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 of Malt Samples & Extraction of Alpha Amylase
Chapter 7: Weighing of Wheat & Barley Flour & Extraction of Alpha Amylase
Chapter 8: Extraction/Dilution of Microbial Enzyme
Chapter 9: Assay Procedure
Chapter 10: Calculations
100 / 200 assays per kit
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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|
|Assay Format:||Spectrophotometer, Auto-analyser|
|Limit of Detection:||0.05 U/mL|
|Reproducibility (%):||~ 3%|
|Total Assay Time:||~ 30 min|
|Application examples:||Cereal flours, fermentation broths and other materials.|
|Method recognition:||AACC Method 22-02.01, AOAC Method 2002.01, ICC Standard No. 303, RACI Standard Method and CCFRA (Flour Testing Working Group Method 0018)|
The Ceralpha Method: α-Amylase test kit is suitable for the specific measurement and analysis of α-amylase in cereal grains and fermentation broths (fungal and bacterial).
Browse the complete list of our enzyme activity assay kits.
- Very cost effective
- All reagents stable for > 2 years after preparation
- Very specific
- Simple format
- Mega-Calc™ software tool is available from our website for hassle-free raw data processing
- Standard included
Measurement of available carbohydrates, digestible, and resistant starch in food ingredients and products.
McCleary, B. V., McLoughlin, C., Charmier, L. M. J. & McGeough, P. (2019). Cereal Chemistry, 97(1), 114-137.
Background and objectives: The importance of selectively measuring available and unavailable carbohydrates in the human diet has been recognized for over 100 years. The levels of available carbohydrates in diets can be directly linked to major diseases of the Western world, namely Type II diabetes and obesity. Methodology for measurement of total carbohydrates by difference was introduced in the 1880s, and this forms the basis of carbohydrate determination in the United States. In the United Kingdom, a method to directly measure available carbohydrates was introduced in the 1920s to assist diabetic patients with food selection. The aim of the current work was to develop simple, specific, and reliable methods for available carbohydrates and digestible starch (and resistant starch). The major component of available carbohydrates in most foods is digestible starch. Findings: Simple methods for the measurement of rapidly digested starch, slowly digested starch, total digestible starch, resistant starch, and available carbohydrates have been developed, and the digestibility of phosphate cross‐linked starch has been studied in detail. The resistant starch procedure developed is an update of current procedures and incorporates incubation conditions with pancreatic α‐amylase (PAA) and amyloglucosidase (AMG) that parallel those used AOAC Method 2017.16 for total dietary fiber. Available carbohydrates are measured as glucose, fructose, and galactose, following complete and selective hydrolysis of digestible starch, maltodextrins, maltose, sucrose, and lactose to glucose, fructose, and galactose. Sucrose is hydrolyzed with a specific sucrase enzyme that has no action on fructo‐oligosaccharides (FOS). Conclusions: The currently described “available carbohydrates” method together with the total dietary fiber method (AOAC Method 2017.16) allows the measurement of all carbohydrates in food products, including digestible starch. Significance and novelty: This paper describes a simple and specific method for measurement of available carbohydrates in cereal, food, and feed products. This is the first method that provides the correct measurement of digestible starch and sucrose in the presence of FOS. Such methodology is essential for accurate labeling of food products, allowing consumers to make informed decisions in food selection.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
Measurement of Starch: Critical evaluation of current methodology.
McCleary, B. V., Charmier, L. M. J. & McKie, V. A. (2018). Starch‐Stärke, 71(1-2), 1800146.
