Transcriptomics and starch biosynthesis analysis in leaves and developing seeds of mung bean provide a basis for genetic engineering of starch composition and seed quality.

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality.

Physico-chemical properties and digestibility of native and citrate starches change in different ways by synchrotron radiation.

The physico-chemical properties and digestibility of native and citrate cassava starches changed as a result of synchrotron radiation treatment. In this study, the native and citrate starch samples were exposed to radiation doses of 0.1, 0.4, 0.8 and 3.9 kGy. The granular morphology revealed that all samples were rupture and damage after radiation. As increasing radiation doses, the relative crystallinity as determined by WAXS and the ratio of 1047/1015 cm-1 from FTIR result decreased while the degree of degradation and solubility increased for all samples. The swelling power of radiated native starches decreased with higher radiation doses indicating that the cross-linking of starch was induced by synchrotron radiation which was related to an increase in the resistant starch content. On the contrary, for radiated citrate samples, the FTIR peak at 1724 cm-1 was observed. The ratio of 1724/2900 cm-1 and total esterified citric acid did not change. The swelling and degree of di-esterification were reduced while the degree of mono-esterification increased with higher doses. It implied that the cross-linking by ester bonds was broken into mono-ester bonds. This work demonstrated that synchrotron radiation changed the physical and chemical properties of native and citrate starches in different ways.

Pre-treatment of granular rice starch to enhance branching enzyme catalysis.

Effects of different pre-treatments of granular rice starch using ethanol (ET) and maltogenic α-amylase (MA), separately or combined sequentially ET→MA, were performed to enable efficient subsequent modification with branching enzyme (BE). The pre-treated samples were characterized with respect to morphology, molecular structure, physicochemical properties and the rate of digestion to amylolytic enzymes. MA produced pores and also eroded the granular surface whereas ET caused coapted granules, noticeable swelling but no pores. Crystallinity and enthalpy of gelatinization dramatically decreased with ET and ET→MA. Subsequent BE catalysis increased the specific surface area, crystallinity, α-1,6-glucosidic linkage ratio and enthalpy. BE catalyzed branching resulted in more intact granules, less swelling capacity, solubility and granular separation as compared to their control. These effects were related to reduced amylolytic susceptibility. Pre-treatment prior to BE catalysis offers an efficient alternative way to modify granular starch with different structure and properties depending on the pre-treatment protocol.

Mechanical strength, structural and hydration properties of ethanol-treated starch tablets and their impact on the release of active ingredients.

Ethanol-treated starch (ETS) shows potentiality to be used for binder of pharmaceutical tablets. This study was aimed to evaluate the mechanical strength, structural and hydration properties of ETS tablets and ETS tablets containing lauric acid and ascorbic acid and their release behavior. ETS was prepared from cassava starch at the temperatures of 80, 90, and 100 °C. The active compounds were entrapped within the ETS tablets by two methods, including dry mixing and ethanol solubilisation. The results indicated that ETS tablets from temperatures of 80 °C showed granular shapes, had high friability and low crushing strength indexes, and dispersed and released active ingredients rapidly upon contact with water. Meanwhile, ETS tablets from temperatures of 90 and 100 °C exhibited non-granular particles, had low friability and high crushing strength indexes. Upon hydration, the tablets of non-granular ETS containing lauric acid eroded gradually and released active ingredients during tablet's erosion, meanwhile ascorbic acid diffused out gradually from the swelled tablets.

Porous high amylose rice starch modified by amyloglucosidase and maltogenic α-amylase.

Porous starch is attractive by providing high surface area for many applications. In this study amyloglucosidase (AMG) and maltogenic α-amylase (MA) were investigated in direct comparison to elucidate potential effects in producing porous starch using high amylose rice starch as a substrate. Both enzymes generated pores at the surface as illustrated by Scanning Electron Microscopy (SEM). The enzyme-treated granules had higher relative crystallinity as deduced from Wide Angle X-ray Scattering (WAXS). MA treatment increased the number of short amylopectin chains and decreased the molecular weight with extended incubation time. The MA-treated starch had higher solubility whereas swelling capacity, amylose content, peak viscosity, final viscosity, breakdown and setback of both treatments were decreased compared to the control. Enzymatic treatments produced starch with delayed gelatinization temperature and increased the enthalpy. The results demonstrate that porous rice starch can provide different functionalities depending on the enzyme mechanisms, extending the range of applications.

