Structural And Functional Properties Of Starch Granule In Endosperm Of High-amylose Rice

High-amylose starch is the main source of natural food containing resistant starch, which can effectively improve the health of human intestinal, reduce the incidence of diabetes and cardiovascular diseases. Rice is one of the main food crops in our country, but conventional endosperm starch in rice contains little resistant starch. It has been proved that amylose content in rice could be significantly improved and new rice cultivars abundant of high-amylose resistant starch could be cultivated with antisense RNA technology suppressing the expression of starch branching enzyme gene Sbel/Ⅱb. The cultivation, processing and application of rice with high-amylose starch was based on the research results of structural characteristics of genetically modified rice starch. In this article, the changes of structural characteristics were studied based on a series of rice cultivars with different content of high-amylose and their transgenic cultivars with Sbel/Ⅱb suppressed. From which a transgenic cultivar TRS was selected as it changed the most from its parent cultivar TQ with the highest content of amylose. High-amylose rice starch were studied and analyzed on the mechanism of acid hydrolysis, enzymatic hydrolysis and in vivo digestion using a series of research methods. Heterogeneous starch granules were isolated from different regions of TRS mature endosperm to learn the differences on physical and chemical properties. The main research contents and results are as follows:1. Effect of simultaneous inhibition of starch branching enzymes Ⅰ and Ⅱb on the crystalline structure of rice starches with different amylose contentsStarch branching enzyme is the key enzyme in the formation of branched amylopectin, the mutation or inhibition of whose gene expression can effectively improve the anylose content of cereal endosperms. In this study, three conventional rice cultivars GLXN. WX 9915 and Teqing with waxy rice with low and high amylose content respectively, and their transgenic cultivars with SBEⅠ/Ⅱb gene inhibited (named corresponding parent cultivars:GLXN-SBEl/Ⅱb-, WX-SBEI/Ⅱb-, TQ-SBEⅠ/Ⅱb-(TRS)) were selected to study the effect of the expression of SBE genes on the structural characteristics of starch from rice cultivars with different amylase content. The results showed that the amylase content of transgenic varieties was significantly higher than the parents, especially for the transgenic line TRS, genetically modified from the high-amylose variety TQ, whose amylose content increased from 28.1% of the parents to 58.1%. Starch granules from transgenic lines of waxy and low-amylose rice have almost the same shape like their parents although slightly smaller. However, heterogeneous starch granules were produced in the endosperm of TQ-SBEI/Ⅱb- with the shape changed significantly. Several research works have been done to study the ordered structure properties of starch granules mentioned with X-ray powder diffractionv (XRD), attenuated total reflectance fourier transform infrared (ATR-FTIR), solid-state 13C cross-polarization magic-angle spinning nuclear magnetic resonance (13C CP/MAS NMR), and small-angle X-ray scattering (SAXS). And the results showed that in transgenic rice cultivars, the thickness of crystal lamellae increases, the degree of crystallinity, the double helix content and the peak intensity of crystalline lamellae scattering reduce, and short-range ordered structure in the outer region of starch granules change. The structural discrepancies mentioned above were embodied most obviously between high-amylose rice cultivar TQ and its transgenic cultivar TQ-SBEI/Ⅱb-. Inhibiting both SEE Ⅰ and Ⅱb changed the crystalline structure of starch from A-type to CA-type in lines derived from waxy and low-AC cultivars, and from A-type to C-type in that derived from the high-AC cultivar.2. Structural analyses of heterogeneous starch granules separated from TRS endosperm using a glycerol centrifugation methodHigh-amylose starch crops usually contain heterogeneous starch granules which differ in morphology and structural properties. Previous studies showed that the grains of high-amylose rice TRS is rich of heterogeneous granules. Heterogeneous starch granules in the grains showed regional distribution in endosperm. In this study, the oval, polygonal, small size, and hollow starch granules were isolated and purified from different regions of TRS grains using the method of centrifugal sedimentation and standing sedimentation in the glycerol medium, and their structures were investigated. The results showed that the polygonal starch granules had low amylose content and high short branch-chain and branching degree of amylopectin, and exhibited A-type crystallinity. The aggregate starch granules had high long branch-chain of amylopectin, relative crystallinity. and double helix content, and