Starch phosphorylation regulates starch granule morphological homogeneity in Arabidopsis thaliana.

Starch granule morphological homogeneity presents a gap in starch research. Transitory starch granules in wild-type plants are discoid, regardless of species. Notably, while the shape of starch granules can differ among mutants, it typically remains homogeneous within a genotype. We found an Arabidopsis thaliana mutant, dpe2sex4, lacking both the cytosolic disproportionating enzyme 2 (DPE2) and glucan phosphatase SEX4, showing an unprecedented bimodal starch granule diameter distribution when grown under a light/dark rhythm. dpe2sex4 contained 2 types of starch granules: large granules and small granules. In contrast to the double starch initiation in wheat (Triticum aestivum) endosperm, where A-type granules are initiated first and B-type granules are initiated later, dpe2sex4 small and large granules developed simultaneously in the same chloroplast. Compared with the large granules, the small granules had more branched amylopectin and less surface starch-phosphate, thus having a more compact structure that may hinder starch synthesis. During plant aging, the small granules barely grew. In in vitro experiments, fewer glucosyl residues were incorporated in small granules. Under continuous light, dpe2sex4 starch granules were morphologically homogeneous. Omitting the dark phase after a 2-wk light/dark cycle by moving plants into continuous light also reduced morphological variance between these 2 types of granules. These data shed light on the impact of starch phosphorylation on starch granule morphology homogeneity.

Beneficial glycaemic effects of high-amylose barley bread compared to wheat bread in type 2 diabetes.

BACKGROUND: Cereals foods with a high content of dietary fibres or amylose have potential to lower postprandial glucose levels. Optimisation of cereal foods may improve management of type 2 diabetes (T2D). METHODS: We investigated the impact on 4 h postprandial glucose responses given as incremental area under curve (iAUC) of bread made of either 50% RNAi-based (genetically modified) amylose-only barley flour (AmOn) (and 50% wheat flour), 50% hulless barley flour (and 50% wheat flour) or 75% hulless barley (and 25% wheat flour) in subjects with T2D compared with 100% wheat flour bread. DESIGN: Twenty adults with T2D were randomly allocated to one of four breads at four separate visits. We measured fasting and 4 h postprandial responses of glucose, insulin, glucagon, triacylglycerol (TG), free fatty acids (FFA), glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP). Mixed model ANOVA was used to examine the differences. RESULTS: Bread made from 50% AmOn lowered the 4 h postprandial glucose by 34%, 27%, 23% (P < 0.05) compared with 100% wheat, 50% or 75% hulless barley, respectively. Bread made from 75% hulless barley reduced the postprandial glucose response (iAUC) by 11% (P < 0.05) compared to 100% wheat bread. Postprandial insulin responses (iAUC) were reduced for 50% AmOn compared with 100% wheat and 50% hulless barley and for 75% hulless compared to 50% hulless barley bread (P < 0.05). 4 h postprandial glucagon (tAUC) did not differ between the four bread types (P > 0.05). Lower postprandial GIP (iAUC) was observed after all barley breads compared to 100% wheat (P < 0.05), whereas no difference was seen in postprandial GLP-1. Postprandial TG and FFA (tAUC) were difficult to judge due to differences in fasting values. CONCLUSIONS: Bread made by replacing wheat flour with either 50% high-amylose or 75% hulless barley flour lowered postprandial glucose responses compared to 100% wheat bread indicating a beneficial impact on glucose regulation in T2D subjects. This trial was registered at clinicaltrials.gov as NCT04646746.

High-pressure pasting performance and multilevel structures of short-term microwave-treated high-amylose maize starch.