Most commonly used methods for the measurement of starch in food, feeds and ingredients employ the combined action of α‐amylase and amyloglucosidase to hydrolyse the starch to glucose, followed by glucose determination with a glucose oxidase/peroxidase reagent. Recently, a number of questions have been raised concerning possible complications in starch analytical methods. In this paper, each of these concerns, including starch hydrolysis, isomerisation of maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase, enzyme purity and hydrolysis of sucrose and β‐glucans have been studied in detailed. Results obtained for a range of starch containing samples using AOAC Methods 996.11 and 2014 .10 are compared and a new simpler format for starch measurement is introduced. With this method that employs a thermostable α-amylase (as distinct from a heat stable α-amylase) which is both stable and active at 100°C and pH 5.0, 10 samples can be analysed within 2 h, as compared to the 6 h required with AOAC Method 2014.10.Hide Abstract
Measurement of α-Amylase in Cereal, Food and Fermentation Products.
McCleary, B. V. & Sturgeon, R. (2002). Cereal Foods World, 47, 299-310.
In General, the development of methods for measuring α-amylase is pioneered in the clinical chemistry field and then translated to other industries, such as the cereals and fermentation industries. In many instances, this transfer of technology has been difficult or impossible to achieve due to the presence of interfering enzymes or sugars and to differences in the properties of the enzymes being analysed. This article describes many of the commonly used methods for measuring α-amylase in the cereals, food, and fermentation industries and discusses some of the advantages and limitations of each.Hide Abstract
Measurement of α-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the ceralpha assay: Collaborative study.
McCleary, B. V., McNally, M., Monaghan, D. & Mugford, D. C. (2002). Journal of AOAC International, 85(5), 1096-1102.
This study was conducted to evaluate the method performance of a rapid procedure for the measurement of α-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing α-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable α-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, α-glucosidase, and β-amylase. When the substrate is cleaved by endo-acting α-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of α-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 µmol p-nitrophenyl (in the presence of excess quantities of α-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal α-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.Hide Abstract
Analysis of feed enzymes.
McCleary, B. V. (2001). “Enzymes in Farm Animal Nutrition”, (M. Bedford and G. Partridge, Eds.), CAB International, pp. 85-107.
Enzymes are added to animal feed to increase its digestibility, to remove anti-nutritional factors, to improve the availability of components, and for environment reasons (Campbell and Bedford, 1992; Walsh et al., 1993). A wide-variety of carbohydrase, protease, phytase and lipase enzymes find use in animal feeds. In monogastric diets, enzyme activity must be sufficiently high to allow for the relatively short transit time. Also, the enzyme employed must be able to resist unfavourable conditions that may be experienced in feed preparation (e.g. extrusion and pelleting) and that exist in the gastrointestinal tract. Measurement of trace levels of enzymes in animal feed mixtures is difficult. Independent of the enzyme studied, many of the problems experienced are similar; namely, low levels of activity, extraction problems inactivation during feed preparation, non-specific binding to other feed components and inhibition by specific feed-derived inhibitors, e.g. specific xylanase inhibitors in wheat flour (Debyser et al., 1999).Hide Abstract
Sheehan, H. & McCleary, B. V. (1988). Biotechnology Techniques, 2(4), 289-292.
A procedure for the measurement of fungal and bacterial α-amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end) p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and α-glucosidase, and is absolutely specific for α-amylase. The assay procedure is simple, reliable and accurate.Hide Abstract
Measurement of cereal α-Amylase: A new assay procedure.
McCleary, B. V. & Sheehan, H. (1987). Journal of Cereal Science, 6(3), 237-251.
A new procedure for the assay of cereal α-amylase has been developed. The substrate is a defined maltosaccharide with an α-linked nitrophenyl group at the reducing end of the chain, and a chemical blocking group at the non-reducing end. The substrate is completely resistant to attack by β-amylase, glucoamylase and α-glucosidase and thus forms the basis of a highly specific assay for α-amylase. The reaction mixture is composed of the substrate plus excess quantities of α-glucosidase and glucoamylase. Nitrophenyl-maltosaccharides released on action of α-amylase are instantaneously cleaved to glucose plus free p-nitrophenol by the glucoamylase and α-glucosidase, such that the rate of release of p-nitrophenol directly correlates with α-amylase activity. The assay procedure shows an excellent correlation with the Farrand, the Falling Number and the Phadebas α-amylase assay procedures.Hide Abstract
Tomić, J., Torbica, A., Popović, L., Hristov, N. & Nikolovski, B. (2016). Food Chemistry, 199, 565-572.
