Chapter 1: Introduction
Chapter 2: Theory of the Analytical Procedure
Chapter 3: Kit content
Chapter 4: Reagent Preparation
Chapter 5: Milling of Samples
Chapter 6: Weighing Samples
Chapter 7: Enzyme Extraction
Chapter 8: Assay of Beta Amylase
Chapter 9: Calculations
100 / 200 assays per kit
Prices exclude VAT
Available for shipping
|Content:||100 / 200 assays per kit|
Short term stability: 2-8oC,
Long term stability: See individual component labels
|Stability:||> 2 years under recommended storage conditions|
|Limit of Detection:||0.05 U/mL|
|Reproducibility (%):||~ 3%|
|Reaction Time (min):||~ 10 min|
|Application examples:||Cereal flours, malts and other materials.|
|Method recognition:||Modification of RACI Standard Method|
The Betamyl-3; β-Amylase test kit is suitable for the specific measurement and analysis of β-amylase in malt flour.
See more of our amylase assay kits and other kit products for measurement of enzyme activities.
- Very cost effective
- All reagents stable for > 2 years as supplied
- Only enzymatic kit available
- Very specific
- Simple format
- Rapid reaction
- Mega-Calc™ software tool is available from our website for hassle-free raw data processing
- Standard included
Diastatic power and maltose value: a method for the measurement of amylolytic enzymes in malt.
Charmier, L. M., McLoughlin, C. & McCleary, B. V. (2021). Journal of the Institute of Brewing, In Press.
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.Hide Abstract
Prediction of potential malt extract and beer filterability using conventional and novel malt assays.
Cornaggia, C., Evans, D. E., Draga, A., Mangan, D. & McCleary, B. V. (2019). Journal of Institute of Brewing, 125(3), 294-309.
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 65oC (MIM 65oC). 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.Hide Abstract
McCleary, B. V. & Codd, R. (1989). Journal of Cereal Science, 9(1), 17-33.
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.Hide Abstract
Impact of exogenous maltogenic α-amylase and maltotetraogenic amylase on sugar release in wheat bread.
Rebholz, G. F., Sebald, K., Dirndorfer, S., Dawid, C., Hofmann, T. & Scherf, K. A. (2021). European Food Research and Technology, 247(6), 1425-1436.
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.Hide Abstract
Analysis of β-amylase gene (Amyβ) variation reveals allele associated with low enzyme activity and increased firmness in cooked sweetpotato (Ipomoea batatas) from East Africa.
Banda, L., Kyallo, M., Entfellner, J. B. D., Moyo, M., Swanckaert, J., Mwanga, R. O., Onyango, A., Magiri, E., Gemenet, D. C., Yao, N., Pelle, R. & Muzhingi, T. (2021). Journal of Agriculture and Food Research, 4, 100121.
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.Hide Abstract
Ethylene regulates post-germination seedling growth in wheat through spatial and temporal modulation of ABA/GA balance.
Sun, M., Tuan, P. A., Izydorczyk, M. S. & Ayele, B. T. (2020). Journal of Experimental Botany, 71(6), 1985-2004.
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.Hide Abstract
High amylose wheat starch structures display unique fermentability characteristics, microbial community shifts and enzyme degradation profiles.
Bui, A., Williams, B., Hoedt, E., Morrison, M., Mikkelsen, D. & Gidley, M. (2020). Food & Function, 6.
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.Hide Abstract
Exploring diverse sorghum (Sorghum bicolor (L.) Moench) accessions for malt amylase activity.
Disharoon, A., Boyles, R., Jordan, K. & Kresovich, S. (2020). Journal of the Institute of Brewing, In Press.
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.Hide Abstract
Starch hydrolysis during mashing: A study of the activity and thermal inactivation kinetics of barley malt α-amylase and β-amylase.
De Schepper, C. F., Michiels, P., Buvé, C., Van Loey, A. M. & Courtin, C. M. (2020). Carbohydrate Polymers, 117494.
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.Hide Abstract
Effects of post-heading high temperature on some quality traits of malt barley.
Ni, S. J., Zhao, H. F. & Zhang, G. P. (2020). Journal of Integrative Agriculture, 19(11), 2674-2679.
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.Hide Abstract
Effect of Sprouting on Proteins and Starch in Quinoa (Chenopodium quinoa Willd.).
Suárez-Estrella, D., Bresciani, A., Iametti, S., Marengo, M., Pagani, M. A. & Marti, A. (2020). Plant Foods for Human Nutrition, 75(4), 635-641.
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.Hide Abstract
Transcriptome and metabolome analysis reveals regulatory networks and key genes controlling barley malting quality in responses to drought stress.
Hong, Y., Ni, S. J. & Zhang, G. P. (2020). Plant Physiology and Biochemistry, 152, 1-11.
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.Hide Abstract
Symplasmic phloem unloading and post-phloem transport during bamboo internode elongation.
Deng, L., Li, P., Chu, C., Ding, Y. & Wang, S. (2020). Tree Physiology, 40(3), 391-412.
