β-Amylase Assay Kit (Betamyl-3)

The primary quality assessor of wheat grain is the Hagberg Falling Number (FN) method. This is a viscometric test surrogate for α-amylase activity. Despite being used for over sixty years, FN has been increasingly scrutinised due to its low throughput, poor reproducibility and inability to differentiate between the causes of low FN including Pre-harvest Sprouting (PHS) and Late Maturity α-Amylase (LMA). Our study describes initial efforts to analyse a specific wheat flour set tailored for the identification of enzymatic candidates that would allow discrimination between PHS and LMA affected grains. Using the sensitive enzyme-coupled assay substrate R-AMGR3, results suggest that α-glucosidase (exo-α-glucosidase) is a potential enzyme marker candidate to specifically detect sprouted but not LMA-affected grain.

A simple method for measurement of the amylolytic activity of malt has been developed and fully evaluated. The method, termed the Maltose Value (MV) is an extension of previously reported work. Here, the MV method has been studied in detail and all aspects of the assay (sample grinding and extraction, starch hydrolysis, maltose hydrolysis and determination as glucose) have been optimised. The method is highly correlated with other dextrinising power methods. The MV method involves extraction of malt in 0.5% sodium chloride at 30°C for 20 minutes followed by filtration; incubation of an aliquot of the undiluted filtrate with starch solution (pH 4.6) at 30°C for 15 min; termination of reaction with sodium hydroxide solution; dilution of sample in an appropriate buffer; hydrolysis of maltose with a specific α-glucosidase; glucose determination and activity calculation. Unlike all subsequent reducing sugar methods, the maltose value method measures a defined reaction product, maltose, with no requirement to use equations to relate analytical values back to Lintner units. The maltose value method is the first viable method in 130 years that could effectively replace the 1886 Lintner method.

Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α-amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65^oC (MIM 65^oC). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times.

A procedure previously developed for the assay of cereal-flour β-amylase has been improved and standardised. The improved procedure uses the substrate p-nitrophenyl maltopentaose (PNPG5) in the presence of near saturating levels of α-glucosidase. PNPG5 is rapidly hydrolysed by β-amylase but less readily by cereal α-amylases. The substrate is hydrolysed by β-amylase to maltose and p-nitrophenyl maltotriose (PNPG3). With the levels of α-glucosidase used in the substrate mixture, PNPG3 is rapidly cleaved to glucose and p-nitrophenol, whereas PNPG5 is resistant to hydrolysis by the α-glucosidase. The assay procedure has been standardised for several β-amylases and the exact degree of interference by cereal α-amylases determined. The procedure can be readily applied to the selective measurement of β-amylase activity in cereal and malted cereal-flours.

β-Amylase hydrolyzes polysaccharides, such as starch, into maltose. It is used as an industrial enzyme in the production of food and pharmaceuticals. The eukaryotic red alga Cyanidioschyzon merolae is a unicellular alga that grows at an optimum pH of 2.0-3.0 and an optimum temperature of 40-50°C. By focusing on the thermostability and acid resistance of the proteins of C. merolae, we investigated the properties of β-amylase from C. merolae (hereafter CmBAM) and explored the possibility of using CmBAM as an industrial enzyme. CmBAM showed the highest activity at 47°C and pH 6.0. CmBAM had a relatively higher specificity for amylose as a substrate than for starch. Immobilization of CmBAM on a silica gel carrier improved storage stability and thermostability, allowing the enzyme to be reused. The optimum temperature and pH of CmBAM were comparable to those of existing β-amylases from barley and wheat. C. merolae does not use amylose, but CmBAM has a substrate specificity for both amylose and amylopectin but not for glycogen. Among the several β-amylases reported, CmBAM was unique, with a higher specificity for amylose than for starch. The high specificity of CmBAM for amylose suggests that isoamylase and pullulanase, which cleave the α-1,6 bonds of starch, may act together in vivo. Compared with several reported immobilized plant-derived β-amylases, immobilized CmBAM was comparable to β-amylase, with the highest reusability and the third-highest storage stability at 30 days of storage. In addition, immobilized CmBAM has improved thermostability by 15-20°C, which can lead to wider applications and easier handling.