The objective of this study was to evaluate albumins profile, proteolytic and amylolytic activity level and baking performance of wheat varieties grown in two production years with different climate conditions (2011 and 2012) in four locations. The results of ANOVA showed that variety, location, production year, and their interactions all had significant effects on all tested wheat quality parameters. The enzymatic activity and specific bread volume were mainly influenced by the variety. The samples from 2012 production year, had the lower values of albumin content, proteolytic and amylolytic activity, and bread specific volume. The correlation analysis, performed for 2011 production year, showed that albumin fraction (15-30 kDa) and proteolytic activity were negatively correlated with bread specific volume indicating the role of this fraction on lowering the crucial bread quality parameter. In 2012 production year, albumin fractions (5-15 kDa; 50-65 kDa) showed the most correlations, especially with parameters of bread quality.Hide Abstract
Wu, T., Yang, C., Ding, B., Feng, Z., Wang, Q., He, J., Tong, J., Xiao, L., Jiang, L. & Wan, J. (2016). Plant Physiology and Biochemistry, 99, 27-38.
Seed dormancy in rice is an important trait related to the pre-harvest sprouting resistance. In order to understand the molecular mechanisms of seed dormancy, gene expression was investigated by transcriptome analysis using seeds of the strongly dormant cultivar N22 and its less dormant mutants Q4359 and Q4646 at 24 days after heading (DAH). Microarray data revealed more differentially expressed genes in Q4359 than in Q4646 compared to N22. Most genes differing between Q4646 and N22 also differed between Q4359 and N22. GO analysis of genes differentially expressed in both Q4359 and Q4646 revealed that some genes such as those for starch biosynthesis were repressed, whereas metabolic genes such as those for carbohydrate metabolism were enhanced in Q4359 and Q4646 seeds relative to N22. Expression of some genes involved in cell redox homeostasis and chromatin remodeling differed significantly only between Q4359 and N22. The results suggested a close correlation between cell redox homeostasis, chromatin remodeling and seed dormancy. In addition, some genes involved in ABA signaling were down-regulated, and several genes involved in GA biosynthesis and signaling were up-regulated. These observations suggest that reduced seed dormancy in Q4359 was regulated by ABA-GA antagonism. A few differentially expressed genes were located in the regions containing qSdn-1 and qSdn-5 suggesting that they could be candidate genes underlying seed dormancy. Our work provides useful leads to further determine the underling mechanisms of seed dormancy and for cloning seed dormancy genes from N22.Hide Abstract
Lazo-Vélez, M. A., Avilés-González, J., Serna-Saldivar, S. O. & Temblador-Pérez, M. C. (2016). LWT-Food Science and Technology, 65, 1080-1086.
Response surface methodology and desirability function were used as tools to optimize wheat (Triticum aestivum L) germination conditions in the presence of SeO3Na2 to obtain selenium (Se)-enriched kernels. The effects of Se concentration (32.85-54.76 mg SeO3Na2/L), germination time (24-48 h) and germination temperature (18-25°C) were investigated. The best Se-enriched wheats were selected based on selenomethionine (SeM) content, α-amylase activity (AC), and sprouting features such as kernel moisture (KM) content, germinated grains (GG) and radicle growth (RG). A second order polynomial model produced a satisfactory fitting of the experimental data considering the total kernel SeM content (R2adj = 58.8, S = 14.29), AC (R2adj = 59.80, S = 48.78), KM (R2adj = 71.30, S = 2.94), GG (R2adj = 66.70, S2 = 1.20) and RG (R2adj = 84.60, S = 23.38). Total desirability (δ) value was 67.6% in operational conditions (35 mg Se/L, 25°C and 24 h germination). Under these conditions sprouted kernels contained 41.8 g/100 g, 95.5% and 98% for KM, GG and RG respectively, and 54 mg Se/kg as SeM and 54.8 CU of AC. In conclusion, the generated models with desirability methodology could be useful to optimize SeM content and alpha amylase activity in Se-enriched sprouted wheat kernels.Hide Abstract
Adachi, Y., Sugiyama, M., Sakagami, J. I., Fukuda, A., Ohe, M. & Watanabe, H. (2015). Plant Production Science, 18(4), 471-475.