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.Hide Abstract
The influence of drought stress on malt quality traits of the wild and cultivated barleys.
Hong, Y. & Zhang, G. P. (2020). Journal of Integrative Agriculture, 19(8), 2009-2015.
As a major abiotic stress, drought causes instability and deterioration of malt barley quality. There is distinct difference among barley cultivars in the responses of the main malt quality traits to drought stress. In the previous study, we identified some Tibetan wild barley accessions with relatively less change of malt quality traits under drought. In this study, we examined the impact of drought stress during grain filling stage on grain weight and several important malt quality traits, including total protein content, β-glucan content, limit dextrinase activity, β-amylase activity, and protein fractions in four barley genotypes (two Tibetan wild accessions and two cultivars). Drought treatment reduced grain weight, β-glucan content, and increased total protein content, β-amylase activity. These changes differed among barley genotypes and treatments, and are closely associated with grain filling process and kernel weight. All the results indicated Tibetan wild barley had great potential for developing drought tolerant barley cultivars. Relatively stable kernel weight or filling process under water stress should be highlighted in malt barley breeding in order to reduce the effect of water stress on malt barley quality.Hide Abstract
Identification of Proteomic Components Associated with Resistance to Fusarium Head Blight in Rye.
Perlikowski, D., Wiśniewska, H., Góral, T., Ochodzki, P., Majka, M., Pawłowicz, I., Belter, J. & Kosmala, A. (2019). The Plant Pathology Journal, 35(4), 313.
Rye was used here to dissect molecular mechanisms of resistance to Fusarium head blight (FHB) and to go deeper with our understanding of that process in cereals. F. culmorum-damaged kernels of two lines different in their potential of resistance to FHB were analyzed using two-dimensional gel electrophoresis and mass spectrometry to identify resistance markers. The proteome profiling was accompanied by measurements of α- and β-amylase activities and mycotoxin content. The proteomic studies indicated a total of 18 spots with clear differences in protein abundance between the more resistant and more susceptible rye lines after infection. Eight proteins were involved in carbohydrate metabolism of which six proteins showed a significantly higher abundance in the resistant line. The other proteins recognized here were involved in stress response and redox homeostasis. Three remaining proteins were associated with protease inhibition/resistance and lignin biosynthesis, revealing higher accumulation levels in the susceptible rye line. After inoculation, the activities of α- and β-amylases, higher in the susceptible line, were probably responsible for a higher level of starch decomposition after infection and a higher susceptibility to FHB. The presented results could be a good reference for further research to improve crop resistance to FHB.Hide Abstract
NTRC and Thioredoxin f Overexpression Differentially Induces Starch Accumulation in Tobacco Leaves.
Ancín, M., Larraya, L., Millán, F. S., Veramendi, J., Burch-Smith, T. & Farran, I. (2019). Plants, 8(12), 543.
Thioredoxin (Trx) f and NADPH-dependent Trx reductase C (NTRC) have both been proposed as major redox regulators of starch metabolism in chloroplasts. However, little is known regarding the specific role of each protein in this complex mechanism. To shed light on this point, tobacco plants that were genetically engineered to overexpress the NTRC protein from the chloroplast genome were obtained and compared to previously generated Trx f-overexpressing transplastomic plants. Likewise, we investigated the impact of NTRC and Trx f deficiency on starch metabolism by generating Nicotiana benthamiana plants that were silenced for each gene. Our results demonstrated that NTRC overexpression induced enhanced starch accumulation in tobacco leaves, as occurred with Trx f. However, only Trx f silencing leads to a significant decrease in the leaf starch content. Quantitative analysis of enzyme activities related to starch synthesis and degradation were determined in all of the genotypes. Zymographic analyses were additionally performed to compare the amylolytic enzyme profiles of both transplastomic tobacco plants. Our findings indicated that NTRC overexpression promotes the accumulation of transitory leaf starch as a consequence of a diminished starch turnover during the dark period, which seems to be related to a significant reductive activation of ADP-glucose pyrophosphorylase and/or a deactivation of a putative debranching enzyme. On the other hand, increased starch content in Trx f-overexpressing plants was connected to an increase in the capacity of soluble starch synthases during the light period. Taken together, these results suggest that NTRC and the ferredoxin/Trx system play distinct roles in starch turnover.Hide Abstract
Gene Expression Profiling in Short‐Term Imbibition of Wheat: Tools for Dissecting of Pasting Properties of Imbibed Wheat Seeds.
Tamura, T., Akuzawa, S. & Mura, K. (2019). Journal of Food Science, 84(5), 946-953.