Porous rice starch produced by combined ultrasound-assisted ice recrystallization and enzymatic hydrolysis.

The effects of multicycle ultrasound-assisted ice recrystallization (US+IR) combined with amyloglucosidase (AMG) or maltogenic α-amylase (MA) catalyzed hydrolysis on structure were investigated. Scanning electron microscopy (SEM) showed that the US+IR produced shallow indentations and grooves on the exterior of granules while the combination US+IR and enzyme hydrolysis created additional pores on starch granules. MA displayed a higher number of pores than AMG. The highest values of specific surface area (SBET) and the total pore volume were obtained for US+IR→MA (1.96 m2 g-1 and 7.26 × 10-3 cm3 g-1, respectively). The US+IR treatment significantly decreased the relative crystallinity, amylose content and swelling capacity. Those parameters were further efficiently decreased following enzymatic hydrolysis. The combined treatments generated products with higher initial gelatinization temperature (Ti) compared to the corresponding controls. The US+IR increased the digestion rate constant (k-value) compared to native starch. However, the combined treatment, US+IR→AMG, significantly decreased the k-value from 2.97 × 10-3 to 2.50 × 10-3 min-1 compared to its control. Our study demonstrates that US+IR treatment in combination with enzyme hydrolysis is a useful method to produce specifically functionalized porous rice starch that can be used as e.g. absorbents and for further chemical modifications.

Starch digestion kinetics of extruded reformed rice is changed in different ways with added protein or fiber.

White rice is considered a high glycemic index (GI) food and extruded reformed rice offers the opportunity to design a lower GI product. This study tested the effect of added soy protein isolate (SPI) or dietary fibers [corn bran (CB), resistant maltodextrin (RMD)] to delay in vitro digestion, and on human gastric emptying rate and satiety scoring. Starch digestion using a human simulated system showed a single-phase kinetic pattern for regular reformed rice and two-phase kinetic patterns for 20% supplemented extruded rice, which lowered the estimated glycemic index (eGI). For 20% SPI extruded rice (20SPI-ER), the first-phase rate constant (k1) of cooked extruded rice was higher than its second-phase (k2). Conversely, for 20% corn bran extruded rice (20CB-ER), a low value of k1 and high for k2 and C∞2 were found. The 20% RMD extruded rice (20RMD-ER) and the control exhibited a single-phase kinetic digestion with similar k values. The end-point of the second-phase digestion (C∞2) and the eGI of cooked 20SPI-ER were lower than for fiber addition. These differences were speculated to be due to the heterogeneous matrices of the SPI and CB incorporated kernels, opposed to the homogeneous matrices of the RMD and control. Thus, extruded rice compositions were found that resulted in different starch digestion kinetics [single phase and two-phase (k1/k2) high/low, low/high] and were medium eGI with added protein or fiber. Compared to the control, only 20RMD-ER had lower gastric half-emptying time and 20CB-ER had higher subject satisfaction.

Structural transformations at different organizational levels of ethanol-treated starch during heating.

Heating ethanol-treated starch (ETS) is the simplest method to produce granular cold-water swelling starch. Structural transformations of ethanol-treated maize and potato starch (ETMS and ETPS) at the crystalline, lamellae and granular structural levels during heating were investigated through in situ wide- and small-angle X-ray scattering (WAXS and SAXS) combined with light microscopy (LM). The result of in situ WAXS indicated that the native crystalline structure was slowly disrupted up to 82 and 60 °C for ETMS and ETPS, respectively. The initial temperature for the formation of a V-type crystalline structure of ETMS was observed to be 86 °C. The result of paracrystalline analysis suggested that the crystalline lamellae of ETS realigned toward a more ordered register when heated to 80 °C and 70 °C for ETMS and ETPS, respectively. The granular forms of ETMS and ETPS were still preserved at 100 °C, although the characteristic of Maltese cross was not observed. A model was proposed to elucidate transformations of ETS at the three structural levels of starch during heating.

Macromolecular characteristics and fine structure of amylomaltase-treated cassava starch.

