Evaluation of pea/rice and amylopectin/chromium as an alternative protein source to improve muscle protein synthesis in rats.

BACKGROUND: A preclinical study reported that the combination of an amylopectin/chromium complex (ACr) of branched-chain amino acids (BCAA) significantly enhanced muscle protein synthesis (MPS). This study was conducted to determine the effects of the addition of ACr complex to a pea/rice (PR) protein on MPS, insulin, muslin levels, and the mTOR pathway in exercised rats. METHODS: Twenty-four rats were divided into three groups: (i) exercise (Ex); (ii) Ex + PR 1:1 blend (0.465 g/kg BW); (iii) Ex + PR + ACr (0.155 g/kg BW). On the day of single-dose administration, after the animals were exercised at 26/m/min for 2 h, the supplement was given by oral gavage. The rats were injected with a bolus dose (250 mg/kg BW, 25 g/L) of deuterium-labeled phenylalanine to determine the protein fractional synthesis rate (FSR) one h after consuming the study product. RESULTS: The combination of PR and ACr enhanced MPS by 42.55% compared to the Ex group, while Ex + PR alone increased MPS by 30.2% over the Ex group (p < 0.0001) in exercised rats. Ex + PR plus ACr significantly enhanced phosphorylation of mTOR and S6K1 (p < 0.0001), and 4E-BP1 (p < 0.001) compared to the Ex (p < 0.0001). PR to ACr also significantly increased insulin and musclin levels (p < 0.0001) in exercised rats. Additionally, compared to Ex + PR alone, Ex + PR + ACr enhanced mTOR (p < 0.0001) and S6K1 (p < 0.0001) levels. CONCLUSION: These data suggested that PR + ACr may provide an alternative to animal proteins for remodeling and repairing muscle by stimulating MPS and mTOR signaling pathways in post-exercised rats. More preclinical and clinical human studies on combining pea/rice and amylopectin/chromium complex are required.

Suitable nitrogen fertilizer application drives the endosperm development and starch synthesis to improve the physicochemical properties of common buckwheat grain.

The effects of nitrogen (N) fertilizer on endosperm development, starch component, key enzyme activity and grain quality of common buckwheat were investigated in this study. The results showed that N fertilization significantly enhanced the number and area of endosperm cells, and significant increases were also observed in the contents of amylose, amylopectin and total starch. With increasing N level, the activities of key enzyme significantly increased showing the maximum under the N2 level (180 kg N ha-1), and then decreased under high N level. As N level increased, the ash, crude protein and amylose content varied from 1.36 to 2.25 %, from 7.99 to 15.84 % and from 22.69 to 27.64 %, respectively. The gelatinization enthalpy significantly increased with the range of 3.46-5.66 J/g, while no change was found in crystalline structure of common buckwheat flour. These results indicated that appropriate N application could effectively improve the endosperm development, starch synthesis and accumulation, and grain properties of common buckwheat, with the best effect under the level of 180 kg N ha-1.

Further slowing down of hydrolysis of amylose heated with black soybean extract by treating with nitrite under gastric conditions.

Black soybean (BSB), which contains cyanidin-3-O-glucoside (C3G) and procyanidins, is cooked with rice in Japan. The color of the cooked rice is purplish red due to the binding of C3G and reddish oxidation products of procyanidins. These components can slowdown pancreatin-induced hydrolysis of amylose more significantly than the hydrolysis of amylopectin, and can react with nitrous acid in the stomach. This manuscript deals with the effects of nitrous acid on pancreatin-induced hydrolysis of amylose heated with BSB extract. The hydrolysis of amylose heated with BSB extract was slow, and the slowdown was due to the binding of C3G/its degradation products and degradation products of procyanidins. The amylose hydrolysis was slowed down further by treating with nitrite under gastric conditions. The further slowdown was discussed to be due to the binding of the products, which were formed by the reaction of procyanidins with nitrous acid, to amylose. In the products, dinitroprocyanidins were included. In this way, the digestibility of amylose heated with BSB extract can be slowed down further by reacting with nitrous acid in the stomach.

CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.

Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4.

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.

Potato starch modified by Streptococcus thermophilus GtfB enzyme has low viscoelastic and slowly digestible properties.