Bambara groundnut (BGN) is termed a complete food due to its nutritional composition and has been researched often for its nutritional constituents. Malting BGN seeds have shown improved nutritional and functional characteristics, which can be used to produce an amylase-rich product as a functional ingredient for food and beverage production in homes and industries. The aim of this study was to investigate the enzyme and antioxidant activities of malted BGN affected by steeping and sprouting times. BGN was malted by steeping in distilled water at 25-30°C for 36 and 48 h and then sprouted for 144 h at 30°C. Samples were drawn every 24 h for drying to study the effect of steeping and sprouting times on the moisture, sprout length, pH, colour, protein content, amylase, total polyphenols, and antioxidant activities of the BGN seeds. The steeping and sprouting times significantly affected the BGN malt colour quality and pH. The protein content of the malted BGN seeds was not significantly different based on steeping and sprouting times. Steeping and sprouting times significantly affected the α- and β-amylase activities of the BGN seeds. The activity of amylases for 36 and 48 h steeping times were 0.16 and 0.15 CU/g for α-amylase and were 0.22 and 0.23 BU/g for β-amylase, respectively. Amylase-rich BGN malt was produced by steeping for 36 h and sprouting for 96 h. Amylase-rich BGN malt can be useful as a functional food ingredient in food and beverage formulations.

Rutin was nano-encapsulated in date [En-Ru(D)] and mushroom [En-Ru(M)] β-glucan matrix to protect it from the harsh gastrointestinal environment and to enhance its bioavailability and biological activity upon digestion. The encapsulation was carried using green technology i.e., ultra-sonication. The En-Ru(D) and En-Ru (M) showed the hydrodynamic diameter of 314.04 and 482.21 nm with polydispersity index of 0.21 and 0.33. The in vitro release behaviour followed the Higuchi model. The antimicrobial activity of En-Ru(D) and En-Ru(M) were evaluated against gram negative E. coli (ATCC 25922) and gram positive (Staphylococcus aureus) bacteria. Furthermore, En-Ru(D) and En-Ru(M) exhibited increased bioavailability of rutin in intestinal fluid with retention of anti-obesity and antioxidant activities after digestion (p < 0.05). Therefore, β-glucan matrix can efficiently encapsulate flavonoids and regulate the release of functional bioactive ingredients in the simulated human digestive conditions.

Retention of bioactive compounds (beta-glucans, lignans and gamma oryzanol) was analyzed after extrusion and during storage period of three months under four relative humidities using saturated salt solutions such as potassium carbonate (43.26%), magnesium nitrate (52.60%), potassium chloride (84.36%) and potassium nitrate (93.58%). The control sample comprising a corn and rice flour blend (50 g each) was substituted with beta-glucans at 3 g/100 g and 6 g/100 g, flaxseed lignans at 6.67 g/kg and 11.67 g/kg and gamma oryzanol at 1.5 g/100 g and 3.0 g/100 g at low and high levels, respectively. After extrusion, beta-glucan was retained up to 82.67 and 90.83%, lignans were retained at 86.31 and 66.66% whereas retention of gamma oryzanol was 71.33 and 51.67% at low and high level of substitution, respectively. Retention of bioactive compounds was the lowest along with a decrease in L* and b* values and an increase in a* value was observed under higher relative humidity (84.36% and 93.58%) storage conditions.

A largely defined series of hydrolytic enzymes active during malting and/or mashing, substantially determine the quality, profitability, and efficiency of the brewing process. These enzymes potentially hydrolyze starch, proteins and cell wall non-starch polysaccharides including β-glucan and arabinoxylan. Commercial malts (94) were assayed for the DP enzymes (limit dextrinase, beta/α-amylase), and NSP hydrolyzing enzymes (β-glucanase, xylanase, arabinofuranosidase, β-glucosidase). The levels of enzyme activity were related to conventional measures of malt quality such as extract, fermentability, protein, KI, DP, friability, wort viscosity, FAN, and β-glucan. These parameters were interrelated with less conventional measures of malt quality including coarse extract and fermentability (modified infusion mash 65 °C), lautering efficiency, the Small-scale Wort ‘I’ Filtration Test (SWIFT), and viscosity. Substantial variation was observed between the malt samples for all enzymes assayed. Australian barley, whether malted in Australia (n = 61) or China (n = 24), was observed to be of comparable quality. A limited set of Canadian barley samples (n = 9) were malted in China and produced malts with somewhat higher levels of extract, AAL, and some enzymes. Remarkably, the level of limit dextrinase was observed to be almost double that from previous investigations. Greater levels of steep water aeration were proposed to explain this dramatic increase. The interrelationships between the enzyme activities and malt quality identified, enable potential selection of novel malt quality parameters that are more predictive of a malt’s brewing performance (efficiency and quality) than current measures to provide a malt quality assessment system based on ‘functional’ malt quality.