This work investigates the macromolecular structure of native and amylomaltase (AM) treated cassava starch at various reaction times. AM-treated starches showed lower amylose content compared to their parental starch. Long chain proportions (DP 25-80) increased with reaction time up to 4 h and then slightly decreased at 24 h. Macromolecular and fine structure of AM-treated starches for 5 min (AM5min) and 4 h (AM4 h) were characterized more deeply by using HPSEC-MALLS and H1 NMR combined with ß-amylolysis and MALDI-TOF-MS. Molar mass and dispersity of both AM-treated starches were lowered. Nevertheless, AM5min had longer external chains whereas AM4 h exhibited a larger and denser macromolecular core. A cyclo-structure with DP 8 was found in both AM-treated starches; however, AM4 h contained cyclo-structures with various sizes (DP 8-32). Finally, by controlling reaction time and substrate constituents, which are important factors affecting the AM action modes, AM-treated starch with various structural features can be obtained.

Structural transformation of crystallized debranched cassava starch during dual hydrothermal treatment in relation to enzyme digestibility.

Structural transformation of crystallized debranched cassava starch prepared by temperature cycling (TC) treatment and then subjected to annealing (ANN), heat-moisture treatment (HMT) and dual hydrothermal treatments of ANN and HMT was investigated. The relative crystallinity, lateral crystal size, melting temperature and resistant starch (RS) content increased for all hydrothermally treated samples, but the slowly digestible starch (SDS) content decreased. The RS content followed the order: HMT → ANN > HMT > ANN → HMT > ANN > TC, respectively. The HMT → ANN sample showed a larger lateral crystal size with more homogeneity, whereas the ANN → HMT sample had a smaller lateral crystal size with a higher melting temperature. After cooking at 50% moisture, the increased RS content of samples was observed, particularly for the ANN → HMT sample. These results suggest that structural changes of crystallized debranched starch during hydrothermal treatments depend on initial crystalline characteristics and treatment sequences, influencing thermal stability, enzyme digestibility, and cooking stability.

Phenolic compounds and antioxidant properties of arabinoxylan hydrolysates from defatted rice bran.

BACKGROUND: The water unextractable arabinoxylans (WUAX) contain beneficial phenolic compounds that can be used for food rather than for animal feed. The antioxidant activities of defatted rice bran obtained by xylanase-aided extraction is reported herein. The chemical and molecular characteristics of extracted fractions were investigated. RESULTS: The WUAX hydrolysate precipitated by 0-60% ethanol (F60), 60-90% ethanol (F6090), and more than 90% ethanol (F90) had decreased molar masses with increasing ethanol concentration. The fractions of interest, F60 and F6090, contained 75% arabinoxylans with ferulic acid as the major bound phenolic acid, followed by p-coumaric acid. According to chemical-based antioxidant assays F60 and F6090 exhibited higher diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric iron reducing ability than F90 which contained minor contents of small sugars and free phenolic acids. In cell-based antioxidant assays, using the fluorescent 2',7'-dichlorofluorescein diacetate probe, all three fractions were potent intracellular scavengers. CONCLUSION: The high molar mass of WUAX hydrolysates with high amount of bound phenolics contributes to the chemical-based antioxidant activity. All fractions of WUAX hydrolysates showed high potent intracellular scavenging activity regardless of molar mass, content and the component of bound phenolics. © 2017 Society of Chemical Industry.

Structure of branching enzyme- and amylomaltase modified starch produced from well-defined amylose to amylopectin substrates.

Thermostable branching enzyme (BE, EC 2.4.1.18) from Rhodothermus obamensis in combination with amylomaltase (AM, EC 2.4.1.25) from Thermus thermophilus was used to modify starch structure exploring potentials to extensively increase the number of branch points in starch. Amylose is an important constituent in starch and the effect of amylose on enzyme catalysis was investigated using amylose-only barley starch (AO) and waxy maize starch (WX) in well-defined ratios. All products were analysed for amylopectin chain length distribution, α-1,6 glucosidic linkages content, molar mass distribution and digestibility by using rat intestinal α-glucosidases. For each enzyme treatment series, increased AO content resulted in a higher rate of α-1,6 glucosidic linkage formation but as an effect of the very low initial branching of the AO, the final content of α-1,6 glucosidic linkages was slightly lower as compared to the high amylopectin substrates. However, an increase specifically in short chains was produced at high AO levels. The molar mass distribution for the enzyme treated samples was lower as compared with substrate WX and AO, indicating the presence of hydrolytic activity as well as cyclisation of the substrate. For all samples, increased amylose substrate showed decreased α- and ß-amylolysis. Surprisingly, hydrolysis with rat intestinal α-glucosidases was higher with increasing α-1,6 glucosidic linkage content and decreasing M¯w indicating that steric hindrance towards the α-glucosidases was directed by the molar mass rather that the branching density of the glucan per se. Our data demonstrate that a higher amylose content in the substrate starch efficiently produces α-1,6 glucosidic linkages and that the present of amylose generates a higher M¯w and more resistant product than amylopectin. The combination of BE→AM→BE provided somewhat more resistant α-glucan products as compared to BE alone.