Potato starch with high viscosity and digestibility cannot be added into some foods. To address this issue, a novel starch-acting enzyme 4,6-α-glucosyltransferase from Streptococcus thermophilus (StGtfB) was used. StGtfB decreased the iodine affinity and the molecular weight, but increased the degree of branching of starch at a mode quite different from glycogen 1,4-α-glucan branching enzyme (GBE). StGtfB at 5 U/g substrate mainly introduced DP 1-7 into amylose (AMY) or DP 1-12 branches into amylopectin (AMP), and increased the ratio of short- to long-branches from 0.32 to 2.22 or from 0.41 to 2.50. The DP 3 branch chain was the most abundant in both StGtfB-modified AMY and StGtfB-modified AMP. The DP < 6 branch chain contents in StGtfB-modified AMY were 42.68%, much higher than those of GBE-modified AMY. StGtfB significantly decreased viscoelasticity but still kept pseudoplasticity of starch. The modifications also slowed down the glucose generation rate of products at the mammalian mucosal α-glucosidase level. The slowly digestible fraction in potato starch increased from 34.29% to 53.22% using StGtfB of 5 U/g starch. This low viscoelastic and slowly digestible potato starch had great potential with respect to low and stable postprandial blood glucose.

Formation and structural evolution of starch nanocrystals from waxy maize starch and waxy potato starch.

The formation and structural evolution of starch nanocrystals from waxy maize starch (WMS) and waxy potato starch (WPS) by acid hydrolysis were studied. The relative crystallinity, the short-range molecular order, and the double-helix content of WMS and WPS increased significantly during the initial stage of acid hydrolysis, indicating that acid preferentially eroded the amorphous regions of starch granules. With time, there was increased destruction of lamellar structures, causing the granules to completely disintegrate to form nanocrystals. WMS and WPS displayed different hydrolysis mechanisms. WPS was more susceptible to acid hydrolysis than WMS, and WMS exhibited an endo-corrosion pattern and WPS showed an exo-corrosion pattern. WMS nanocrystals had a parallelepiped shape, and WPS nanocrystals were round. This difference in shape is likely due to the different packing configuration of double helices in native starches.

Highly phosphorylated functionalized rice starch produced by transgenic rice expressing the potato GWD1 gene.

Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato GWD1 in rice. Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges. The gelatinization temperatures of both rice flour and extracted starch were significantly lower than those of the control and hence negatively correlated with the starch phosphate content. The 6-P content was positively correlated with amylose content and relatively long amylopectin chains with DP25-36, and the 3-P content was positively correlated with short chains of DP6-12. The starch pasting temperature, peak viscosity and the breakdown were lower but the setback was higher for transgenic rice flour. The 6-P content was negatively correlated with texture adhesiveness but positively correlated with the cohesiveness of rice flour gels. Our data demonstrate a way forward to employ a starch bioengineering approach for clean modification of starch, opening up completely new applications for rice starch.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

BACKGROUND: Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers. RESULTS: A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern. CONCLUSION: This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

Molecular insights into how a deficiency of amylose affects carbon allocation--carbohydrate and oil analyses and gene expression profiling in the seeds of a rice waxy mutant.

BACKGROUND: Understanding carbon partitioning in cereal seeds is of critical importance to develop cereal crops with enhanced starch yields for food security and for producing specified end-products high in amylose, ß-glucan, or fructan, such as functional foods or oils for biofuel applications. Waxy mutants of cereals have a high content of amylopectin and have been well characterized. However, the allocation of carbon to other components, such as ß-glucan and oils, and the regulation of the altered carbon distribution to amylopectin in a waxy mutant are poorly understood. In this study, we used a rice mutant, GM077, with a low content of amylose to gain molecular insight into how a deficiency of amylose affects carbon allocation to other end products and to amylopectin. We used carbohydrate analysis, subtractive cDNA libraries, and qPCR to identify candidate genes potentially responsible for the changes in carbon allocation in GM077 seeds. RESULTS: Carbohydrate analysis indicated that the content of amylose in GM077 seeds was significantly reduced, while that of amylopectin significantly rose as compared to the wild type BP034. The content of glucose, sucrose, total starch, cell-wall polysaccharides and oil were only slightly affected in the mutant as compared to the wild type. Suppression subtractive hybridization (SSH) experiments generated 116 unigenes in the mutant on the wild-type background. Among the 116 unigenes, three, AGP, ISA1 and SUSIBA2-like, were found to be directly involved in amylopectin synthesis, indicating their possible roles in redirecting carbon flux from amylose to amylopectin. A bioinformatics analysis of the putative SUSIBA2-like binding elements in the promoter regions of the upregulated genes indicated that the SUSIBA2-like transcription factor may be instrumental in promoting the carbon reallocation from amylose to amylopectin. CONCLUSION: Analyses of carbohydrate and oil fractions and gene expression profiling on a global scale in the rice waxy mutant GM077 revealed several candidate genes implicated in the carbon reallocation response to an amylose deficiency, including genes encoding AGPase and SUSIBA2-like. We believe that AGP and SUSIBA2 are two promising targets for classical breeding and/or transgenic plant improvement to control the carbon flux between starch and other components in cereal seeds.

Inhibitory effects of Citrus flavonoids on starch digestion and antihyperglycemic effects in HepG2 cells.