The current trend in craft breweries is to carry out heavy dry-hopping by increasing the hopping rate. This practice sometimes leads to uncontrolled and aberrant aroma profile production. The aim of this work was to determine whether part of the enzymatic content of hop (α-amylase and β-amylase) could impact yeast metabolism, resulting in aroma profile modification during secondary fermentation. In this research, spectrophotometric methods were used to assess the amylase activity within hop. Moreover, liquid chromatographic methods (HPLC-ELSD) showed modification of the beer sugar profile by production of glucose and maltose as well as by the degradation of a higher degree of polymerization sugar by hop enzymes. Furthermore, gas chromatographic techniques (GC-ECD/FID) were used to assess yeast metabolism using vicinal diketones (diacetyl/pentanedione) as a marker of the secondary fermentation. Finally, a principal component analysis (PCA) of the yeast main aromas (esters, higher alcohols, and aldehydes) demonstrated the significance of this yeast-hop interaction on the beer’s aroma profile.

The use of exogenous maltogenic α-amylases or maltotetraogenic amylases of bacterial origin is common in wheat bread production, mainly as antistaling agents to retard crumb firming. To study the impact of maltogenic α-amylase and maltotetraogenic amylase on straight dough wheat bread, we performed a discovery-driven proteomics approach with commercial enzyme preparations and identified the maltotetraogenic amylase P22963 from Pelomonas saccharophila and the maltogenic α-amylase P19531 from Geobacillus stearothermophilus, respectively, as being responsible for the amylolytic activity. Quantitation of mono-, di- and oligosaccharides and residual amylase activity in bread crumb during storage for up to 96 h clarified the different effects of residual amylase activity on the sugar composition. Compared to the control, the application of maltogenic α-amylase led to an increased content of maltose and especially higher maltooligosaccharides during storage. Residual amylase activity was detectable in the breads containing maltogenic α-amylase, whereas maltotetraogenic amylase only had a very low residual activity. Despite the residual amylase activities and changes in sugar composition detected in bread crumb, our results do not allow a definite evaluation of a potential technological function in the final product. Rather, our study contributes to a fundamental understanding of the relation between the specific amylases applied, their residual activity and the resulting changes in the saccharide composition of wheat bread during storage.

β-amylase is a thermostable enzyme that hydrolyses starch during cooking of sweetpotato (Ipomoea batatas) storage roots, thereby influencing eating quality. Its activity is known to vary amongst genotypes but the genetic diversity of the beta-amylase gene (Amyβ) is not well studied. Amyβ has a highly conserved region between exon V and VI, forming part of the enzyme's active site. To determine the gene diversity, a 2.3 kb fragment, including the conserved region of the Amyβ gene was sequenced from 25 sweetpotato genotypes. The effect of sequence variation on gene expression, enzyme activity, and firmness in cooked roots was determined. Six genotypes carrying several SNPs within exon V, linked with an AT or ATGATA insertion in intron V were unique and clustered together. The genotypes also shared an A336E substitution in the amino acid sequence, eight residues upstream of a substrate-binding Thr344. The genotypes carrying this allele exhibited low gene expression and low enzyme activity. Enzyme activity was negatively correlated with firmness (R = -0.42) in cooked roots. This is the first report of such an allele, associated with low enzyme activity. These results suggest that genetic variation within the AmyB locus can be utilized to develop markers for firmness in sweetpotato breeding.

