α-Amylase SD Assay Kit

α-Amylase content in milled wheat is a key quality parameter in the baking industry. This metric is usually determined using the Hagberg Falling Number (FN) method, the results of which can be used to segment grains into a range of quality categories. The authors describe herein an alternative to the traditional FN assay, namely the Amylase SD assay, as a predictor of baked bread quality. 144 Wheat grain samples with known FN values were analysed using the Amylase SD assay and a simple model for the conversion of Amylase SD units into “predicted FN” (PFN) values has been described. To test the usefulness of this approach, 11 separate wheat grain samples were analysed using the FN method, the Amylase SD assay, and also for gluten content. These grain samples were then milled to produce flour which was re-analysed using the Amylase SD assay and then formulated into dough that was subjected to Mixolab analysis, before being baked to produce pan bread. Correlations of baked bread volume and common Mixolab parameters to both FN and PFN values have been investigated for each sample. The Amylase SD assay has been shown to be a potentially promising alternative to the FN method for use as a baked bread quality predictor.

Background The measurement of α-amylase (EC 3.2.1.1) in sprout-damaged grains is a crucial analysis yet a problematic one owing to the typically low α-amylase levels in ground wheat samples. A number of standardised methods such as the Falling Number method and the Ceralpha method exist which are routinely used for the assay of α-amylase. These methods, however, are either highly substrate-dependent or lack the required sensitivity to assess sprout damage. Results Novel colorimetric and fluorometric reagents have been prepared (Amylase HR, Amylase SD, BzCNPG₇ reagent and BzMUG₇ reagent) for the direct and specific assay of α-amylase activity in sprout-damaged wheat. Assays employing these reagents have been developed and optimised to include a decolourisation step using activated charcoal. When used in a convenient assay format, Amylase SD – containing EtNPG₇ (II) as the colorimetric substrate and α-glucosidase as the ancillary enzyme – was found to be an excellent reagent for the assessment of sprout damage in wheat with incubation times as short as 5 min. Conclusion The assay using Amylase SD is completely specific for α-amylase. The use of the Amylase SD assay represents a sensitive and valid alternative to the traditionally used Falling Number values for the assessment of sprout damage in wheat samples.

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.

Dormancy in barley has been thoroughly studied and shown to negatively impact malt quality, resulting in selection against dormancy. However, reduced dormancy coincides with increased preharvest sprout (PHS) risk, thus sparking a new interest in integrating dormancy back into American barley lines if the negative effects of dormancy on malt quality can be overcome. We evaluated the dormancy and hydration index (HYI) in a biparental mapping population to determine the genotypes that would protect against PHS but have good malt quality. We found 4 HYI QTLs and 4 dormancy QTLs, one of which was near the well-described SD2 QTL. The HYI QTLs were pleiotropically related to seed size (1H), dormancy (5H) and malt quality (2H). Lines with dormancy (5H) and increased HYI (2H and 3H) had malt quality similar to nondormant lines while maintaining PHS resistance, suggesting improvements in HYI could be the key to overcoming the negative effects of dormancy in malting.

(1) Background: Chayote [Sechium edule Jacq. (Sw.)] is a non-traditional export product; recently, demand has increased due to its nutritional and functional properties. There is a wide diversity of varietal groups (VGs) within this species. Despite this, only virens levis and nigrum spinosum varieties are commercialized on a large scale, while the rest are underutilized and poorly studied, so the genetic pool of this species is at risk. (2) Methods: The following variables were evaluated in the fruits of 10 chayote groups of varieties: shape, size, weight, stomatal frequency (SF), stoma size, stomatal index (SI), color index (CO*), pigments, titratable acidity (TA), total soluble solids (TSS), total sugars and moisture content. In addition, the postharvest behavior of the ten VGs stored at room temperature and the effect of 1-MCP on fruit quality during cold storage were evaluated. (3) Results: The groups a. minor and n. minor showed rapid weight loss, the albus varieties showed high epidermis oxidation, while v. levis, n. maximum, n. spinosum and n. xalapensis were susceptible to viviparity, blisters and fungal incidence. 1-MCP prevented chilling injury (CI) and weight loss. (4) Conclusions: The diversity of postharvest characteristics allows the use of VGs for different uses such as a fresh fruit, agroindustrial transformation or mixing with other vegetables.