Flavonoids are a class of important bioactive natural products and are being extensively used in functional foods. In the present study, the effects of four Citrus flavonoids (i.e., hesperidin, naringin, neohesperidin, and nobiletin) on amylase-catalyzed starch digestion, major digestive enzyme activities (e.g., pancreatic α-amylase and α-glucosidase), and glucose use in HepG2 cells were investigated. The results showed that all of the tested Citrus flavonoids significantly inhibited amylase-catalyzed starch digestion. Moreover, naringin and neohesperidin mainly inhibited amylose digestion, whereas hesperidin and nobiletin inhibited both amylose and amylopectin digestion. However, these flavonoids showed weak inhibitory activities against digestive enzymes. Furthermore, glucose consumption, glycogen concentration, and glucokinase activity were significantly elevated, and glucose-6-phosphatase activity was markedly decreased by Citrus flavonoids. These results demonstrate that Citrus flavonoids play important roles in preventing the progression of hyperglycemia, partly by binding to starch, increasing hepatic glycolysis and the glycogen concentration, and lowering hepatic gluconeogenesis. This work suggests that Citrus flavonoids might be potentially used for the prevention of postprandial hyperglycemia.

Purification of pectin methylesterase from Lycopersicon esculentum and its application.

Pectin methylesterase (PME) (3.1.1.11) is the pectin degrading enzyme which catalyses the hydrolysis of pectin methylester group, resulting in de-esterification. PME is widely distributed in plants, fungi, yeast and bacteria. In the present study, PME was extracted from tomato by using 8.8% NaCl (4°C). The crude enzyme precipitated with 60% ammonium sulphate resulted in 1.02 fold purification of the enzyme. The purification was done by ion exchange chromatography using DEAE-Cellulose column. This resulted in 1.82 fold purification of the enzyme. The molecular weight of purified enzyme was determined by SDS-PAGE which was found to be 34.0 kDa. During characterization of the purified enzyme, the maximum activity was found at temperature 50°C, pH 6.5, reaction time 45 min. Citrus pectin was the best substrate for maximum enzyme activity. The enzyme did not require any metal ion to express its activity, enzyme was found to be very stable at 4°C and at 50°C the enzyme was stable upto 2 h as it retained 70% of its activity. The K(m) and V(max) values of the enzyme were found to be 0.115 mg/ml and 1.03 µmol/ml/min. PME enhanced the pectin degradation process in apple juice clarification in combination with polygalacturonase and increased %T(650) from 1.7% to 5.6%.

13C pulse-labeling assessment of the community structure of active fungi in the rhizosphere of a genetically starch-modified potato (Solanum tuberosum) cultivar and its parental isoline.

• The aim of this study was to gain understanding of the carbon flow from the roots of a genetically modified (GM) amylopectin-accumulating potato (Solanum tuberosum) cultivar and its parental isoline to the soil fungal community using stable isotope probing (SIP). • The microbes receiving (13)C from the plant were assessed through RNA/phospholipid fatty acid analysis with stable isotope probing (PLFA-SIP) at three time-points (1, 5 and 12 d after the start of labeling). The communities of Ascomycota, Basidiomycota and Glomeromycota were analysed separately with RT-qPCR and terminal restriction fragment length polymorphism (T-RFLP). • Ascomycetes and glomeromycetes received carbon from the plant as early as 1 and 5 d after labeling, while basidiomycetes were slower in accumulating the labeled carbon. The rate of carbon allocation in the GM variety differed from that in its parental variety, thereby affecting soil fungal communities. • We conclude that both saprotrophic and mycorrhizal fungi rapidly metabolize organic substrates flowing from the root into the rhizosphere, that there are large differences in utilization of root-derived compounds at a lower phylogenetic level within investigated fungal phyla, and that active communities in the rhizosphere differ between the GM plant and its parental cultivar through effects of differential carbon flow from the plant.

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch.

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.

Effects of genetically modified starch metabolism in potato plants on photosynthate fluxes into the rhizosphere and on microbial degraders of root exudates.

A high percentage of photosynthetically assimilated carbon is released into soil via root exudates, which are acknowledged as the most important factor for the development of microbial rhizosphere communities. As quality and quantity of root exudates are dependent on plant genotype, the genetic engineering of plants might also influence carbon partitioning within the plant and thus microbial rhizosphere community structure. In this study, the carbon allocation patterns within the plant-rhizosphere system of a genetically modified amylopectin-accumulating potato line (Solanum tuberosum L.) were linked to microbial degraders of root exudates under greenhouse conditions, using (13)C-CO(2) pulse-chase labelling in combination with phospholipid fatty acid (PLFA) analysis. In addition, GM plants were compared with the parental cultivar as well as a second potato cultivar obtained by classical breeding. Rhizosphere samples were obtained during young leaf developmental and flowering stages. (13)C allocation in aboveground plant biomass, water-extractable organic carbon, microbial biomass carbon and PLFA as well as the microbial community structure in the rhizosphere varied significantly between the natural potato cultivars. However, no differences between the GM line and its parental cultivar were observed. Besides the considerable impact of plant cultivar, the plant developmental stage affected carbon partitioning via the plant into the rhizosphere and, subsequently, microbial communities involved in the transformation of root exudates.