This study aimed to gain insights into the molecular mechanisms underlying the role of ethylene in regulating germination and seedling growth in wheat by combining pharmacological, molecular, and metabolomics approaches. Our study showed that ethylene does not affect radicle protrusion but controls post-germination endospermic starch degradation through transcriptional regulation of specific α-amylase and α-glucosidase genes, and this effect is mediated by alteration of endospermic bioactive gibberellin (GA) levels, and GA sensitivity via expression of the GA signaling gene, TaGAMYB. Our data implicated ethylene as a positive regulator of embryo axis and coleoptile growth through transcriptional regulation of specific TaEXPA genes. These effects were associated with modulation of GA levels and sensitivity, through expression of GA metabolism (TaGA20ox1, TaGA3ox2, and TaGA2ox6) and signaling (TaGAMYB) genes, respectively, and/or the abscisic acid (ABA) level and sensitivity, via expression of specific ABA metabolism (TaNCED2 or TaCYP707A1) and signaling (TaABI3) genes, respectively. Ethylene appeared to regulate the expression of TaEXPA3 and thereby root growth through its control of coleoptile ABA metabolism, and root ABA signaling via expression of TaABI3 and TaABI5. These results show that spatiotemporal modulation of ABA/GA balance mediates the role of ethylene in regulating post-germination storage starch degradation and seedling growth in wheat.

A slower rate of starch digestion in the small intestine increases the amount of resistant starch (RS) entering the large intestine, which is associated with health benefits. Although increasing the amylose (AM) content of dietary starch intake is one way to increase RS, the processes involved in gut microbial hydrolysis and fermentation of high AM-RS substrates are poorly understood. In this study, five high AM wheat (HAW) starches ranging from 47% AM to 93% AM and a wild type (37% AM), in both native granular and cooked forms, were subjected to in vitro fermentation with a porcine faecal inoculum. Fermentation kinetics, temporal microbial changes, amylolytic enzyme activities and residual starch were determined. All granular starches showed similar fermentation characteristics, independent of AM level, whereas cooking accelerated fermentation of lower AM but slowed fermentation of high AM starches. HAW starches with a very high AM content (>85%) all had similar fermentation kinetics and short-chain fatty acid end-product profiles. Microbial α-amylase, β-amylase, pullulanase and amyloglucosidase enzymatic activities were all detected and followed fermentation kinetics. HAW starch promoted shifts in the microbial community, with increases of the family Lachnospiraceae and the genus Treponema observed, while the genera Prevotella and Streptococcus were reduced in comparison to 37% AM. Overall, these findings suggest that any HAW starch incorporated into high RS food products would be expected to have beneficial microbiota-mediated effects in terms of fermentation kinetics and end products.

Sorghum is a climate resilient grain cereal crop originating from Africa that has traditionally been used to make a variety of fermented beverages such as pito and baijiu. In Western markets, the use of sorghum to produce beers and beverages has recently risen due to the visibility of a gluten sensitive/intolerant market and a growing interest in unique inputs for beverage production. As such, there is a developing body of research on sorghum as a malted input into beverages. A major limitation to the wider adoption of sorghum as a substrate in mashing is its low activity of amylolytic enzymes, either the result of insufficient activity or inhibition by endogenous compounds. A collection of 42 diverse accessions representing the grain sorghum diversity panel, was evaluated for associations between alpha and beta amylase content, race, origin, and seed colour as well as two classes of amylase inhibitors, phenols and tannins. Among these accessions are several commonly used genetic resources, including reference line BTx623. Notable findings include accessions with high alpha amylase content, sources that may harbour additional high amylase sorghums, associations with grain colour, and populations which may be used to genetically map the trait.

Hydrolysis of starch is key in several industrial processes, including brewing. Here, the activity and inactivation kinetics of amylases throughout barley malt mashing are investigated, as a prerequisite for rational optimisation of this process. Varietal differences were observed in the activity of α- and β-amylases as a function of temperature for six barley and malt varieties. These differences were not reflected in the resulting wort composition after mashing, using three isothermal phases of 30 min at 45°C, 62°C and 72°C with intermediate heating by 1°C/min. Thermal inactivation kinetics parameters determined for α- and β-amylases of an industrially relevant malt variety in a diluted system showed that enzymes were inactivated at lower temperatures than expected. The obtained kinetic parameters could predict α-amylase, but not β-amylase inactivation in real mashing conditions, suggesting that β-amylase stability is enhanced during mashing by components present or formed in the mash.