We investigated the morpho-physiological traits of rice (Oryza sativa L.) during the germination and post-germination phases to explore avoidance of hypoxic conditions. We compared four lines selected for anaerobic germination (AG lines) with the variety IR42. The germination capacity of AG lines was higher than that of IR42. The germination percentages and coleoptile elongation differed among the four AG lines; IR06F459 showed the fastest germination and rapid coleoptile elongation. The coleoptiles of IR06F459 were significantly longer than those of IR42. The α-amylase activity in germinating seeds was significantly higher in IR06F459 than in IR42. At 2 days after sowing, the sucrose and glucose concentrations in germinating seeds were higher in IR06F459 than in IR42. These results show that IR06F459, an AG line with a long coleoptile, has high α-amylase activity and high sucrose and glucose concentrations in germinating seeds. These attributes partly explain its vigorous germination and coleoptile growth under hypoxic conditions.Hide Abstract
Kolawole, A. N. & Kolawole, A. O. (2015). African Journal of Biochemistry Research, 9(6), 81-88.
This study sought to understand the importance of variation of steeping and germination conditions (temperature, pH and salts) on the quality of African finger millet malt in terms of diastic power (a-amylase and β-amylase), endo- (1,3) (1,4)-β-D-glucanase, b-glycan content and protein profile. The results show that the physiological responses of African finger millet malted seeds are correlated to pH (acidity and alkalinity) but inversely correlated to temperature stress. The effect of the stresses on the activity of a-amylase, b-amylase and endo-(1,3)(1,4)-b-D-glucanase as well as b-glycan content was significantly different in magnitude except for the β-amylase activities obtained after acidic and alkaline treatment at 40°C which are not statistically different. Alkaline pH and heat stress at 30°C were the dominant factors for malting optimization from the result of diastic power indices. a-Amylase activity is a better predictor of diastic power. The grains subjected to the steeping and germination process carried out in Tris-HCl buffer solution (25 mM, pH 9) containing 100 mM NaCl at 30°C during 96 h showed higher α-amylase and β-amylase activity. This shows that for a salt–alkali-heat mix stress, a reciprocal enhancement among salt stress, alkali and heat stress was a characteristic feature with no significant change in the hordein protein expression. The influential effect of the stress conditions indicate that alkaline pH steeping and 30°C malting is the most effective condition for producing malted African millet flour with a promising potential of distinct malting quality metrics.Hide Abstract
Sugimura, Y., Michiyama, H. & Hirano, T. (2015). Plant Production Science, 18(3), 277-283.
Identifying the mechanisms regulating starch remobilization after heading in rice leaf sheaths is essential to understand the capability of the source for grain filling. In the present study, the changes in starch content and expression levels of α-amylase genes in the third leaf sheaths of Takanari, a high-yielding indica cultivar, were compared with those of Nipponbare, a standard japonica cultivar, during the post-heading stage to examine the starch remobilization characteristics in the leaf sheath of a high-yielding cultivar. Starch content in Takanari tended to decrease at a faster rate than in Nipponbare starting 3 days after heading. The decrease in starch content during 12 days after heading was greater in Takanari than in Nipponbare. Of eight genes predicted to encode α-amylase in the rice genome, RAmy2A and RAmy3C were primarily expressed in the leaf sheaths after heading. Moreover, RAmy2A mRNA level peaked at 9 days after heading in both cultivars. Particularly in Takanari, the RAmy2A mRNA levels rapidly increased from 3 to 9 days after heading. In addition, α-amylase activity was significantly higher in Takanari than in Nipponbare at 9 days after heading. Our results suggest that the rapid degradation of starch in the leaf sheaths of Takanari at the post-heading stage may be attributed, at least in part, to the enhancement of α-amylase activity caused by an increase in RAmy2A transcription level.Hide Abstract
Mestres, C., Nguyen, T. C., Adinsi, L., Hounhouigan, J., Fliedel, G. & Loiseau, G. (2015). Journal of Cereal Science, 63, 8-13.