Germination of wheat maximizes phytochemical content and antioxidant activity while altering chemical composition, gluten content, and pasting properties. This study investigated the effect of short‐term imbibition on gene expression profiles and the physical and functional characteristics of wheat. Changes in gene expression profiles of wheat during short‐term imbibition (0, 16, and 24 hr) were evaluated by DNA microarray analysis. Gene Ontology (GO) analysis was carried out to categorize the function of genes with altered expression. Genes related to cellulose and cell wall synthesis were upregulated by imbibition for 16 hr, whereas those associated with polysaccharide catabolism and nucleosome assembly were upregulated in the subsequent 8 hr. The genes related to proteases and gluten were expressed in dry seeds but disappeared after 16 hr of imbibition. Genes encoding α‐amylase were not expressed in dry seeds whereas those encoding β‐amylase were expressed in dry seeds and downregulated by imbibition. According to quantitative real‐time PCR and enzymatic activity assay, α‐Amylase expression increased by imbibition and reached a maximum 24 hr after imbibition, with a corresponding increase in enzymatic activity. Pasting properties of flour made from wheat seeds imbibed for different times were decreased when seeds were imbibed for over 16 hr, by examination with Rapid Visco Analyzer. Gluten content did not significantly change until 24‐hr imbibition, although expression of genes encoding gliadin and glutenin disappeared by 16‐hr imbibition. The data indicated that it was possible to use 16‐hr imbibed wheat, with up to the 50% w/w replacement of nonimbibed wheat.Hide Abstract
Simultaneous enhancement of barley β-amylase thermostability and catalytic activity by R115 and T387 residue sites mutation.
Wang, X., Niu, C., Bao, M., Li, Y., Liu, C., Yun, Z., Li. Q. & Wang, J. (2019). Biochemical and Biophysical Research Communications, 514(1), 301-307.
Objective: To simultaneously increase the thermostability and catalytic activity of barley β-amylase. Methods: The amino acid sequences of various barley β-amylases with different enzyme properties were aligned, two amino acid residues R115 and T387 were identified to be important for barley β-amylase properties. R115C and T387V were then generated using site-directed and saturation mutagenesis. Results: R115C and T387V mutants increased the enzyme catalytic activity and thermostability, respectively. After combinational mutagenesis, the T50 value and t(1/2,60oC) value of R115C/T387V mutant reached 59.4°C and 48.8 min, which were 3.6°C higher and 29.5 min longer than those of wild-type. The kcat/Km value of mutant R115C/T387V were 59.82/s·mM, which were 54.7% higher than that of wild-type. The increased surface hydrophobicity and newly formed strong hydrogen bonds and salt bridges might be responsible for the enzyme thermostability improvement while the two additional hydrogen bonds formed in the active center may lead to the catalytic property enhancement. Conclusions: The mutant R115C/T387V showed high catalytic activity and thermostability indicating great potential for application in industry.Hide Abstract
Rice malting optimization for the production of top‐fermented gluten‐free beer.
Ceccaroni, D., Marconi, O., Sileoni, V., Wray, E. & Perretti, G. (2019). Journal of the Science of Food and Agriculture, 99(6), 2726-2734.
Background: A safe method to obtain gluten‐free beer led to the use of naturally gluten‐free grains, such as rice, but the specific malting program for rice is long and requires a large amount of water, and the resulting beer showed a flat flavour profile. In this study, an optimization of the malting and brewing procedure is proposed to overcome the aforementioned issues. Different steeping conditions and kilning temperatures are considered, and a top‐fermented beverage from rice malt is obtained for the first time. Results: The malting procedure has been optimized by assessing the use of short‐time steeping as an alternate to long air rest to obtain sufficient moisture content in the green malt, saving water consumption. The malt obtained allowed a regular fermentation, as confirmed by the sensorial analysis, which did not reveal any off‐flavours. The use of a top‐fermenting yeast formed high content of higher alcohol and relatively low amount of esters. Conclusion: This study confirms the potential of rice for the production of malt and beer. The optimized malting programme allowed water saving. The production of a top‐fermented rice malt beer was a successful attempt to introduce a new flavoured product for consumption by individuals affected by coeliac disease.Hide Abstract
Optimization of malting conditions for two landraces of West African sorghum and influence of mash bio-acidification on saccharification improvement.
Tokpohozin, S. E., Fischer, S. & Becker, T. (2019). Journal of Cereal Science, 85, 192-198.
Traditional sorghum beer processing based on uncontrolled malting and mashing conditions limits significant saccharification. We optimized the amylase activity of west African sorghum to improve saccharification. Furthermore, we bio-acidified the mash with Lactobacillus plantarum (ND-32, ND-130), Lactobacillus paracasei ND-34, and studied its impact on wort nitrogen, especially the branched amino acid content. The β-amylase content of the red sorghum malt used for traditional beer processing was 261 U/g of dried malt when sorghum seeds were steeped to 42% and germinated at 27°C for 5 days. Despite the optimization of hydrolases, protein hydrolysis was still limited. Sorghum mash pre-heating and bio-acidification increased the wort free amino nitrogen content up to 27%. The contents of several amino acids, including the branched amino acids valine, leucine, and isoleucine, were highly improved. Our new approach based on mash pre-heating and biological acidification helped to increase the wort branched amino acid content by up to 50%. The branched amino acids valine, isoleucine, and leucine are respective precursors for isobutanol, amyl alcohol, and isoamyl alcohol, all of which are critical components of beer aroma.Hide Abstract