Microwave treatment is an environmentally friendly method for modification of high-amylose maize starch (HAMS). Here, the effects of short-time (≤120 s) microwave treatment on the structure and pasting of two types of HAMSs, Gelose 50 (HAMSI) and Gelose 80 (HAMSII), with apparent amylose content (AAC) of 45 % and 58 %, respectively, was studied using a multiscale approach including X-ray scattering, surface structures, particle size distribution, molecular size distributions and high temperature/pressure Rapid Visco Analysis (RVA)-4800 pasting. As compared to starch with no amylose (waxy maize starch, WMS) and 25 % amylose content (normal maize starch, NMS), HAMSI underwent similar structural and pasting changes as WMS and NMS upon microwave treatment, and it might primarily be attributed to the amylopectin fraction that was affected by cleavage of the connector chains between double helices and backbone chains, which decreased the crystallinity and thickness of the crystalline lamellae. However, the multi-scale structure of HAMSII was almost unaffected by this treatment. The pasting properties of fully gelatinized HAMSI starch showed a decrease in RVA-4800 peak and final viscosities after microwave treatment. In contrast, for HAMSII starch, the microwave treatment led to an increase in these viscosities. The combined results highlight the influence of varying AAC on the effects of microwave-mediated modification, leading to diverse alterations in the structure and functionality of starches.

Strategies for starch customization: Agricultural modification.

Raw starch is commonly modified to enhance its functionality for industrial applications. There is increasing demand for 'green' modified starches from both end-consumers and producers. It is well known that environmental conditions are key factors that determine plant growth and yield. An increasing number of studies suggest growth conditions can expand affect starch structure and functionality. In this review, we summarized how water, heat, high nitrogen, salinity, shading, CO2 stress affect starch biosynthesis and physicochemical properties. We define these treatments as a fifth type of starch modification method - agricultural modification - in addition to chemical, physical, enzymatic and genetic methods. In general, water stress decreases peak viscosity and gelatinization enthalpy of starch, and high temperature stress increases starch gelatinization enthalpy and temperature. High nitrogen increases total starch content and regulates starch viscosity. Salinity stress mainly regulates starch and amylose content, both of which are genotype-dependent. Shading stress and CO2 stress can both increase starch granule size, but these have different effects on amylose content and amylopectin structure. Compared with other modification methods, agricultural modification has the advantage of operating at a large scale and a low cost and can help meet the ever-rising market of clean-label foods and ingredients.

Periodic changes in chain lengths distribution parameters of wheat starch during endosperm development.

This study analyzed the molecular structure of developing wheat endosperm starch at different stages after anthesis (DAA) using chain length distribution analysis by size exclusion chromatography (SEC) and fluorophore-assisted carbohydrate electrophoresis. Our results revealed periodic changes in the content of both amylose and amylopectin fractions. Specifically, the content of amylose chains with a degree of polymerization (DP) > 100 significantly decreased from 5 to 10 DAA (28% to 21%) and from 15 to 20 DAA (29% to 26%), but increased between 10 and 15 DAA (21% to 29%) and 20 to 25 DAA (30.0% to 33%). Conversely, the content of short amylopectin chains with DP ≤ 32 showed the opposite trend. Interestingly, mRNA expression levels of key starch biosynthesis genes did not exhibit periodic changes. These findings contribute to our understanding of starch biosynthesis and provide important insights for the development of starch-based products.

Improved Hydrolysis of Granular Starches by a Psychrophilic α-Amylase Starch Binding Domain-Fusion.

Degradation of starch granules by a psychrophilic α-amylase, AHA, from the Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 was facilitated by C-terminal fusion to a starch-binding domain (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3) via a decapeptide linker. Depending on the waxy, normal or high-amylose starch type and the botanical source, the AHA-SBD fusion enzymes showed up to 3 times higher activity than AHA wild-type. The SBD-fusion thus increased the density of enzyme attack-sites and binding-sites on the starch granules by up to 5- and 7-fold, respectively, as measured using an interfacial catalysis approach that combined conventional Michaelis-Menten kinetics, with the substrate in excess, and inverse kinetics, having enzyme in excess, with enzyme-starch granule adsorption isotherms. Higher substrate affinity of the SBDGA compared to SBDGWD3 was accompanied by the superior activity of AHA-SBDGA in agreement with the Sabatier principle of adsorption limited heterogenous catalysis.

Interfacial Catalysis during Amylolytic Degradation of Starch Granules: Current Understanding and Kinetic Approaches.