Gowé is a traditional fermented Beninese soft cooked paste made from a blend of malted and non-malted cereals that is diluted with water/ice and sugar just before consumption as a thirst-quenching drink. Major differences in the processes used for the preparation of gowé, which includes natural lactic fermentation, result in variable quality. Acidity and free sugar content have been linked to the process parameters but also to the type of strains that can be used for inoculation. The aim of this study was to investigate the starch degradation mechanism in relation with the activity of degrading enzymes during the preparation of gowé and enzyme impact on the quality (sugar content and viscosity) of the final product. Our results point to a key role for malt α-amylase and its susceptibility to acidic conditions in the sequence of the preparation process and in the final quality of gowé. Pre-cooking and inoculation speeds up and increases acidification of the product thereby favoring its safety, but reduces the final free sugar content and increases the final viscosity of gowé, which are both organoleptic defects of the product.Hide Abstract
Hattingh, M., Alexander, A., Meijering, I., van Reenan, C. A. & Dicks, L. M. T. (2015). African Journal of Biotechnology, 14(4), 310-318.
Two α-amylase-producing strains of Lactobacillus plantarum were isolated from South African barley. The extracellular α-amylase activity produced by strain A.S1.2 coincided with cell growth, while strain B.S1.6 produced α-amylase mainly during stationary growth. Cell wall α-amylases in both strains were approximately five times higher than recorded for extracellular α-amylases. Both strains demonstrated highest extracellular α-amylase activity in 2% (w/v) maltose, followed by 2% (w/v) malt extract and 2% (w/v) starch, respectively. The α-amylase produced by the two strains functioned optimally at 50°C and under alkaline conditions. The two strains of L. plantarum fermented carbohydrates naturally present in barley, and produced cell-bound and cell-free α-amylase at alkaline conditions. The two strains may be developed into starter cultures to facilitate the germination of barley and produce malt with a higher fermentable sugar content.Hide Abstract
Wolter, A., Hager, A. S., Zannini, E., Galle, S., Gänzle, M. G., Waters, D. M. & Arendt, E. K. (2014). Food Microbiology, 37, 44-50.
This study determined exopolysaccharide (EPS) production by Weissella cibaria MG1 in sourdoughs prepared from gluten-free flours (buckwheat, oat, quinoa and teff), as well as wheat flour. Sourdoughs (SD) were fermented without sucrose, or by replacing 10% flour with sucrose to support EPS production. The amount of EPS depended on the substrate: high amounts of EPS corresponding to low amounts of oligosaccharides were found in buckwheat (4.2 g EPS/kg SD) and quinoa sourdoughs (3.2 g EPS/kg SD); in contrast, no EPS but panose-series oligosaccharides (PSO) were detected in wheat sourdoughs. Organic acid production, carbohydrates and rheological changes during fermentation were compared to the EPS negative control without added sucrose. Corresponding to the higher mineral content of the flours, sourdoughs from quinoa, teff and buckwheat had higher buffering capacity than wheat. Fermentable carbohydrates in buckwheat, teff and quinoa flours promoted W. cibaria growth; indicating why W. cibaria failed to grow in oat sourdoughs. Endogenous proteolytic activity was highest in quinoa flour; α-amylase activity was highest in wheat and teff flours. Protein degradation during fermentation was most extensive in quinoa and teff SD reducing protein peaks 18-29, 30-41 and 43–55 kDa extensively. Rheological analyses revealed decreased dough strength (AF) after fermentation, especially in sucrose-supplemented buckwheat sourdoughs correlating with amounts of EPS. High EPS production correlated with high protein, fermentable sugars (glucose, maltose, fructose), and mineral contents in quinoa flour. In conclusion, W. cibaria MG1 is a suitable starter culture for sourdough fermentation of buckwheat, quinoa and teff flour.Hide Abstract
Hu, S., Yu, J., Dong, J., Evans, D. E., Liu, J., Huang, S., Huang, S., Fan W., Yin, H. & Li, M. (2014). Starch‐Stärke, 66(7-8), 615-623.
