A rapid,automated method for measuring α-amylase in pre-harvest sprouted (sprout damaged) wheat

The quality of wheat for baking is critically dependent on the level of α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1), which can be present as “late maturity α-amylase” (LMA), or due to pre-harvest sprouting due to high rainfall and humidity at the time of harvesting. The most commonly used method to measure α-amylase in wheat grain is the Hagberg Falling Number method, but values are also influenced by rheological properties of starch in the grain. In this study we describe a simple, rapid, automated method (Amylase SD) for measurement of α-amylase in pre-harvest sprouted (sprout damaged) wheat grain. The method (Amylase SD) measures the release of p-nitrophenol from 4,6-O-ethylidene-α-4-nitrophenyl-maltoheptaoside by α-amylase in the presence of α-glucosidase. The absorbance of p-nitrophenolate measured at 405 nm in a ChemWell^®-T auto-analyser is directly related to the level of α-amylase activity present in the milled wheat grain extract. The Amylase SD method generated <6%CV and correlation to the Falling Number method was represented by an inflection point at ∼160 s. The precision, sensitivity and speed of this method provides an ideal alternative to the Falling Number method for measurement of α-amylase (sprout damage) in wheat grain in wheat breeding programmes or at grain receival points.     

The heterogeneous distribution of α-amylase and endoxylanase activity over a population of preharvest sprouted wheat kernels and their localization in individual kernels

To develop targeted approaches to improve the quality of preharvest sprouted (PHS) wheat as a raw material for food manufacturing, knowledge on the nature and distribution of hydrolytic enzymes in PHS wheat is crucial. Results of the present study indicate that α-amylase and endoxylanase activities are heterogeneously distributed among a population of PHS kernel. Within individual severely sprouted kernels, the enzyme activities are heterogeneously distributed throughout the different tissues. α-Amylase activity, almost exclusively of endogenous nature, is mainly detected in the germ region and to a lesser extent in the aleurone layer. Endoxylanase activity is predominantly of microbial origin and located on the kernel surface. In spite of this, light and epifluorescence microscopy show decreased kernel integrity and cell wall breakdown in the crushed cells layer, the endosperm, and the aleurone layer in a selection of kernels upon preharvest sprouting. This knowledge offers opportunities for the development of treatments to reduce the enzyme load in PHS wheat at postharvest level to improve its flour quality.     

Pre-maturity α-amylase in wheat: The role of abscisic acid and gibberellins

The occurrence of pre-maturity α-amylase (PMA) is a major cause of poor bread-making quality (low Hagberg Falling Number) in wheat grain. In susceptible genotypes, it involves the excessive accumulation of high isoelectric point (pI) α-amylase in mature grain prior to germination and in the absence of pre-harvest sprouting. Several factors regulate PMA formation in developing grain, including genotype, agronomy, and environmental conditions. In particular, a cold period during mid-grain development has been found to be a major stimulus for PMA induction. Although the factors affecting the PMA occurrence are well known, little is known about the molecular mechanism governing its induction. The plant hormones abscisic acid (ABA) and gibberellins (GAs) influence various aspects of grain development, and it has been suggested that PMA involves changes in the amount of these hormones or the sensitivity of the grain to these hormones. This review summarizes recent studies investigating the role of ABA and GAs in PMA induction and PMA occurrence.     

Content of starch and sugars and in vitro digestion of starch by α-amylase in five minor millets

Five varieties of minor millets were studied for their amylose, soluble amylose, amylopectin, soluble amylopectin, reducing sugar, total sugar and starch contents. Pure starch was isolated from each variety and the enzymic degradation of starch by porcine pancreatic -amylase were examined with and without gelatinisation. Gelatinised sample ofEchinochloa frumentacea (var. K2) showed minimal hydrolysis and gelatinised sample ofPanicum miliaceum (var. CO3) showed maximum hydrolysis of starch by porcine pancreatic -amylase. Gelatinised starch was highly susceptible to enzymic digestion when compared to ungelatinised starch. The extent of starch degradation varied from 71 to 85 percent in gelatinised samples and starch degradation in ungelatinised sample varied from 10 to 18 percent.     

Inhibition study of red rice polyphenols on pancreatic α-amylase activity by kinetic analysis and molecular docking

This study sought to investigate the possible inhibition mechanism of red rice polyphenols (RRP) on pancreatic α-amylase (PA) activity. RRP showed strong inhibition against PA activity and the half-inhibitory concentration (IC₅₀) value was 3.61 μg/mL. The fluorescence quenching of PA by RRP was a combination of static quenching and dynamic quenching. RRP could aggregate with PA and the physiochemical properties of the aggregates were closely related to the concentration of RRP. Kinetic analysis suggested that the inhibition mode of RRP on PA was reversible inhibition, which was a mixing of competitive inhibition and noncompetitive inhibition. Molecular docking speculated that RRP could form hydrogen bonds with PA by binding to the catalytic active sites (ASP197, GLU233 and ASP300) and the microenvironments of TRP58 and TRP59 were altered, thus inhibiting PA activity.     

