. Dynamic oscillatory analysis showed that SSIII deficiency decreased G' at 90 °C during heating and increased it when the paste was cooled to 25 °C at 40 Hz during a frequency sweep. Additional GBSS deficiency further decreased the G'. Structural and compositional bases responsible for these changes in physical properties of the starches are discussed. This study highlighted the relationship between SSIII and some physicochemical properties of maize starch.

On the molecular structure of the amylopectin fraction isolated from "high-amylose" ae maize starches.

The amylopectin fractions from starch of a series of amylose-extender (ae) maize samples (HYLON(®) V, VII and VIII starches) were isolated and analysed for their molecular composition and structure. The fractions from all samples contained both a high and a low molecular weight fraction (HMF and LMF), of which LMF increased with the amylose content of the starch and appeared to have substantially more of long chains than HMF. A normal amylose-containing maize starch (NMS), which served as a reference sample, contained very little LMF, which suggested that LMF was the inherent result of the effect of the loss of starch branching enzyme IIb activity in the ae mutants. Clusters were isolated from the amylopectin fractions using Bacillus amyloliquefaciens α-amylase, which effectively hydrolyses long internal chain segments between clusters. During the hydrolysis process, clearly more of small dextrins were released from the ae starches in comparison to NMS. It appeared that some of these small dextrins did not precipitate in methanol together with the majority of the clusters. Nevertheless, isolated clusters from the HYLON starch samples were smaller than in NMS and the clusters possessed a lower density of branches with longer chains. The composition of small, branched building blocks was also clearly different: HYLON starch samples possessed much more of single-branched blocks and less multiple-branched blocks than NMS.

Structure of clusters and building blocks in amylopectin from African rice accessions.

Enzymatic hydrolysis in combination with gel-permeation and anion-exchange chromatography techniques were employed to characterise the composition of clusters and building blocks of amylopectin from two African rice (Oryza glaberrima) accessions-IRGC 103759 and TOG 12440. The samples were compared with one Asian rice (Oryza sativa) sample (cv WITA 4) and one O. sativa×O. glaberrima cross (NERICA 4). The average DP of clusters from the African rice accessions (ARAs) was marginally larger (DP=83) than in WITA 4 (DP=81). However, regarding average number of chains, clusters from the ARAs represented both the smallest and largest clusters. Overall, the result suggested that the structure of clusters in TOG 12440 was dense with short chains and high degree of branching, whereas the situation was the opposite in NERICA 4. IRGC 103759 and WITA 4 possessed clusters with intermediate characteristics. The commonest type of building blocks in all samples was group 2 (single branched dextrins) representing 40.3-49.4% of the blocks, while groups 3-6 were found in successively lower numbers. The average number of building blocks in the clusters was significantly larger in NERICA 4 (5.8) and WITA 4 (5.7) than in IRGC 103759 and TOG 12440 (5.1 and 5.3, respectively).

Impact of full range of amylose contents on the architecture of starch granules.

The effects of amylose deposition on crystalline regions of barley starch granules were studied in granules containing zero to 99.1% amylose using "waxy" (WBS, 0% amylose), normal (NBS, 18% amylose) and amylose-only barley lines (AOS, 99.1% amylose). The effects were probed after hydrolysis of amorphous regions of starch granules in dilute HCl generating lintners, which typically represent the crystalline lamella of starch granules. Compared to NBS and WBS, AOS granules exhibited an irregular, multilobular morphology with a rough surface texture. AOS displayed lower rates of acid hydrolysis than WBS, and AOS reached a plateau at ∼45wt% acid hydrolysis. High-performance anion-exchange chromatography of lintners at equivalent levels of hydrolysis (45wt%) revealed the average degree of polymerization (DP) of AOS lintners was 21, substantially smaller than that of NBS and WBS (DP 42). AOS lintners contained the lowest number of chains (NC) per molecule (1.1) compared to NBS (2.8) and WBS (3.3) and the average chain length of AOS, NBS and WBS lintners was 19, 15 and 13, respectively. Hence, both NC and the average chain length correlated with amylose content. The size distribution profile of AOS lintners revealed a repeat motif in the molecules corresponding to 5-6 glucose residues.

Small differences in amylopectin fine structure may explain large functional differences of starch.

Four amylose-free waxy rice starches were found to give rise to gels with clearly different morphology after storage for seven days at 4°C. The thermal and rheological properties of these gels were also different. This was remarkable in light of the subtle differences in the molecular structure of the amylopectin in the samples. Addition of iodine to the amylopectin samples suggested that not only external chains, but also the internal chains of amylopectin, could form helical inclusion complexes. It is suggested that these internal helical segments participate in the retrogradation of amylopectin, thereby stabilising the gels through double helical structures with external chains of adjacent molecules. Albeit few in number, such interactions appear to have important influences on starch functional properties.

Unit and internal chain profile of African rice (Oryza glaberrima) amylopectin.