The fermentable carbohydrate composition of wort has a direct influence on yeast fermentation efficiency and resultant beer quality. In this study, the relationship between diastatic power enzymes (DPE) and their wort sugars products during the course of small-scale, emulated commercial mashing was investigated. Malts derived from 13 barley cultivars were mashed and assayed at five time points during mashing for the levels of DPE and fermentable sugars. Comparisons of the patterns of DPE activity and wort sugar production showed that the activity levels of β-amylase and limit dextrinase (LD) during mashing were variable between the 13 cultivars, in comparison to the level of α-amylase and resultant composition of wort sugars. Moreover, comparison of peak DPE activities indicated that α-amylase correlated positively and significantly with LD, while no obvious correlation was found between β-amylase and either α-amylase or LD, indicating that activity pattern of α-amylase and LD was closely related during mashing. Multiple linear regression models, based on levels of the DPE as various time points during mashing, thermostability of β-amylase and malt Kolbach index, were able to explain 42.9%, 91.9%, 94%, and 73.2% of wort maltotriose, maltose, glucose, and fermentable sugar composition, respectively. A combination of these insights into the dynamics of starch hydrolysis during mashing will assist brewers in malt cultivar selection and the adjustment of mashing conditions so as optimize the sugar content for the efficient production of high quality beer.Hide Abstract
Gebremariam, M. M., Zarnkow, M. & Becker, T. (2013). Food and Bioprocess Technology, 6(12), 3462-3472.
Teff is a gluten-free cereal with attractive nutritional profile. This research was aimed to study the influence of kilning on the enzyme activities and dimethyl sulphide (DMS) level of DZ-Cr-387 teff variety and suggest a kilning condition that yields teff malt with low DMS with no or little damage on its enzyme activities. The malts were dried using isothermal conditions at 30, 40, 50, 60 and 70°C for 40 h with sampling in certain time interval. To set up kilning program, two temperature regimens 18 h at 30°C + 1h at 60°C + 3 or 5 h at 65°C (R1) and 18 h at 30°C + 1 h at 60°C + 3 or 5 h at 80°C (R2) were selected. Results from isothermal kilning indicated that enzyme activities, DMS and moisture contents were affected (P < 0.05) by time and temperature. The values of α-amylase, β-amylase, limit dextrinase activities and DMS content while using the first regimen (R1) with 3 h curing at 65°C were 68 U/g, 440 U/g, 1,072 U/kg and 3.3 mg/kg, respectively. Whereas in the second regimen with 3 h curing at 80°C, the values were 42 U/g, 406 U/g, 736 U/kg and 2.15 mg/kg, respectively. Prolonged curing in both kilning regimens caused an adverse effect on the amylolytic enzyme activities. R1 with shorter curing time is considered to be the best condition in preserving enzymes. The enzyme activities and DMS level show that teff can be an alternative raw material for production of gluten-free malt.Hide Abstract
Tyo, K. E., Liu, Z., Petranovic, D. & Nielsen, J. (2012). BMC Biology, 10(1), 16.
Background: The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase). Results: HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress. Conclusions: Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.Hide Abstract