Enzymatic hydrolysis of starch granules forms the fundamental basis of how nature degrades starch in plant cells, how starch is utilized as an energy resource in foods, and develops efficient, low-cost saccharification of starch, such as bioethanol and sweeteners. However, most investigations on starch hydrolysis have focused on its rates of degradation, either in its gelatinized or soluble state. These systems are inherently more well-defined, and kinetic parameters can be readily derived for different hydrolytic enzymes and starch molecular structures. Conversely, hydrolysis is notably slower for solid substrates, such as starch granules, and the kinetics are more complex. The main problems include that the surface of the substrate is multifaceted, its chemical and physical properties are ill-defined, and it also continuously changes as the hydrolysis proceeds. Hence, methods need to be developed for analyzing such heterogeneous catalytic systems. Most data on starch granule degradation are obtained on a long-term enzyme-action basis from which initial rates cannot be derived. In this review, we discuss these various aspects and future possibilities for developing experimental procedures to describe and understand interfacial enzyme hydrolysis of native starch granules more accurately.

A new insight into the biosynthesis, structure, and functionality of waxy maize starch under drought stress.

BACKGROUND: Drought stress (DS) is the main abiotic stress that maize suffers during its whole growth period, and maize is also sensitive to DS. It had been demonstrated that DS could improve the quality of normal maize starch. However, waxy maize, which has special properties, has not been explored in depth, which limits the breeding and cultivation of waxy maize varieties and the application of waxy maize starch. Therefore, in this study, we investigated the effects of DS on the biosynthesis, structure, and functionality of waxy maize starch. RESULTS: The results showed that DS decreased the expression level of SSIIb, SSIIIa, GBSSIIa, SBEI, SBEIIb, ISAII, and PUL, but increased the expression level of SSI and SBEIIa. DS did not change the average chain length of amylopectin, while increased the relative content of fa chains (RCfa ) and decreased the RCfb1 and RCfb3 . Furthermore, DS decreased the amylose content, amorphous lamellar distance da , semi-crystalline repeat distance, and average particle size, whereas it increased the relative crystallinity, crystalline distance dc , the content of rapidly digested starch in the uncooked system and resistant starch content in both the uncooked and cooked system. CONCLUSIONS: For waxy maize, DS could raise the relative expression level of SSI and SBEIIa, thus increasing RCfa . The larger number of RCfa could create steric hindrance, which can lead to producing more resistant starch in waxy maize starch. © 2023 Society of Chemical Industry.

Starch granular size and multi-scale structure determine population patterns in bivariate flow cytometry sorting.

Bivariate flow cytometry (FC) sorting with forward scatter (FSC) and side scatter (SSC) is a recently established novel technique to separate starch granules. However, the forming mechanism of starch FC-dependent population patterns (i.e. the number of subgroups (NS) and FSC/SSC-dependent distribution patterns) remain partly elusive. For this, the correlation of granular size and multi-scale structure of native starches and FC-dependent population patterns was investigated through employing a wide range of native starches originating from different species involving cereal-, pulse-, and tuber crops. Results showed NS was pertinent with particle size, amylose content (AC), amylopectin chains length distribution, lamellar structure, short-range ordered structure. The distinct NS was determined by impacts of native starch FSC / SSC-dependent distribution patterns. Specifically, starch granular size significantly correlated with both FSC and SSC-dependent distribution patterns. The proportion of chains with DP 6-12 was the intra-molecular decisive factor to influence short-range ordered structure, finally leading to FSC-dependent distribution patterns. By contrast, AC was another intra-molecular index to determine SSC-dependent distribution patterns through affecting lamellar structure and short-range ordered structure.

The effects of different types of high-amylose maize starches on viscosity and digestion of acidified milk gels.