Synergistic amylomaltase and branching enzyme catalysis to suppress cassava starch digestibility.

Starch provides our main dietary caloric intake and over-consumption of starch-containing foods results in escalating life-style disease including diabetes. By increasing the content of α-1,6 branch points in starch, digestibility by human amylolytic enzymes is expected to be retarded. Aiming at generating a soluble and slowly digestible starch by increasing the content and changing the relative positioning of the branch points in the starch molecules, we treated cassava starch with amylomaltase (AM) and branching enzyme (BE). We performed a detailed molecular analysis of the products including amylopectin chain length distribution, content of α-1,6 glucosidic linkages, absolute molecular weight distribution and digestibility. Step-by-step enzyme catalysis was the most efficient treatment, and it generated branch structures even more extreme than those of glycogen. All AM- and BE-treated samples showed increased resistance to degradation by porcine pancreatic α-amylase and glucoamylase as compared to cassava starch. The amylolytic products showed chain lengths and branching patterns similar to the products obtained from glycogen. Our data demonstrate that combinatorial enzyme catalysis provides a strategy to generate potential novel soluble α-glucan ingredients with low dietary digestibility assets.

Crystallization and chain reorganization of debranched rice starches in relation to resistant starch formation.

The effects of chain distribution, concentration, temperature and hydrothermal treatments on the recrystallization behavior and formation of resistant starch (RS) were investigated. Waxy and normal rice starches were debranched at 10 and 21% w/w solid concentrations, incubated at 25 or 50 °C, and further subjected to annealing or heat moisture treatment (HMT) to enhance RS formation. The crystallization at 25 °C favored the formation of the B-type structure, whereas crystallization at 50 °C led to the A-type structure with a higher melting temperature (100-120 °C) and a higher RS content (52%). All incubated samples showed an increase in RS content after subsequent hydrothermal treatments. The sample incubated at a high temperature contained the highest RS content (74.5%) after HMT with larger/perfect crystallites. These results suggested that the RS formation could be manipulated by crystallization conditions and improved by hydrothermal treatments which are dependent on the initial crystalline perfection.

Chemical, molecular, and structural characterization of alkali extractable nonstarch polysaccharides from Job's tears.

The polished Job's tears ( Coix lachryma-jobi L.) seeds, dark and white husk types, were sequentially extracted by hot water (75 degrees C) and 0.5 M NaOH solution. Nonstarch polysaccharides were not found in the water extract but were present in the alkali extract. The major components of the alkali extract from both Job's tears were protein, ash, and nonstarch polysaccharides, mainly arabinoxylans. The high arabinose to xylose ratio of 1.25 and 1.24 indicated a highly substituted structure. The average molecular weight (MW) of arabinoxylans of the dark and white husk types were 741,000 Da (Pd 1.5) and 1,449,000 Da (Pd 2.6), respectively, and their average MW reduced after treatment with protease. The alkali extractable arabinoxylans were elucidated to have a (1,4)-linked beta- d-xylan main chain highly substituted with single arabinose units. The results showed that the alpha- l-arabinofuranosyl residues (Ara f) were attached to the main chain mostly at O-3, followed by both O-2 and O-3 of xylopyranosyl residues (Xyl p).

Effect of extrusion parameters on conjugated linoleic acids of corn extrudates.

The effects of extrusion temperature, 150-190 degrees C, and torque, 50-70%, on the content and configuration of conjugated linoleic acids (CLA) in corn extrudates were analyzed by GC and HPLC. At a temperature of 150 degrees C, CLA content increased from 1.2 mg/g of oil in feed to 7.8 mg/g of oil in corn extrudates. A decrease in total CLA (P < 0.05) was obtained when the product temperature was further increased to 190 degrees C. Alteration of CLA geometrical configuration was observed at higher extrusion temperatures. trans,trans-CLA significantly increased (P < 0.05) from 10.2% in feed to 11.9% of CLA at the extrusion condition of 190 degrees C and 70% torque. The highest expansion of extrudates was found at the product temperature of 150 degrees C and 70% torque. This extrusion condition also gave the maximum total CLA content and minimum trans,trans-CLA formation.