Dough retardation is commonly used to extend dough shelf-life, but it poses a challenge for flatbreads due to their large surface. This study explored the sourdough fermentation of oats and barley, addressing challenges in the retardation of dough for flatbread. Sourdough, using flour only or flour blended with bran (3:1), was fermented with a LIVENDO LV1 starter at 30°C for 24 h. The pH value, microbial viable cell count, total titratable acidity and organic acids concentration of the sourdough were measured. The properties of dough and flatbread, depending on the retardation time (24 h and 48 h), sourdough type (oat or barley) and sourdough level (30% or 50% dough weight), were investigated. Oat flour’s limited acidification improved with the inclusion of bran, resulting in a desirable pH, TTA, and lactic to acetic acid ratio after 15 h of fermentation, which were comparable to results achieved with barley sourdough. The sourdough addition slowed down the enzymatic browning of dough during retardation. Dough retardation at 24 h reduced the phytates content (32-38%) and crumb hardness (9-16%), depending on the sourdough type and level. In dough retardation, β-glucans were degraded by up to 9% in the case of oats and by up to 28% in the samples with barley. Overall, adding oat or barley sourdough at a 30% dough weight can be recommended to enhance flatbread’s nutritional value and prolong its shelf life.

This study examined the effects of pulsed electric field (PEF) treatment on enzymes, non-starch polysaccharides, and bread-making potential of oat and barley flour. Enzyme activity, microstructure, β-glucan extractability, molecular weight (Mw) and structure of non-starch polysaccharides, dough rheology, and flat bread properties were determined. An exponential decay model explained better the residual activity of oat β-glucanase across electric field intensity than barley β-glucanase. PEF treatment of flour at 12 kV/cm for 162 ms significantly reduced β-glucanase activity (40.2-76.5%) while increasing the concentration of total β-glucans (33.5%) and water-extractable arabinoxylans (36-41%). Mw of linear β-d-glucans decreased (9%) while Mw of branched arabinoxylans increased (6-33%). Scanning electron microscopy showed changes in microstructure of barley proteins. Blending wheat flour (70%) with oat or barley flour (30% weight) after PEF treatment enhanced gluten aggregation energy (29-19%) and breakdown viscosity (18-43%) of dough, as well as increased β-glucan content (21-32%) but reduced specific volume (11-24%). The findings of this study provide a comprehensive insight into the PEF’s potential for retarding enzymatic reactions and preserving integrity of cereal non-starch polysaccharides.

In the present study, yellow pea (CDC Amarillo) and faba bean (CDC Snowdrop) seeds were soaked overnight and then germinated in the dark at ambient temperature for 24, 48 and 72 h. During the short-term germination, germination percentages higher than 96.6% were achieved and progressive growth of radicles was observed for both varieties. The soaked and germinated seeds were dried at 55°C and milled into flours, and their chemical compositions, physicochemical properties and in vitro starch and protein digestibility were systematically examined. Overall, soaking and germination did not noticeably alter the chemical compositions of each flour. The most obvious changes in the physicochemical properties were found in the pasting, emulsifying and foaming properties of the pulse flours. Soaking and 24-h germination greatly enhanced the pasting viscosities of the flours; as the germination proceeded, their viscosities gradually decreased, resulting from the degradation of starch by endogenous amylase(s) during pasting. Germination progressively improved the emulsion activity and stability, foaming capacity and foam stability of both pulse flours. In addition, germination enhanced the in vitro digestibility of starch and protein of the flours; however, the treatment did not improve their in vitro protein digestibility corrected amino acid scores (IV-PDCAAS). Short-term germination of 24-72 h has been demonstrated to be an effective approach to generating pulse flours possessing diverse functional properties and enhanced digestibility of macronutrients.

Background and objectives: There are few methods in wheat to directly measure α-amylase, which is the result of pre-harvest sprouting (PHS) and late-maturity α-amylase (LMA). Both of these conditions are associated with reduced wheat and flour quality. Those methods that do exist for measuring α-amylase in wheat are based on enzyme activity. The objective of this study was to evaluate commercially available enzyme linked immunosorbent assay (ELISA) kits on sound grain, sprouted grain, and grain that had both PHS and LMA. Findings: Three kits were evaluated; human AMY1 and AMY2, and a “plant α-amylase” kit. The wheat samples were also assayed for α-amylase activity using the Megazyme SD α-amylase method. All three ELISA kits functioned very well based on calibration curves (R² > 0.99). Of the three, the AMY1 and plant α-amylase kits appeared to show no useful detection of wheat α-amylase. The AMY2 kit, however, detected a 2.7 fold increase in α-amylase protein in the PHS and PHS/LMA grain lots, similar to a 2.7 fold difference in activity. Conclusions: It appears that the particular human AMY2A ELISA kit, and perhaps others, can detect α-amylase in wheat and may be a useful alternative technology to enzyme kits. These kits may also be useful in predicting PHS and LMA potential damage. Significance and novelty: Thus far, α-amylase studies have focused on enzyme kits, whereas this study examined three commercially available ELISA kits to measure α-amylase content in sound, sprouted, and PHS/LMA grain. Both PHS and LMA are highly detrimental to grain and result in low prices paid to growers.