High-amylose maize starch (HAMS) can provide dietary fiber to foods. In this study, we investigated the effects of three HAMSs (Gelose 50, Hylon VII, and NAFU50) on the functionality of casein (CA) and/or whey protein (WP) networks in acidified milk gels using normal maize starch (NMS) as a control thickener. When compared with NMS, HAMSs performed better in increasing the resistant starch content (RS), storage modulus, loss modulus, and complex viscosity of all the milk gels. The results are attributed to the retention of the starch granular integrity of HAMSs during the preparation of the milk gels, as observed by microscopy. HylonVII exhibited the highest RS and viscosity in all milk gel systems, especially in WP gels (71.8 % RS and >3000 Pa.s at 1 Hz viscosity). Our data provide support for the potential of using HAMS to develop healthier yogurt products using functional thickeners from natural sources.

High-amylose starch: Structure, functionality and applications.

Starch with a high amylose (AM) content (high AM starch, HAS) has attracted increasing research attention due to its industrial application potential, such as functional foods and biodegradable packaging. In the past two decades, HAS structure, functionality, and applications have been the research hotspots. However, a review that comprehensively summarizes these areas is lacking, making it difficult for interested readers to keep track of past and recent advances. In this review, we highlight studies that benefited from rapidly developing techniques, and systematically review the structure, functionality, and applications of HAS. We particularly emphasize the relationships between HAS molecular structure and physicochemical properties.

Effects of growth temperature on multi-scale structure of root tuber starch in sweet potato.

Sweet potato was planted at three soil and air temperatures (21, 25 and 28 °C) with the same humidity and light time/intensity. Root tuber starches were isolated, and their multi-scale structures were investigated to reveal the effects of growth temperature on starch properties. Growth temperature did not change the morphology and amylose content of starch, but markedly increased the size of starch from volume-weighted mean diameter 12.2 µm to 17.0 µm. Starch grown at high growth temperature exhibited less A branch-chains and lower branching degree of amylopectin and more B2 and B3+ branch-chains of amylopectin than at low growth temperature. With increasing growth temperature, starch changed from CC-type to CA-type, its relative crystallinity and lamellar peak intensity increased, and the thickness of crystalline and amorphous lamellae did not significantly change. Starch grown at high growth temperature exhibited significantly higher gelatinization temperature than at low growth temperature, but had similar gelatinization enthalpy.

The effects of drought treatments on biosynthesis and structure of maize starches with different amylose content.

We investigated the effects of drought stress (DS) on maize varieties with different amylose content (AC). In starches with AC of 33 %, DS increased the contents of amylopectin (AP) chains with a degree of polymerization (DP) > 36 and decreased the AP chains with DP ≤ 36, while the AC was unchanged. DS decreased the crystallinity, the thickness of both amorphous and crystalline lamellae, and average granular size. In contrast, the digestibility increased. For starches with AC of 45 %, DS increased the content of AP chains with DP > 24 and AC, while the contents of AP chains with DP ≤ 24 decreased. DS produced starch with thinner crystalline lamellae, thicker amorphous lamellae, more elongated and larger granules. The digestibility of the starches decreased. In starches with AC of 53 %, moderate DS led to similar structural and functional changes as found for starches with AC of 45 %. Finally, severe DS resulted in the decrease of AC.

The effects of fermentation of Qu on the digestibility and structure of waxy maize starch.

The fermentation of Qu (FQ) could efficiently produce enzymatically modified starch at a low cost. However, it is poorly understood that how FQ influences the waxy maize starch (WMS) structure and the digestion behavior. In this study, WMS was fermented by Qu at different time and starches were isolated at each time point, and its physico-chemical properties and structural parameters were determined. Results showed that the resistant starch (RS), amylose content (AC), the average particle size [D(4,3)] the ratio of peaks at 1,022/995 cm-1, and the onset temperature of gelatinization (T o ) were increased significantly after 36 h. Conversely, the crystallinity, the values of peak viscosity (PV), breakdown (BD), gelatinization enthalpy (ΔH), and the phase transition temperature range (ΔT) were declined significantly after 36 h. It is noteworthy that smaller starch granules were appeared at 36 h, with wrinkles on the surface, and the particle size distribution was also changed from one sharp peak to bimodal. We suggested that the formation of smaller rearranged starch granules was the main reason for the pronounced increase of RS during the FQ process.