High-performance anion-exchange chromatography was used to study the unit chain profiles of amylopectins and their φ,ß-limit dextrins from two African rice (Oryza glaberrima) accessions-TOG 12440 and IRGC 103759. The samples were compared with two Asian rice (Oryza sativa) samples (cv Koshihikari and cv WITA 4) and one O. sativa × O. glaberrima cross (NERICA 4). The ratio of short:long chains ranged between 12.1 and 13.8, and the ratio of A:B-chains was ∼ 1.0 in all samples. A significant difference was observed in the distribution of internal chains with regards to the proportion of short "fingerprint" B-chains (Bfp-chains), which in the φ,ß-limit dextrins have a degree of polymerization (DP) 3-7. The African rice starches and NERICA 4 had higher levels of Bfp-chains, but the major group of short B-chains (DP 8-25) was similar to that of the Asian rice samples. The average chain length (CL), internal chain length (ICL), and total internal chain length (TICL) were similar in all samples. However, the external chain length (ECL) was longer in the African rice samples and NERICA 4. ECL correlated positively and significantly (p<0.05) with gelatinization transition temperatures and enthalpy suggesting differences between the two rice types in cooking properties.

Starch structure in developing barley endosperm.

Barley spikes of the cultivars/breeding lines Gustav, Karmosé and SLU 7 were harvested at 9, 12 and 24 days after flowering in order to study starch structure in developing barley endosperm. Kernel dry weight, starch content and amylose content increased during development. Structural analysis was performed on whole starch and included the chain-length distribution of the whole starches and their ß-limit dextrins. Karmosé, possessing the amo1 mutation, had higher amylose content and a lower proportion of long chains (DP ≥38) in the amylopectin component than SLU 7 and Gustav. Structural differences during endosperm development were seen as a decrease in molar proportion of chains of DP 22-37 in whole starch. In ß-limit dextrins, the proportion of Bfp-chains (DP 4-7) increased and the proportion of BSmajor-chains (DP 15-27) decreased during development, suggesting more frequent activity of starch branching enzymes at later stages of maturation, resulting in amylopectin with denser structure.

Thermal properties of barley starch and its relation to starch characteristics.

Amylopectin fine structure and starch gelatinization and retrogradation were studied in 10 different barley cultivars/breeding lines. Clusters and building blocks were isolated from the amylopectin by α-amylase from Bacillus amyloliquefaciens and their structure was characterized. Gelatinization was studied at a starch:water ratio of 1:3, and retrogradation was studied on gelatinized starch at starch:water ratio of 1:2, by differential scanning calorimetry. Three barley cultivars/breeding lines possessed the amo1 mutation, and they all had a lower molar proportion of chains of DP ≥38 and more of large building blocks. The amo1 mutation also resulted in a higher gelatinization temperature and a broader temperature interval during gelatinization. Overall, small clusters with a dense structure resulted in a higher gelatinization temperature while retrogradation was promoted by short chains in the amylopectin and many large building blocks.

Branching patterns in leaf starches from Arabidopsis mutants deficient in diverse starch synthases.

This is the first report on the cluster structure of transitory starch from Arabidopsis leaves. In addition to wild type, the molecular structures of leaf starch from mutants deficient in starch synthases (SS) including single enzyme mutants ss1-, ss2-, or ss3-, and also double mutants ss1-ss2- and ss1-ss3- were characterized. The mutations resulted in increased amylose content. Clusters from whole starch were isolated by partial hydrolysis using α-amylase of Bacillus amyloliquefaciens. The clusters were then further hydrolyzed with concentrated α-amylase of B. amyloliquefaciens to produce building blocks (α-limit dextrins). Structures of the clusters and their building blocks were characterized by chromatography of samples before and after debranching treatment. While the mutations increased the size of clusters, the reasons were different as reflected by the composition of their unit chains and building blocks. In general, all mutants contained more of a-chains that preferentially increased the number of small building blocks with only two chains. The clusters of the double mutant ss1-ss3- were very large and possessed also more of large building blocks with four or more chains. The results from transitory starch are compared with those from agriculturally important crops in the context that to what extent the Arabidopsis can be a true biotechnological reflection for starch modifications through genetic means.

Structure of Arabidopsis leaf starch is markedly altered following nocturnal degradation.

Little is known about the thermal properties and internal molecular structure of transitory starch. In this study, granule morphology, thermal properties, and the cluster structure of Arabidopsis leaf starch at beginning and end of the light period were explored. The structural properties of building blocks and clusters were evaluated by using diverse chromatographic techniques. On the granular level, starch from end of day had larger granule size, thinner crystalline lamellae thickness, lower free surface energy of crystals, and lower tendency to retrograde than that from end of night. On the molecular level, the starch had lower amylose content, larger cluster size, and higher number of blocks per cluster at the end of day than at end of night. It is concluded that the core of the granules contains a more permanent molecular and less-ordered physical structure different from the transitory layers laid down around the core at daytime.