Global change is bringing barley with more frequency of suffering from high temperature. However, little has been known about the influence of high temperature on malt quality traits. In this study, we investigated the impact of 1-wk heat stress (32°C/26°C, day/night, 12 h/12 h) initiating from the 7th (HT7) and 14th (HT14) days after heading on some grain and malt quality traits of two barley cultivars. In comparison with normal temperature (24°C/18°C, day/night, 12 h/12 h), heat stress significantly reduced kernel weight, seed setting rate and grains per spike: HT7 having a larger effect than HT14. Meanwhile, total protein and β-glucan contents, and β-amylase and limit dextrinase activities were significantly increased under high temperature, with HT7-treated plants showing larger changes. Moreover, the different changes of four protein fractions under heat stress were found in the two barley cultivars, indicating the possibility of breaking positive association between protein content and enzyme activity.

This study aims at understanding the relation among sprouting time (from 12 up to 72 h), changes in protein and starch components, and flour functionality in quinoa. Changes related to the activity of sprouting-related proteases were observed after 48 h of sprouting in all protein fractions. Progressive proteolysis resulted in relevant modification in the organization of quinoa storage proteins, with a concomitant increase in the availability of physiologically relevant metals such as copper and zinc. Changes in the protein profile upon sprouting resulted in improved foam stability, but in impaired foaming capacity. The increased levels of amylolytic enzymes upon sprouting also made starch less prompt to gelatinize upon heating. Consequently, starch re-association in a more ordered structure upon cooling was less effective, resulting in low setback viscosity. The nature and the intensity of these modifications suggest various possibilities as for using flour from sprouted quinoa as an ingredient in the formulation of baked products.

Malting quality will be greatly deteriorated when barley plants suffer from post-anthesis drought stress, however there is a marked difference among barley genotypes in the responses of malting quality to drought stress, and the molecular mechanisms underlying the genotypic difference remain unclear. We made transcriptome and metabolome analysis on the developing grains of two barley genotypes differing in the responses to drought stress. Post-anthesis drought treatments led to decreased grain weight and β-glucan content, increased grain protein content and β-amylase activity. Drought stress enhanced H₂O₂ and heat-shock protein accumulation in the two barley genotypes, with the drought-tolerant genotype showing higher capacity of scavenging H₂O₂ and reducing misfolded protein accumulation than the drought-susceptible genotype. Moreover, the drought-tolerant genotype was more efficient in redistributing assimilates stored in the vegetative tissues into the developing grains. After re-watering to relieve drought stress, the drought-tolerant genotype can further modify auxin transport and ethylene signaling, enhancing redistribution of assimilates into grains. Transcriptome comparisons and weighted correlation network analysis (WGCNA) identified some key genes regulating the responses of malting quality traits to drought stress, such as RLK-LRR, β-glucosidase and HSP . In conclusion, less change of main malting quality traits in the drought-tolerant genotype under post-anthesis drought stress is attributed to its higher capacity of alleviating the stress injury through scavenging ROS and redistributing the metabolites stored in the vegetative organs into the developing grains.

In traditional opinions, no radial transportation was considered to occur in the bamboo internodes but was usually considered to occur in the nodes. Few studies have involved the phloem unloading and post-phloem transport pathways in the rapid elongating bamboo shoots. Our observations indicated a symplastic pathway in phloem unloading and post-unloading pathways in the culms of Fargesiayunnanensis Hsueh et Yi, based on a 5,6-carboxyfluorescein diacetate tracing experiment. Significant lignification and suberinization in fiber and parenchyma cell walls in maturing internodes blocked the apoplastic transport. Assimilates were transported out of the vascular bundles in four directions in the inner zones but in two directions in the outer zones via the continuum of parenchyma cells. In transverse sections, assimilates were outward transported from the inner zones to the outer zones. Assimilates transport velocities varied with time, with the highest values at 0):00 h, which were affected by water transport. The assimilate transport from the adult culms to the young shoots also varied with the developmental degree of bamboo shoots, with the highest transport velocities in the rapidly elongating internodes. The localization of sucrose, glucose, starch grains and the related enzymes reconfirmed that the parenchyma cells in and around the vascular bundles constituted a symplastic pathway for the radial transport of sugars and were the main sites for sugar metabolism. The parenchyma cells functioned as the ‘rays’ for the radial transport in and between vascular bundles in bamboo internodes. These results systematically revealed the transport mechanism of assimilate and water in the elongating bamboo shoots.