Amylopectin Molecules Research Articles

Starch phosphorylation—A needle in a haystack

Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities.

In situ starch and water modification using atmospheric plasma: Structure, functionality and in vitro digestibility

Starch modification has become necessary to develop functional foods. Sustainable technologies like plasma-activated water (PAW) have been combined with hydrothermal treatments; however, the scaling-up process is challenging. Therefore, a simultaneous treatment of starch and water in a plasma reactor and a 24 h soaking of starch-water after the treatment were compared. The effect of the treatments on the structural, functional, and digestibility properties of starches (normal (NS), waxy (WS), and high amylose (HAS)) with different amylose/amylopectin content was assessed. The results show that simultaneous treatment mainly affected the water properties. Nevertheless, the contact time (24 h) after treatment between starch and water promoted the alteration of amylose chains, branching amylopectin molecules and hydrated crystals of HAS, increasing its rapidly (RDS) and slowly (SDS) digestible starch content. Meanwhile, in NS, the contact time favored the high re-engagement of amylose-amylopectin chains inside starch granules, significantly improving the resistant starch (RS) fraction from 31 % to 75 %.

Relationship between the nanometer-scale structures of amylopectin molecules and temperature dependence of internal structures of starch granules in endosperm of starch branching enzyme 2b (be2b) allelic mutant lines from japonica rice

We analyzed the relationship between the nanometer-scale structure of amylopectin and physicochemical properties such as thermal properties of starch granules and higher-order internal structure of starch granules evaluated by using synchrotron radiation X-ray scattering and scanning electron microscopy. In this study, a line of allelic mutants of rice lacking in starch branching enzyme IIb (BEIIb) activity in their endosperm were used as starch sources. The crystal lattice changed from A-type to C- and B-type. The nanoscale cluster size, and higher-order structures of the starch granules varied among mutant lines. The thermal gelatinization properties of the rice-derived starch samples, as well as the dependence of this process on their crystal structure, were evaluated using differential scanning calorimetry and synchrotron X-ray scattering. After gelatinization, several nm-scaled density fluctuations owing to the formation of gel balls were observed. These fluctuations of all starch samples correlated with the cluster size decreased with the temperature.

Unraveling the mechanisms of pea protein isolate in modulating retrogradation behavior of pea starch

The study investigated the impact of pea protein isolate (PPI) on the short-term and long-term retrogradation behavior of pea starch (PS). Dynamic time scanning and water migration analysis revealed that the presence of PPI enhanced water mobility within the PS gel during short-term storage (4 °C, 8 h) and impeded amylose gelation, effectively suppressing the short-term retrogradation of amylose. Over long-term storage (4 °C, 14 d), PPI reduced the retrogradation enthalpy and the ordered structure of starch molecules. The relative crystallinity and retrogradation degree reduced by 40.6% and 29.9%, respectively, as the PPI content increased from 0 to 12%. Textural profile analysis showed PPI decreased the gel hardness and induced the formation of a weak gel of PS, thereby delaying the long-term retrogradation of amylopectin. Scanning electron microscopy analysis further confirmed that the PS-PPI mixed gel exhibited a more disordered and loose structure compared to the PS gel, which resulted from PPI inhibiting the rearrangement and aggregation of amylose and amylopectin molecules. These findings suggested that PPI exhibited the potential to inhibit the short-term and long-term retrogradation of PS, thus providing valuable theoretical evidence for enhancing the development and research of pea-based products.

Effects of purple potato anthocyanins on the in vitro digestive properties of starches of different crystalline types

This study explored the potential of purple potato anthocyanins (PPAs) in regulating the digestive properties of starches of various crystalline types. In vitro digestion experiments indicated that PPAs inhibit the hydrolysis of rice starch (A-type) better than that of garden pea starch (C-type) and potato starch (B-type). Further structural assessment of different PPA–starch systems showed that PPAs and starch likely interact through non-covalent bonds, resulting in structural changes. Microstructural changes observed in the starches were consistent with the in vitro digestion results, and the chain length and proportions of short/long chains in amylopectin molecules affected the binding strengths and interaction modes between PPAs and starch. Hence, the three starches differed in their PPA loading efficiency and digestibility. These discoveries contribute to a deeper understanding of the mechanisms underlying the inhibition of starch digestibility by PPAs. They can aid the formulation of value-added products and low-glycemic-index foods.

Influence of Long-Chain Amylopectin on Physicochemical and External Digestion Properties of Glutinous Rice in Zongzi.