Fatty acids, epicatechin-dimethylgallate, and rutin interact with buckwheat starch inhibiting its digestion by amylase: implications for the decrease in glycemic index by buckwheat flour.

Glycemic indexes of bread made from mixtures of wheat flour and buckwheat flour are lower than those made from wheat flour. To discuss the mechanism of the buckwheat flour-dependent decrease in glycemic indexes, the formation of a starch-iodine complex and amylase-catalyzed digestion of starch were studied using buckwheat flour itself and buckwheat flour from which fatty acids, rutin, and proanthocyanidins including flavan-3-ols had been extracted. Absorbance due to the formation of a starch-iodine complex was larger in extracted than control flour, and starch in extracted flour was more susceptible to pancreatin-induced digestion than starch in control flour. Fatty acids, which were found in the buckwheat flour extract, bound to amylose in the extracted flour, inhibiting its digestion by pancreatin. Rutin and epicatechin-dimethylgallate, which were also found in the extract, bound to both amylose and amylopectin in the extracted flour, inhibiting their digestion induced by pancreatin. We discussed from these results that the lower glycemic indexes of bread made from mixtures of wheat flour and buckwheat flour were due to binding of fatty acids, rutin, and epicatechin-dimethylgallate, which were contained in buckwheat flour, to wheat flour starch.

Precision breeding for novel starch variants in potato.

Potato can be used as a source of modified starches for culinary and industrial processes, but its allelic diversity and tetraploid genome make the identification of novel alleles a challenge, and breeding such alleles into elite lines is a slow and difficult process. An efficient and reliable strategy has been developed for the rapid introduction and identification of new alleles in elite potato breeding lines, based on the ethylmethanesulphonate mutagenesis of dihaploid seeds. Using the granule-bound starch synthase I gene (waxy) as a model, a series of point mutations that potentially affect gene expression or enzyme function was identified. The most promising loss-of-function allele (waxy(E1100)) carried a mutation in the 5'-splice donor site of intron 1 that caused mis-splicing and protein truncation. This was used to establish elite breeding lineages lacking granule-bound starch synthase I protein activity and producing high-amylopectin starch. This is the first report of rapid and efficient mutation analysis in potato, a genetically complex and vegetatively propagated crop.

Study on alpha-amylase hydrolysis of potato amylopectin by a quartz crystal microbalance.

Potato amylopectin with phosphate groups was immobilized on a quartz crystal microbalance with dissipation monitoring (QCMD) using the attractive interaction between opposite charges, and enzymatic starch hydrolysis was monitored directly. Poly( L-lysine) (PLL) proved to be an appropriate cationic linker between the QCMD silica sensor and potato amylopectin. Increased mass and dissipation were observed when amylopectin was adsorbed onto the PLL layer and reversed when alpha-amylase was added. The effect of chitosan with cationic property on the hydrolysis of amylopectin was studied. Chitosan was observed to be adsorbed onto the amylopectin surface and to suppress hydrolysis by alpha-amylase. The formation of alternating layers of amylopectin and chitosan was monitored by QCMD. Amylopectin-chitosan trilayers increased resistance to digestion by alpha-amylase compared to one layer and to control without chitosan.

Determination of the maximum water solubility of eight native starches and the solubility of their acidic-methanol and -ethanol modified analogues.

The maximum water solubilities of eight native starches from potato, shoti, tapioca, maize, waxy maize, amylomaize-7, wheat, and rice and their acid-methanol and acid-ethanol modified analogues have been determined. Maximum solubilities of 18.7 and 17.4 mg/mL were obtained for waxy maize and tapioca and 12.4 mg/mL for potato and maize starches by autoclaving 220 mg/10 mL at 121 degrees C; 8.7 mg/mL was obtained for shoti starch by stirring in 85:15 (v/v) Me(2)SO-H(2)O at 20 degrees C; and 7.0 and 5.2mg/mL for rice and amylomaize-7 starches by stirring in 1M NaOH at 20 degrees C. The acid-alcohol treated starches were 4-9 times more soluble than their native starches. The compositions of the solubilized starches had, in general, much higher ratios of amylose to amylopectin than the ratios in their native granules. A major exception to this was the acid-methanol treated potato, shoti, and rice starches that had much lower ratios of amylose to amylopectin than the ratios in their granules.