exhibited C-type crystallinity. The elongated starch granules had high amylose content and low branching degree of amylopectin and relative crystallinity,and exhibited C-type crystallinity. The hollow starch granules had very high amylose content, proportion of amorphous conformation, and amylose-lipid complex, and very low branch-chain of amylopectin, branching degree of amylopectin, and double helix content, and exhibited no crystallinity. The different structures of heterogeneous starch granules from high-amylose rice resulted in significantly different thermal properties.3. Ordered structure and thermal characteristics of acid-modified high-amylose starchAcid-modification of starch granule is an important necessary method of studying the internal structure of starch granules. In this study, the ordered structure and thermal properties of acid-modified high-amylose rice starch was investigated by gel permeation chromatography (GPC), high-performance anion-exchange chromatograph (HPAEC),13C CP/MAS NMR, XRD and differential scanning calorimetry (DSC). The results show that acid hydrolysis of starch can be separated into the early stage of rapid degradation and subsequent slow degradation phase. Acid preferentially degraded the amylose, then A chain and short B chain of amylopectin. Relative double helix content and crystallinity both initially increased sharply and then progressively with acid hydrolysis. The relative crystallinity of starches obtained from 13C CP/MAS NMR was higher than that from XRD. The onset gelatinisation temperature decreased, while the peak and conclusion temperatures increased with increasing hydrolysis time. The endothermic value initially increased and then decreased with acid hydrolysis. The swelling power decreased while solubility increased after acid hydrolysis. These results add to our understanding of the effect of acid hydrolysis on the high-amylose rice starch.4. Ordered structure characteristics of enzyme-hydrolyzed high-amylose starch by a-amylase and amyloglucosidaseEnzyme hydrolysis of starch is involved in many biological and industrial processes.The morphological and structural changes of starch residues following porcine pancreatic a-amylase (PPA) and black Aspergillus glucosidase (AAG) hydrolysis were characterized by SEM, DSC, XRD,13C CP/MAS NMR, and ATR-FTIR. The results of structural properties of PPA-hydrolyzed starch showed that:TQ starch was hydrolyzed faster than TRS starch. PPA pitted the starch granule surface first, then penetrated into the interior and hydrolyzed the granule from the inside out. The peripheral region of subgranule and the surrounding band of TRS starch were more resistant to PPA hydrolysis than the external region of TQ starch. Both amorphous and crystalline (the long- and shortrange ordered) structures were simultaneously hydrolyzed in TQ starch. A-type polymorph of TRS C-type starch was hydrolyzed faster than B-type polymorph. The long-range ordered structure was hydrolyzed faster than the amorphous structure, whereas the short-range ordered structure was hydrolyzed slower than the amorphous structure in TRS starch in the initial hydrolysis stage.The results of structural properties of PPA-hydrolyzed starch showed that:AAG hydrolysed TQ starch from the granule surface, and TRS starch from the granule interior. During AAG hydrolysis, the content of amorphous structure increased, the contents of ordered structure and single helix decreased, and gelatinization enthalpy decreased in TQ and TRS starch residues. The A-type polymorph of TRS C-type starch was hydrolysed faster than the B-type polymorph. The above results indicated that B-type crystallinity and short-range ordered structure in the peripheral region of subgranule and the surrounding band of TRS starch increased the resistance of TRS starch to PPA and AAG hydrolyses.5. Structural changes of high-amylose rice starch residues following in vitro and in vivo digestionHigh-amylose cereal starch has a great benefit on human health through its resistant starch content. In this study, the structural changes of digestive starch residues were characterized using DSC, XRD,13C CP/MAS NMR, and ATR-FTIR. TQ starch both in vivo and in vitro digestion is more easily digested than TRS, but ordered structure characteristic did not change significantly after digestion, indicating that the crystal structure and the amorphous structure is simultaneously degraded. Both amorphous and the long-range order structures were also simultaneously hydrolyzed in TRS starch, but the short-range order (double helix) structure in the external region of TRS starch granule increased with increasing digestion time. The A-type polymorph of TRS C-type starch was hydrolyzed more rapidly than the B-type polymorph. These results suggested that B-type crystallinity and short-range order structure in the external region of starch granule made TRS starch resistant to digestion.