Zongzi, made from glutinous rice, is usually thought to stay in the stomach for a long time, causing many people to shy away. In our research, Zongzi was prepared from three indica glutinous rice samples, and three japonica glutinous rice samples were digested in vitro in a human gastric simulator (HGS). It was found that digestion performance in HGS (gastric emptying) was mainly related to the hardness and stickiness of texture properties, and surprisingly, the hardness and stickiness of Zongzi were positively correlated, which contradicts past perception. Through the extraction and analysis of the coated layer on the surface of glutinous rice grains in Zongzi, the main source of its stickiness was the entanglement between the long chains of leached amylopectin molecules. The hardness was also mainly due to the high proportion of long chains in its glutinous rice starch, which made it difficult to gelatinize. Studies suggested that stickiness gradually disappeared during digestion, while hardness had a longer impact on digestive performance. The indica glutinous rice Zongzi with a higher long-chain level showed a higher resistant-starch (RS) level and slow hydrolysis in the intestinal digestion stage. Therefore, the texture and digestibility of Zongzi can be adjusted by changing the molecular structure of glutinous rice starch.

A comparative study of grain quality and physicochemical properties of premium japonica rice from three typical production regions.

Grain quality indicates rice commodity value. This research compared grain quality and physicochemical properties of premium japonica rice from three production regions, Yangtze River downstream of China (YRDCN), Northeast region of China (NECN) and Japan. Results showed that there were distinct quality and physicochemical characteristics variance among the three groups of japonica rice, while CVs of most quality parameters from low to high was Japan, YRDCN and NECN. YRDCN rice presented obvious lower apparent amylose content (AAC) and ratio of each chain-length sections of amylopectin, and showed higher protein contents especially glutelin and ratio in short and intermediate amylopectin molecules. Among three rice groups, YRDCN rice presented weaker appearance, whereas did not show inferior cooking and eating properties, which was primarily linked to lower AAC. Rice AAC and starch fine structure significantly correlated with pasting parameters, swelling power and solubility, while protein content had close relation with taste analyzer parameters. Results of this study indicated improvement direction for japonica rice of YRDCN, and also provided reference for consumers' rice purchasing selection in accordance with individual taste preference.

Revealing the Structural Organization of Gamma-irradiated Starch Granules Using Polarization-resolved Second Harmonic Generation Microscopy.

Starch is a semi-crystalline macromolecule with the presence of amorphous and crystalline components. The amorphous amylose and crystalline amylopectin regions in starch granules are susceptible to certain physical modifications, such as gamma irradiation. Polarization-resolved second harmonic generation (P-SHG) microscopy in conjunction with SHG-circular dichroism (CD) was used to assess the three-dimensional molecular order and inherent chirality of starch granules and their reaction to different dosages of gamma irradiation. For the first time, the relationship between starch achirality (χ21/χ16 and χ22/χ16) and chirality (χ14/χ16) determining susceptibility tensor ratios has been elucidated. The results showed that changes in the structure and orientation of long-chain amylopectin were supported by the decrease in the SHG anisotropy factor and the χ22/χ16 ratio. Furthermore, SHG-CD illustrated the molecular tilt angle by revealing the arrangement of amylopectin molecules pointing either upward or downward owing to molecular polarity.

Starch structural reasons for the effects of SSIIIa deficiency on the textural and digestive properties of cooked rice

Due to the urgent requirements for development of healthy diets, foods with slow digestibility combined with superior eating quality attract increasingly more attention. A high amylose indica rice mutant with starch synthase IIIa (SSIIIa) deficiency was reported to have higher resistant starch content, while how the starch structure affects the textural and digestive properties of the cooked rice have not been clarified. The ss3a starch had a significantly higher amylose content (AC), longer amylose intermediate chains but shorter amylose long chains, and also displayed a higher proportion of amylopectin A, B1, and B2 chains but a lower amount of B3 chains and shorter chain length of amylopectin molecules. The gelatinization temperatures and paste viscosity were decreased because of the increased AC and more amylopectin short chains, which further contributed to the harder texture of cooked rice. In addition, more amylose intermediate to long chains and longer amylose intermediate chains of the mutant resulted in the slow digestion rate of the fast-digestible fraction, and less amylopectin intermediate to long chains caused the complete digestion of cooked rice. These findings explained the nutritional benefits of the ss3a rice and its importance to the production of slow-digestive food products with acceptable texture.

Enzymatic Approaches for Structuring Starch to Improve Functionality.

