Authentication of ST25 rice using temperature-perturbed Raman measurement with variable selection by Incremental Association Markov Blanket.

A temperature-perturbed transmission Raman measurement was demonstrated for the discrimination of ST25 and non-ST25 rice samples. ST25 rice is a premium long-grain Vietnamese rice with the aroma of pandan leaves and the scent of early sticky rice. Raman spectra of rice samples were acquired with temperature perturbation ranging from 20 to 50 °C, and the variables (intensities of peaks) with greater discrimination were selected from the spectra using Incremental Association Markov Blanket (IAMB) for authentication. The combination of four, seven, and four variables selected from the spectra at 20, 30, and 50 °C, respectively, yielded the highest accuracy of 97.9%. The accuracies in the single-temperature measurements were lower, suggesting that the combination of mutually complementary spectral features acquired at these temperatures is synergetic to recognize the compositional differences between two sample groups, such as in the amylose/amylopectin ratio and the protein constituent.

Maltoheptaoside hydrolysis with chromatographic detection and starch hydrolysis with reducing sugar analysis: Comparison of assays allows assessment of the roles of direct α-amylase inhibition and starch complexation.

The aim was to determine inhibition of human α-amylase activity by (poly)phenols using maltoheptaoside as substrate with direct chromatographic product quantification, compared to hydrolysis of amylose and amylopectin estimated using 3,5-dinitrosalicylic acid. Acarbose exhibited similar IC50 values (50% inhibition) with maltoheptaoside, amylopectin or amylose as substrates (2.37 ± 0.11, 3.71 ± 0.12 and 2.08 ± 0.01 µM respectively). Epigallocatechin gallate, quercetagetin and punicalagin were weaker inhibitors of hydrolysis of maltoheptaoside (<50% inhibition) than amylose (IC50: epigallocatechin gallate = 20.41 ± 0.25 µM, quercetagetin = 30.15 ± 2.05 µM) or amylopectin. Interference using 3,5-dinitrosalicylic acid was in the order punicalagin > epigallocatechin gallate > quercetagetin, with minimal interference using maltoheptaoside as substrate. The main inhibition mechanism of epigallocatechin gallate and punicalagin was through complexation with starch, especially amylose, whereas only quercetagetin additionally binds to the α-amylase active site. Interference is minimised using maltoheptaoside as substrate with product detection by chromatography, potentially allowing assessment of direct enzyme inhibition by almost any compound.

Quantitative Assessment of the Conformational Heterogeneity in Amylose across Force Fields.

The inherent flexibility and conformational heterogeneity of a carbohydrate pose a challenge for its modeling and sampling by the existing classical force field. This work quantitatively assesses the quality of four popular carbohydrate force fields (CHARMM36, GLYCAM06, OPLS-AA, GROMOS53A6CARBO_R) against their ability to accurately model the conformational landscape of a dodecamer of single-stranded amylose, the key constituent of starch. While past NMR and X-ray studies have hinted at evidence of a helical structure of amylose and its spontaneous helix-coil transition, it remains to be seen how existing force fields fare against modeling its structural transition. Toward this end, we perform a multimicrosecond long extensive molecular dynamics simulation of dodecamer of a single-stranded amylose chain in explicit water in each of the four force fields and assess these force fields' ability to model relative structural transitions via analyzing the radius of gyration, glycosidic linkage orientation, and pyranose ring puckering of the amylose. In particular, the simulations show that while GLYCAM06 and CHARMM36 force fields predict a significant helix-coil transition in the amylose, GROMOS53A6CARBO_R and OPLS-AA majorly favor extended conformation. The Markov State Model (MSM), built using the simulation trajectories, for each force field, provides a comparative quantification of the population of key macrostates of amylose and elucidates an underlying network of pathways of their mutual interconversion. The macrostates obtained from MSM revealed that metastable helixlike and semicoil intermediate conformations are more probable for CHARMM36, whereas elongated or helixlike conformations are more probable in OPLS-AA and GROMOS53A6CARBO_R. GLYCAM06 showed significant probability for both helix and coil conformations along with intermediate conformations. We find that the differences in the conformations across force fields are governed by differences in the kinetics of glycosidic linkages and pyranose ring pucker conformers. All four force fields share one common point that the majority of α(1 → 4) glycosidic linkages preferred syn conformation, which is found to be energetically more favorable than anti. However, except for GROMOS53A6CARBO_R, all other force fields predicted non-negligible minor anti conformation. The multimicrosecond long simulations on the single-chain amylose, in combination with MSM, described here, suggest that sampling of α(1 → 4) linked oligosacharides on microsecond time scales enable quantitative predictions of helix-coil, glycosidic linkage, and pyranose ring exchange kinetics. These exchange kinetics have otherwise remained inaccessible to quantification by experiments or nanosecond time scale simulations which might have hindered the comparison of the possibility of helix-coil exchange across different force fields on equal footing.

Discovery and mapping of genomic regions governing economically important traits of Basmati rice.