The role of sensitivity to abscisic acid and gibberellin in pre-maturity α-amylase formation in wheat grains

To study the role of abscisic acid (ABA) and gibberellin (GA) sensitivity in regulating pre-maturity α-amylase (PMA) in wheat grains, plants were grown in a glasshouse under cold-shock and ambient conditions. α-amylase activity in response to applied ABA and GA was measured in detached-grains with the embryo removed (in vitro) and in intact-grains attached to the plant (in situ). The in vitro experiment was conducted using Spark (low PMA-susceptible genotype) and Rialto (highly PMA-susceptible genotype), with the aim of defining the time point for GA-sensitivity. The results showed an increase in GA-sensitivity at about 640 degree days after anthesis (DAA) in Rialto. There was no evidence for a change in ABA-sensitivity in either variety. The in situ experiments were conducted using genotypes from a Spark × Rialto doubled haploid population segregating for the Rht-D1a (tall) or Rht-D1b allele and for the presence or absence of 1BS/1RS. For Rht-D1a (tall) or Rht-D1b genotypes with or without 1BS/1RS, the cold-shock significantly increased GA-sensitivity, whereas there was no significant change in ABA-sensitivity. These results show PMA is related to an increase in GA-sensitivity that occurs in the aleurone at around 640 degree DAA, and can be enhanced by environmental factors (e.g. cold-shock).     

The role of sensitivity to abscisic acid and gibberellin in pre-maturity α-amylase formation in wheat grains

To study the role of abscisic acid (ABA) and gibberellin (GA) sensitivity in regulating pre-maturity α-amylase (PMA) in wheat grains, plants were grown in a glasshouse under cold-shock and ambient conditions. α-amylase activity in response to applied ABA and GA was measured in detached-grains with the embryo removed (in vitro) and in intact-grains attached to the plant (in situ). The in vitro experiment was conducted using Spark (low PMA-susceptible genotype) and Rialto (highly PMA-susceptible genotype), with the aim of defining the time point for GA-sensitivity. The results showed an increase in GA-sensitivity at about 640 degree days after anthesis (DAA) in Rialto. There was no evidence for a change in ABA-sensitivity in either variety. The in situ experiments were conducted using genotypes from a Spark × Rialto doubled haploid population segregating for the Rht-D1a (tall) or Rht-D1b allele and for the presence or absence of 1BS/1RS. For Rht-D1a (tall) or Rht-D1b genotypes with or without 1BS/1RS, the cold-shock significantly increased GA-sensitivity, whereas there was no significant change in ABA-sensitivity. These results show PMA is related to an increase in GA-sensitivity that occurs in the aleurone at around 640 degree DAA, and can be enhanced by environmental factors (e.g. cold-shock).     

Studies on α-amylase and trypsin inhibitors in legume seeds using agar diffusion and isoelectric focusing techniques

Amylolytic and tryptic inhibitors of faba bean extracts were determined by an agar diffusion test. The amylolytic inhibitor had protein characters. Furthermore, water-soluble trypsin inhibitors ofCicer arietinum, Lens esculenta, Lupinus termis, Phaseolus vulgaris, Pisum sativum, Trigonella foenum-graecum andVicia faba which were separated by polyacrylamide gel isoelectric focusing (PAGIF) in thin-layers, showed species specific patterns. Negative staining showed 10 bands for French beans, 9 for fenugreek seeds, 8 for lentils and chickpeas, 7 for peas and 6 for faba beans. Lupin seeds were free from trypsin inhibitors. Treatments (soaking, germination and heat processing) of faba beans reduced the number of trypsin inhibitors in PAGIF patterns, less after soaking and germination, but more after roasting and frying. No inhibitors were detected after cooking.     

Investigating the impact of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough fermentation and bread sugar levels

Wheat flour is generally supplemented with α-amylases to increase maltose levels in bread dough and increase loaf volume. While the preference of yeast for glucose and fructose over maltose as substrate for fermentation is well documented, the impact of maltose versus glucose producing enzymes on bread dough fermentation kinetics and bread sugar levels is ill documented. Hence the impact of α-amylase, α-glucosidase and glucoamylase action on both aspects was investigated. Glucoamylase and α-amylase increase the total fermentable sugar content of dough, while α-glucosidase only affects the glucose/maltose ratio. Due to their effect on total fermentable sugar levels, addition of α-amylase or glucoamylase prolongs the total productive fermentation time, while this is not the case for α-glucosidase. In contrast to α-amylase, both glucoamylase and α-glucosidase supplementation leads to higher CO₂ production rates during the initial stages of fermentation. In the final bread product, different sugar levels are observed depending on the dosage and type of starch-degrading enzyme. The results of this study imply that long and short fermentation processes benefit from α-amylase and α-glucosidase addition, respectively, while glucoamylase supplementation is suitable for both long and short fermentation times.     