Starch is one of the most abundant renewable biopolymers in nature and is the main constituent in the human diet and a raw material for the food industry. Native starches are limited in most industrial applications and often tailored by structural modification to enhance desirable attributes, minimize undesirable attributes, or create new attributes. Enzymatic approaches for structuring starch have become of interest to the food industry precisely because the reactions minimize the formation of undesirable by-products and coproducts and are therefore considered environmentally friendly methods for producing clean-label starches with better behavioral characteristics. Starches with improved functionalities for various applications are produced via enzyme hydrolysis and transfer reactions. Use of novel, multifunctional, starch-active enzymes to alter the structures of amylose and/or amylopectin molecules, and thus alter the starch's physiochemical attributes in a predictable and controllable manner, has been explored. This review provides state-of-the-art information on exploiting glycosyl transferases and glycosyl hydrolases for structuring starch to improve its functionalities. The characteristics of starch-active enzymes (including branching enzymes, amylomaltases, GH70 α-transglycosylases, amylosucrases, maltogenic amylases, cyclomaltodextrinases, neopullulanases, and maltooligosaccharide-forming amylases), structure-functionality-driven processing strategies, novel conversion products, and potential industrial applications are discussed.

Modification of red rice starch by a combination of hydrothermal pretreatments and α-amylase hydrolysis

The objective of this study was to evaluate the modification of red rice starch by a combination of hydrothermal pretreatments and α-amylase hydrolysis. In vitro digestibility and the morphological, structural, functional, thermal, textural and rheological properties of red rice starch were evaluated. The starch submitted to autoclave (A3) obtained the highest hydrolysis yield (37.66 %) after 300 min. The morphological analysis showed that for the native starch, the granules presented a polyhedral shape and increased in diameter (2.36–394.12 μm) due to hydrothermal pre-treatments. α-Amylase (9 U mg−1) from Aspergillus oryzae modified the structure of red rice starch, presenting technological properties different from native starch. X-ray diffraction (XDR) were altered after the starch granules were cooked, showing a rupture in the amylose and amylopectin molecules, which justifies the greater absorption capacity of oil and milk. Cohesiveness, adhesiveness and apparent viscosity decreased according to HPT temperature and pressure, as well as α-amylase action.

Molecular structure and architectural characteristics of outer shells and inner blocklets of normal and waxy wheat A- and B- starch granules

Outer shells and inner blocklets were isolated from normal and waxy wheat A- and B- starch granules. Microstructural, molecular, and architectural characteristics of the isolated shells and blocklets were investigated using different methods and techniques. The results revealed that the outer shells were flake and net pocket shaped, with a compact surface and mass fractal. On the other hand, inner blocklets exhibited spherical, beaded, and rod shapes with a typical V-type crystalline structure. Crystallinity, degree of short-range order, amylopectin and amylose molecular weight, amount of intramolecular hydrogen bonds, and the average of shorter semicrystalline lamellae repetition distance varied between outer shells and inner blocklets, as well as depending on the type of wheat starch granules, i.e. normal and waxy wheat A- and B- starch granules. The branch chain length distribution of amylopectin affected the thickness of amorphous and crystalline regions, and the crystal order degree influenced the fractal and morphological characteristics of outer shells and inner blocklets. Additionally, amylose and amylopectin of inner blocklets were polymerized to form larger amylose and amylopectin molecules and develop the outer shells. The obtained findings give a theoretical basis for understanding the relationship between molecular structure and architectural characteristics of starch.

Starch adsorption on hematite surfaces: Evidence of the adsorption mechanism dependence on the surface orientation

In this study, the adsorption mechanism of amylopectin molecules on the hematite (112¯0), (11¯00) and (0001) single-crystal surfaces were investigated by surface-sensitive techniques. Our results show evidence of preferential adsorption on the hematite surface planes in the order of (112¯0) > (11¯00) > (0001). Based on these findings, we propose that distinct singly and triply coordinated oxygen atoms on the surface react in pairs by a multisite complexation mechanism to promote the attachment of amylopectin molecules. These results demonstrate the pivotal role of the exposed hematite surface structure–reactivity relationship in starch adsorption and the possible causes of inefficiencies in industrial flotation processes, as well as biomedical and environmentally friendly technologies based on hematite nanoparticles.