BACKGROUND: Basmati rice, originated in the foothills of Himalayas, commands a premium price in the domestic and international markets on account of its unique quality traits. The complex genetic nature of unique traits of Basmati as well as tedious screening methodologies involved in quality testing have been serious constraints to breeding quality Basmati. In the present study, we made an attempt to identify the genomic regions governing unique traits of Basmati rice. RESULTS: A total of 34 Quantitative Trait Loci (QTLs) for 16 economically important traits of Basmati rice were identified employing F(2), F(3) and Recombinant Inbred Line (RIL) mapping populations derived from a cross between Basmati370 (traditional Basmati) and Jaya (semi-dwarf rice). Out of which, 12 QTLs contributing to more than 15 % phenotypic variance were identified and considered as major effect QTLs. Four major effect QTLs coincide with the already known genes viz., sd1, GS3, alk1 and fgr governing plant height, grain size, alkali spreading value and aroma, respectively. For the remaining major QTLs, candidate genes were predicted as auxin response factor for filled grains, soluble starch synthase 3 for chalkiness and VQ domain containing protein for grain breadth and grain weight QTLs, based on the presence of non-synonymous single nucleotide polymorphism (SNPs) that were identified by comparing Basmati genome sequence with that of Nipponbare. CONCLUSIONS: To the best of our knowledge, the current study is the first attempt ever made to carry out genome-wide mapping for the dissection of the genetic basis of economically important traits of Basmati rice. The promising QTLs controlling important traits in Basmati rice, identified in this study, can be used as candidates for future marker-assisted breeding.

Assessment of the influence of amylose-LPC complexation on the extent of wheat starch digestibility by size-exclusion chromatography.

Amylose forms inclusion complexes with lysophosphatidylcholine (LPC), that decrease the susceptibility of amylose to amylase degradation. This study on the influence of complexation on starch susceptibility to amylase explains the nature of this protective effect. Wheat starch suspensions (9% w/w) containing 0.5-5% LPC were subjected to hydrolysis by porcine pancreatic α-amylase at 37 °C for several digestion times. The digesta were analysed by size-exclusion chromatography (SEC). The molar mass distribution was closely dependent on the digestion time and amount of LPC. This study precisely demonstrates the alteration of the digestion profile of starch on a molecular level, influenced by amylose-LPC complexation; however the effect depends on the digestion time. During 15 and 30 min digestion, inclusion complexes not only protect amylopectin in the initial hydrolysis stage, but also demonstrate lower susceptibility of the molecular amylose complexes to amylase hydrolysis. Digestion for 240 min resulted in a lower oligosaccharide peak concentration, in the presence of a high LPC concentration, which is related to less degradation of complexed amylose fraction.

Production of resistant starch by extrusion cooking of acid-modified normal-maize starch.

The objective of this study was to utilize extrusion cooking and hydrothermal treatment to produce resistant starch (RS) as an economical alternative to a batch-cooking process. A hydrothermal treatment (110 degrees C, 3 d) of batch-cooked and extruded starch samples facilitated propagation of heat-stable starch crystallites and increased the RS contents from 2.1% to 7.7% up to 17.4% determined using AOAC Method 991.43 for total dietary fiber. When starch samples were batch cooked and hydrothermally treated at a moisture content below 70%, acid-modified normal-maize starch (AMMS) produced a greater RS content than did native normal-maize starch (NMS). This was attributed to the partially hydrolyzed, smaller molecules in the AMMS, which had greater mobility and freedom than the larger molecules in the NMS. The RS contents of the batch-cooked and extruded AMMS products after the hydrothermal treatment were similar. A freezing treatment of the AMMS samples at -20 degrees C prior to the hydrothermal treatment did not increase the RS content. The DSC thermograms and the X-ray diffractograms showed that retrograded amylose and crystalline starch-lipid complex, which had melting temperatures above 100 degrees C, accounted for the RS contents.

Monte Carlo simulation of multiple attack mechanism of alpha-amylase.

Porcine pancreatic alpha-amylase (EC 3.2.1.1) produces short maltooligosaccharides from a single enzyme-substrate complex without dissociation by multiple or repetitive attack. Multiple attack is caused by relative sliding of the enzyme along the product chain of the enzyme-product complex without dissociation to form another productive complex. The Monte Carlo method was applied to the multiple attack mechanism to predict product distribution from amylose and amylopectin molecules of arbitrary sizes. The position of the initial attack to make the enzyme-substrate complex and branched reaction paths from the enzyme-product complex were selected by random numbers and probabilities. A simulated product distribution from 100,000 samples of amylose of chain length greater than 80 agreed completely with experimental data at the early stage of hydrolysis of amylose of mean chain length 90. On the other hand, the simulated product distribution from amylopectin agreed with experimental data of potato amylopectin when the effective chain length of the A chain was 9. Since the mean chain length of the A chain of potato amylopectin is 15, it is possible that amylopectin is partially compact in solution, so that the enzyme can recognize and act only on the outer side of the A chain at the early stage of digestion.