Interspecific competition,N use and interference with weeds in pea–barley intercropping

Field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) were intercropped and sole cropped to compare the effects of crop diversity on productivity and use of N sources on a soil with a high weed pressure. ¹⁵N enrichment techniques were used to determine the pea–barley–weed-N dynamics. The pea–barley intercrop yielded 4.6 t grain ha⁻¹, which was significantly greater than the yields of pea and barley in sole cropping. Calculation of land equivalent ratios showed that plant growth factors were used from 25 to 38% more efficiently by the intercrop than by the sole crops. Barley sole crops accumulated 65 kg soil N ha⁻¹ in aboveground plant parts, which was similar to 73 kg soil N ha⁻¹ in the pea–barley intercrop and significantly greater than 15 kg soil N ha⁻¹ in the pea sole crop. The weeds accumulated 57 kg soil N ha⁻¹ in aboveground plant parts during the growing season in the pea sole crops. Intercropped barley accumulated 71 kg N ha⁻¹. Pea relied on N₂ fixation with 90–95% of aboveground N accumulation derived from N₂ fixation independent of cropping system. Pea grown in intercrop with barley instead of sole crop had greater competitive ability towards weeds and soil inorganic N was consequently used for barley grain production instead of weed biomass. There was no indication of a greater inorganic N content after pea compared to barley or pea–barley. However, 46 days after emergence there was about 30 kg N ha⁻¹ inorganic N more under the pea sole crop than under the other two crops. Such greater inorganic N levels during early growth phases was assumed to induce aggressive weed populations and interspecific competition. Pea–barley intercropping seems to be a promising practice of protein production in cropping systems with high weed pressures and low levels of available N.     

Biological and chemical evaluation of chick pea seed proteins as affected by germination,extraction and α-amylase treatment

The effects of germination, extraction (double extraction with 70% ethanol and water at isoelectric point) and -amylase treatments of chick pea seed flours on crude protein, total carbohydrate, protein efficiency ratio (PER), biological value (BV), true digestibility (TD), net protein utilization (NPU), essential amino acid composition, in-vitro protein digestibility (IVPD) and actual amino acid indices (essential amino acid index or amino acid score) were evaluated. Crude protein content was increased (8–149%), while total carbohydrate was decreased (11–62%) by germination, extraction and -amylase treatments. Alpha-amylase treatment was more efficient in reducing total carbohydrate and increasing the protein content than that of extraction treatment. The protein quality of chick pea flours as measured by PER, BV, TD, NPU, IVPD and corrected amino acid indices (actual amino acid indices×IVPD) was significantly improved by these treatments. The protein quality of germinated--amylase treatment was comparble with casein, while germinated--amylase treaded seeds appeared nutritionally superior to casein. The results indicate that the germinated--amylase and germinated--amylase-extracted treatments could be used successfully as a source of concentrated high quality protein for baby food production. The corrected amino acid indices gave better prediction of PER, BV, TD and NPU (r=93 to 97) than actual amino acid indices (r=45 to 71). PER was highly correlated with corrected amino acid score (r=0.93). The PER could be predicted from the following simple regression equation: PER=–1.827+0.0561×corrected amino acid score.     

Inhibitory Activity of Cinnamon Bark Species and their Combination Effect with Acarbose against Intestinal α-glucosidase and Pancreatic α-amylase

Inhibition of α-glucosidase and pancreatic α-amylase is one of the therapeutic approaches for delaying carbohydrate digestion, resulting in reduced postprandial glucose. The aim of this study was to evaluate the phytochemical analysis and the inhibitory effect of various cinnamon bark species against intestinal α-glucosidase and pancreatic α-amylase. The results showed that the content of total phenolic, flavonoid, and condensed tannin ranged from 0.17 to 0.21 g gallic acid equivalent/g extract, from 48.85 to 65.52 mg quercetin equivalent/g extract, and from 0.12 to 0.15 g catechin equivalent/g extract, respectively. The HPLC fingerprints of each cinnamon species were established. Among cinnamon species, Thai cinnamon extract was the most potent inhibitor against the intestinal maltase with the IC₅₀ values of 0.58 ± 0.01 mg/ml. The findings also showed that Ceylon cinnamon was the most effective intestinal sucrase and pancreatic α-amylase inhibitor with the IC₅₀ values of 0.42 ± 0.02 and 1.23 ± 0.02 mg/ml, respectively. In addition, cinnamon extracts produced additive inhibition against intestinal α-glucosidase and pancreatic α-amylase when combined with acarbose. These results suggest that cinnamon bark extracts may be potentially useful for the control of postprandial glucose in diabetic patients through inhibition of intestinal α-glucosidase and pancreatic α-amylase.     