High pressure/temperature pasting and gelling of starch related to multilevel structure-analyzed with RVA 4800.

This paper evaluated the relationship between multi-scale structure and high-pressure gelation properties of nine types of starches with different amylose (AM) content and crystalline polymorphic structure by RVA 4800. Higher average chain lengths of long amylopectin (AP) chains (DP > 36) and AM content, and lower relative content of short AP chains (DP ≤ 36) contributed to the higher peak temperature and peak time at 95-140 °C, and lower peak viscosity, through viscosity, and final viscosity at 95-110 °C. Rheological and texture parameters including storage modulus, loss modulus, hardness, and gumminess, had no significant correlation with starch structural parameters at 95-120 °C, but were instead controlled by AM molecular structure at 130-140 °C. AM content was mainly responsible for the rheological behaviors of starch gels at 130-140 °C, and short and intermediate AM chains were mainly associated with the texture of starch gels at the temperature ranges.

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.

High Amylose-Based Bio Composites: Structures, Functions and Applications.

As biodegradable and eco-friendly bio-resources, polysaccharides from a wide range of sources show steadily increasing interest. The increasing fossil-based production of materials are heavily associated with environmental and climate concerns, these biopolymers are addressing such concerns in important areas such as food and biomedical applications. Among polysaccharides, high amylose starch (HAS) has made major progress to marketable products due to its unique properties and enhanced nutritional values in food applications. While high amylose-maize, wheat, barley and potato are commercially available, HAS variants of other crops have been developed recently and is expected to be commercially available in the near future. This review edifies various forms and processing techniques used to produce HAS-based polymers and composites addressing their favorable properties as compared to normal starch. Low toxic and high compatibility natural plasticizers are of great concern in the processing of HAS. Further emphasis, is also given to some essential film properties such as mechanical and barrier properties for HAS-based materials. The functionality of HAS-based functionality can be improved by using different fillers as well as by modulating the inherent structures of HAS. We also identify specific opportunities for HAS-based food and biomedical fabrications aiming to produce cheaper, better, and more eco-friendly materials. We acknowledge that a multidisciplinary approach is required to achieve further improvement of HAS-based products providing entirely new types of sustainable materials.

Biosynthesis, structure and functionality of starch granules in maize inbred lines with different kernel dehydration rate.

In this study, we report important relationships between kernel starch and kernel dehydration rate for eight maize inbred lines with different dehydration characteristics. High-throughput RNA sequencing data of starch biosynthesis-related genes showed that kernel moisture content and dehydration rate were both associated with differential expression of most starch biosynthetic genes. Especially, kernel moisture content was positively correlated with the increased expression of SBEI and SBEIIb, thereby potentially inducing biosynthesis of amylose with low molecular weight and amylopectin with low content of amylopectin chains with degree of polymerization (DP) 6-12 in inbred lines with fast kernel dehydration rate. We found a negative correlation between short amylopectin chains (DP 6-12) and the starch retrogradation rate. Hence, a low amount of amylopectin chains with DP 6-12 in the inbred lines with fast kernel dehydration rate was a plausible reason for their high short- and long-term retrogradation.

Rice starch multi-level structure and functional relationships.

Starch from 15 different rice genotypes with amylose content (AC) ranging 1.5%-30.6% were investigated for relationships between structures and properties. For parameters related to the granular level, the most important relationships were found for AC, average chain lengths (ACL) of the amylopectin (AP) fb1 chains having a length of DP 13-24, crystallinity, and the thickness of the crystalline (dc) and the amorphous lamellae (da) of the starch granule. AC and dc were negatively correlated with the peak gelatinization temperature (Tp), thermal enthalpy (ΔH), and peak viscosity (PV), but positively correlated with swelling power. ACLfb1 and da, as compared to AC and dc, had the opposite effects on these parameters, demonstrating important roles of specific molecular and lamellar structures on the starch granular stability. For the gelatinized systems, increasing ACLfb1 decreased retrogradation, while AC increased retrogradation by increasing the resistant starch (RS) content, storage modulus (G'), and setback (SB).