Edible films to improve quality and shelf life of fresh tortillas

Edible films have emerged as packaging aids to replace synthetic polymers due to their biodegradable nature. The aim of this study was to develop starch-based edible films for application in packaging, to inhibit moisture transfer from the filling to the tortilla, while maintaining physicochemical and organoleptic quality characteristics, and improving shelf life. Twenty-four types of filmogenic solutions were produced by casting from corn, pea, and chestnut starch blended with agar, guar, and potassium alginate, using glycerol as a plasticizer. The films were selected due to its surface properties and water absorption capacity, which allowed selecting the films that best retained moisture. The hydration properties of the selected films allowed to verify that the corn and pea starch films show good resistance to water and microbial development. Microstructural properties showed that the pea starch films exhibit a homogeneous and smooth surface without porosity. Fourier Transform Infrared Micro-Spectroscopy allowed the characterization of the starch films at the molecular level. The mechanical properties of corn and pea starch films were evaluated in tensile strength (21.98–27.5 KPa), elongation at break (59.17–185.96%), and elastic modulus (42.35–17.09 MPa). The results suggest that pea films are more flexible than starch films due to differences in the amylose and amylopectin molecules and molecular masses of the different starches. The sensory analysis concluded that the pea starch film delayed moisture transfer from the filling to the tortilla, maintaining the texture, appearance, and organoleptic characteristics for 6 days, improving the consumption experience of these food products.

Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice.

Down-regulation of starch branching enzymes alters fine structure and starch properties, especially the B-type crystalline pattern and extremely high amylose content identified in the BEIIb-deficiency mutant in the indica rice. The relative importance of the starch branching enzymes in determining the molecular fine structure and starch functional properties were uncovered in this study. An indica rice, Guangluai 4 with high amylose content (AC) and high gelatinization temperature (GT) was used to generate the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) knockout lines. Five mutant lines were identified including be1-1, be1-2, be2a-1, be2a-2 and be2b-1, and analysis of western blot showed the CRISPR/Cas9 system was successful in inducing mutations in the targeted genes. AC of be2b-1 (34.1%) was greater than that of wild type (WT) (27.4%) and other mutants. Mutations of either BEI or BEIIa did not alter the starch crystallite pattern (A-type). The BEIIb deficiency caused an opaque endosperm phenotype, changed the crystallite pattern from A- to B-type, and dramatically increased the degree of ordered structure, the relative proportion of amylose chains and intermediate to long amylopectin chains, average chain length of amylopectin molecules as well as GT. The BEIIa deficiency had no effect on the proportion of amylose chains, the length of amylopectin intermediate-long chains, conclusion temperature and enthalpy of gelatinization. Down-regulation of BEI increased the proportion of shortest amylopectin chains (fa) but decreased the proportion of long amylopectin chains (fb2 and fb3), leading to a lower GT. It is concluded that the relative importance in determining starch fine structures and functionality was in the order of BEIIb > BEI > BEIIa. Our results provide new information for utilizations of BE-deficient mutants in rice quality breeding.

Development of an asymmetrical flow field-flow fractionation system for the size characterization of starch granules

淀粉颗粒粒径与分子尺寸分别在1~100 μm和20~250 nm之间,是影响淀粉功能特性的重要因素之一。非对称场流分离(AF4)是一种基于样品与外力场相互作用机制的分离技术,已应用于表征淀粉分子尺寸分布。商品化的AF4系统的粒径检测范围为1 nm~10 μm,对于淀粉颗粒粒径表征具有一定的局限性。该文研制了AF4分离系统;考察了其在微米尺度下对红薯、莲子和大米淀粉颗粒粒径表征的性能;采用微米尺寸的聚苯乙烯乳化球(PS)标准样品验证了构建的AF4系统的分离性能。实验结果显示,构建的AF4系统对PS混合样品(粒径2、6、12、20 μm)实现了基线分离,同商品化AF4相比提高了检测上线,具有分离表征淀粉颗粒的潜力。此外,该文研究了载液组成对淀粉颗粒分离表征的影响;通过光学显微镜验证了构建的AF4系统在微米尺度上对淀粉颗粒粒径分布的表征能力。最后,采用商品化的AF4系统串联多角度激光光散射检测器和示差折光检测器对3种淀粉分子进行了分离表征,考察了淀粉的溶解温度对其表征结果的影响。在摩尔质量10 6~108 g/mol范围内,红薯和莲子淀粉的回转半径和水合半径的比值(Rg/Rh)在0.9~1.1之间,大米淀粉的Rg/Rh在1.2~1.4之间。实验结果证明构建的AF4系统是一种快速、准确的淀粉颗粒粒径表征方法,与商品化的AF4系统结合可为研究淀粉尺寸分布与其功能性质之间的关系提供技术支持。

Starch molecular fine structure is associated with protein composition in chickpea seed

Chickpea (Cicer arietinum L.) seed is a nutritional food high in starch and protein. This study aims to find the relationships between the molecular fine structure of starch and the composition of storage proteins and metabolic enzymes, using different chickpea varieties. It is found that storage proteins and starch biosynthetic enzymes influence each other. The initial formation of amylopectin molecules is affected by storage proteins, as suggested by the positive correlation (p < 0.01) between the average molecular size of amylopectin and total protein content. In addition, a higher amount of seed globulin could be an indication of higher amylose content and more short – medium amylose chains (degree of polymerization, DP, 118–2000). This study might assist selection of chickpea varieties with desirable qualities, such as low starch digestibility.