Studies on bioactivities of tea (Camellia sinensis L.) fruit peel extracts: Antioxidant activity and inhibitory potential against α-glucosidase and α-amylase in vitro

Tea fruit peel is the main byproduct during manufacture of tea seed oil. The great increase in tea seed oil production in recent years brings the challenge of finding application for tea fruit peel. The aims of this study were to obtain tea fruit peel extracts enriched with bioactive compounds by several solvent extraction methods and to evaluate their antioxidant activity and inhibitory potential against α-glucosidase and α-amylase in vitro. Flavonoids and phenolics were accumulated in ethyl acetate, butanol, and chloroform fractions, and these fractions possessed much better antioxidant activities, including scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) cation radicals (ABTS⁺), and reducing activity, compared with those of the 75% ethanol extract and water fraction. Moreover, the ethyl acetate, butanol, and chloroform fractions exhibited excellent inhibitory activity against α-glucosidase, much stronger than that of the positive control (acarbose). These fractions also showed mild inhibition on α-amylase activity.     

Two β-amylase genes,OsBAM2 and OsBAM3, are involved in starch remobilization in rice leaf sheaths

To identify mechanisms of starch degradation in rice leaf sheaths at the post-heading stage, we investigated the function of OsBAM2 and OsBAM3, which encode plastid-targeted active β-amylase isoforms, in starch remobilization in leaf sheaths. The starch content in the second leaf sheaths below the flag leaf (the third leaf sheaths) peaked at the flag leaf emergence stage and gradually decreased until 15 days after heading. The mRNA levels of OsBAM2 and OsBAM3 in the third leaf sheaths increased from the flag leaf emergence stage to the heading stage when the starch content began to decrease. However, these mRNA levels did not always remain high during post-heading. Overexpression of OsBAM2 or OsBAM3 markedly repressed starch accumulation in the third leaf sheaths, showing that OsBAM2 and OsBAM3 function in starch degradation in rice leaf sheaths. In contrast, no significant differences in starch content in the third leaf sheaths were detected between knockdown plants of OsBAM2 or OsBAM3 and non-transgenic wild-type plants. Our results suggest that reduced expression of the individual genes, OsBAM2 or OsBAM3, does not result in excess accumulation of starch in the leaf sheaths, probably because of the complementary function of another gene or the action of other genes encoding starch-degrading .     

Polymorphism and pedigree analysis of β-amylase isozymes in Australian wheat

β-Amylase encoded by multiple loci on chromosomes of triticeae crop plants is a starch metabolic enzyme with a significant physiological role in flour functionality. In this study, wheat grain β-amylase isozymes were characterized using an extensive Australian wheat population and genetic breeding/mapping lines. β-amylase isozymes were fractionated by high-resolution native polyacrylamide gel electrophoresis (PAGE) and validated by zymogram and peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Specific antibodies were generated against the β-amylase purified through protein chromatography and used for β-amylase identification. Further characterization of wheat β-amylases was carried out with immunoblotting and 2-dimensional electrophoresis (2-DE). The enzyme was found to be polymorphic among the Australian and foreign wheat cultivars, with a total of six isozymes: two common and four varying isozymes, the latter group including one novel β-amylase isozyme of fast-mobility in native PAGE, According to pedigree analysis, the origin of this particular isozyme was from the ancestral genotype, Ciano F 67.     

Effects of roasting on barley β-glucan,thermal, textural and pasting properties

Different hulled barley cultivars were subjected to roasting in hot sand (280 °C) and the effects on β-glucan extractability, physico-chemical, thermal and pasting properties were studied. The grain puffed upon roasting and grain hardness and colour difference ΔE were significantly lowered. The roasted barley flour had significantly higher water absorption and water solubility index. The cultivars DWR-28, RD-2503 and PL-172 had the highest total β-glucan content (up to 5.47%). Roasting brought about a decrease in the soluble β-glucan content in all the cultivars and this decrease ranged from 4.9 to 25.3%. The β-glucan extractability was not affected by the roasting process but roasting lowered the ratio of soluble to insoluble β-glucan content by 8.1–41.9%. Roasting significantly affected the pasting and thermal properties of the flours, together with an increase in the cooked starch content that ranged from 28.8 to 43.1%.     

Correlations between ultrastructure and photosynthesis in leaves of rice and barnyard-grass

The ultrastructure of the leaves of rice(Oryza sativa)and barnyardgrass(Echinochloa crus-galli)in their milking stage were studied withelectron microscopy.The content of chloro-     

Relationships between ultra-drying tolerance and heat-stable proteins in riceseeds

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