Microcalorimetric investigation of amylopectin branching and the content of amylose in potato starches

Hydroxyethylated starches are of particular interest for applications in infusion therapy because of their high volemic effect. The duration of this effect depends on the circulation time of the drug in the blood, i.e., the volume of circulating blood increases with an increase in the concentration, molecular weight and degree of polymer substitution. It is known that hydroxyethylated starches have a wide range of molecular weight and degree of substitution. This allows their solutions to circulate for a long time (up to 10 hours) in the bloodstream without leakage of the active substance into the interstitium of vital organs. The dependence of the melting point of the crystalline starch lamella on the number of glucose residues can be described using the modified Fox - Loshaek ratio, which takes into account the degree of branching of amylopectin molecules and the content of amylose in starch. We proposed an analytical expression for the evaluation of the degree of branching of amylopectin from potato starch based on microcalorimetric data. The proposed methodology allows the genotypes of potatoes with the highest degree of amylopectin branching and a minimum quantity of amylose in starch to be selected. Starches of such potato varieties can be used in the blood plasma substitutes production - hydroxyethylated starches that have good pharmacodynamic properties. Within the developed approach, it is possible to determine the effect of the degree of substitution on the thermodynamic properties of the studied starches through a change in the melting temperature of the lamella, Tm, hence estimating the main pharmacodynamic parameters of substituted starches.

Antagonistic effects of amylopectin and amylose molecules on the starch inter- and intramolecular interactions during retrogradation

Starch retrogradation property is critical for developing functional foods with desirable nutritional and textural attributes, while its nature currently remains elusive. The current study investigated the relations between starch chain-length distributions (CLDs) and interaction patterns (i.e. intermolecular versus intramolecular interactions) of starch molecules during the retrogradation process. CLDs for different waxy potato and sago starch mixtures were characterized by size-exclusion chromatography, and molecular interactions were characterized by differential scanning calorimetry and small amplitude oscillatory shear rheological test. Results for the first time showed that amylose molecules were involved in the formation of short-range intermolecular interactions with amylopectin molecules. Most intermolecularly heterogenous double helices were formed at the 1 : 1 mixing ratio of waxy potato to sago starch. Although the addition of waxy potato starch to sago starch increased the overall amount of short-range double helices, the amount of short-range intermolecular double helices was decreased. It suggests that there was an antagonistic effect between the formation of short-range inter- and intramolecular double helices. These results help a better understanding of the interaction patterns of amylose and amylopectin molecules during the retrogradation process.

Microwave irradiation alters the rheological properties and molecular structure of hull-less barley starch

Hull-less barley kernels and their isolated starches were irradiated under different microwave conditions (power 640, 720, and 800 W, time 60, 120 and 180 s) and changes in rheological properties and molecular structures were studied. Steady shear rheological results showed that, as the microwave energy input increased, K values decreased and n values increased, displaying a less thickening, more thinning effect. Microwave irradiation of in-kernel starches may result in more amylose breakdown than that of isolated starches, resulting in smaller K values. Dynamic rheological results showed starch pastes tended to have lower TGʹmax after treatment. The Gʹmax and Gʹ90°C of most in-kernel MWI HBS and isolated MWI HBS increased significantly. In-kernel MWI HBS had higher strength, harder gel, and higher Gʹ25°C, Gʹ0.1Hz and Gʹ20Hz than untreated starches. However, chain-length distributions of A and B chains showed no significant changes. Furthermore, molecular size distributions of the treated starches showed that peaks (Rh = ~100 nm, due to amylopectin) shifted slightly to the left, indicating that amylopectin molecules were degraded. For all treated starches, no new peaks appeared. When the treatment time was 180 s, the peak (Rh = ~10 nm, due to amylose) of in-kernel MWI HBS increased significantly with power, while for isolated MWI HBS, this peak increased dramatically at relatively low power. The primary effect of microwave irradiation may result from cutting C chains.