Measurement of β-amylase in cereal flours and commercial enzyme preparations.
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.
Modelling the β-amylase activity during red sorghum malting when Bacillus subtilis is used to control mould growth.
Bwanganga Tawaba, J. C., Béra, F. & Thonart, P. (2013). Journal of Cereal Science, 57(1), 115-119.
Steeping in dilute alkaline (0.2% NaOH) followed by resteeping in biocontrol (starters of Bacillus subtilis S499) has been used during red sorghum malting. The effect of steeping and germination conditions has been described using 2 functions: a Weibull 4-parameter model combined with a General Linear Model with Logarithm Link with significant goodness. Steeping conditions (combined use of NaOH and B. subtilis S499) affects the synthesis capacity of grain: when B. subtilis culture used in the steeping step is diluted, ln α increases, suggesting a loss of treatment efficacy. The germination temperature affects the β-amylase synthesis rate during the induction phase: the germination temperature increase is accompanied by a decrease of the β-amylase synthesis rate. During the repression phase of β-amylase synthesis, the effect of malting conditions was found to taper.
Refining the prediction of potential malt fermentability by including an assessment of limit dextrinase thermostability and additional measures of malt modification, using two different methods for multivariate model development.
Evans, D. E., Dambergs, R., Ratkowsky, D., Li, C., Harasymow, S., Roumeliotis, S. & Eglinton, J. K. (2010). Journal of the Institute of Brewing, 116(1), 86-96.
Prediction of malt fermentability (apparent attenuation limit — AAL) by measurement of the diastatic power enzymes (DPE), α-amylase, total limit dextrinase, total β-amylase, β-amylase thermostability, and the Kolbach index (KI or free amino nitrogen — FAN) is superior to the conventional use of diastatic power (DP) alone. The thermostability of β-amylase is known to be an important factor in determining fermentability, thus the thermostability of the other relatively thermolabile enzyme, limit dextrinase, was investigated to determine if it was also useful in predicting fermentability. To facilitate this aim, methods were developed for a rapid and cost efficient assay of both β-amylase and limit dextrinase thermostability. Internationally important Australian and international malting varieties were compared for their total limit dextrinase and β-amylase activity and thermostability. Interestingly, the level of limit dextrinase thermostability was observed to be inversely correlated with total limit dextrinase activity. The prediction of malt fermentability was achieved by both forward step-wise multi-linear regression (MLR) and the partial least squares (PLS) multivariate model development methods. Both methods produced similar identifications of the parameters predicting wort fermentability at similar levels of predictive power. Both models were substantially better at predicting fermentability than the traditional use of DP on its own. The emphasis of this study was on the identification of predictive factors that can be consistently used in models to predict fermentability, because the model parameter estimates will subtly vary depending on mashing conditions, yeast strain/fermentation conditions and malt source. The application of these multivariate model development methods (PLS and MLR) enabled the identification of further potential fermentability predicting factors. The analyses divided the predictive parameters into those defined by DP enzymes and those associated with modification (KI, FAN, fine/coarse difference, wort β-glucan and friability). Surprisingly, limit dextrinase thermostability was not a substantial predictor of fermentability, presumably due to its negative correlation with total limit dextrinase activity. The application of these insights in the malting and brewing industries is expected to result in substantial improvements in brewing consistency and enable more specific quality targets for barley breeder's progeny selection cut-off limits to be more precisely defined.
Gamma irradiation of sorghum flour: effects on microbial inactivation, amylase activity, fermentability, viscosity and starch granule structure.
Mukisa, I. M., Muyanja, C. M. B. K., Byaruhanga, Y. B., Schüller, R. B., Langsrud, T. & Narvhus, J. A. (2012). Radiation Physics and Chemistry, 81(3), 345-351.
Malted and un-malted sorghum (Sorghum bicolor (L.) Moench) flour was gamma irradiated with a dose of 10 kGy and then re-irradiated with 25 kGy. The effects of irradiation on microbial decontamination, amylase activity, fermentability (using an amylolytic L. plantarum MNC 21 strain), starch granule structure and viscosity were determined. Standard methods were used during determinations. The 10 kGy dose had no effect on microbial load of un-malted flour but reduced that of malted flour by 3 log cycles. Re-irradiation resulted in complete decontamination. Irradiation of malt caused a significant (p <0.05) reduction in alpha and beta amylase activity (22% and 32%, respectively). Irradiation of un-malted flour increased the rates of utilization of glucose and maltose by 53% and 100%, respectively, during fermentation. However, microbial growth, rate of lactic acid production, final lactic acid concentration and pH were not affected. Starch granules appeared normal externally even after re-irradiation, however, granules ruptured and dissolved easily after hydration and gelatinization. Production of high dry matter density porridge (200 g dry matter/L) with a viscosity of 3500 cP was achieved by irradiation of un-malted flout at 10 kGy. Gamma irradiation can be used to decontaminate flours and could be utilized to produce weaning porridge from sorghum.
Polyphenol oxidase, alpha-amylase and beta-amylase activities of Triticum monococcum, Triticum turgidum and Triticum aestivum: A two-year study.
Hidalgo, A., Brusco, M., Plizzari, L. & Brandolini, A. (2013). Journal of Cereal Science, 58(1), 51-58.
Enzymatic activity often reduces the nutritional value of wheat flour during food manufacturing, causing compound degradation and/or heat damage. The choice of wheat varieties with low enzymatic activity could therefore help to preserve the nutritional quality of food. The aim of this research was to evaluate polyphenol oxidase, alpha-amylase and beta-amylase activities in whole meal flours of 59 accessions belonging to different wheat species and subspecies, cropped in two years. The extraction pH (7.0), reaction pH (5.5) and reaction temperature (45°C) were determined by preliminary trials. The ANOVA highlighted significant differences for all enzymes among species/subspecies and, for amylases, between cropping years; however, the year influence was overwhelming only for alpha-amylase. Einkorn showed the highest polyphenol oxidase (362.1 ± 9.46 U/g DM) as well as the lowest alpha-amylase (0.20 ± 0.006 CU/g DM) and beta-amylase (12.0 ± 0.36 B3U/g DM) activities. The embryo/scutellum had the highest polyphenol oxidase and alpha-amylase values, followed by the bran and the endosperm; in contrast, beta-amylase was evenly distributed in the bran and the endosperm, and was absent in the embryo/scutellum.
Evaluation of heat damage, sugars, amylases and colour in breads from einkorn, durum and bread wheat flours.
Hidalgo, A. & Brandolini, A. (2011). Journal of Cereal Science, 54(1), 90-97.
To limit nutritional losses and optimise bread processing, heat damage indices (furosine, glucosylisomaltol, hydroxymethylfurfural), sugars, α-amylase, β-amylase and colour were monitored during bread manufacturing from refined flour of three einkorn, three bread and one durum wheat samples. The heat damage indices increased only during the baking step. Furosine was significantly lower in einkorn (on average, 9.3 ± 5.33 and 25.3 ± 10.70 mg/100 g protein in crumb and crust, respectively) than in bread wheat (31.6 ± 3.05 and 115.6 ± 13.53) and durum wheat (36.2 ± 2.82 and 165.0 ± 3.17). Glucosylisomaltol and hydroxymethylfurfural were detected only in the crust, with lower levels in einkorn (on average, 2.3 ± 1.78 and 10.0 ± 7.79 mg/kg DM, respectively) than in bread wheat (13.1 ± 5.57 and 42.8 ± 10.64) and durum wheat (18.9 ± 1.11 and 57.2 ± 0.80). The different behaviour of einkorn was probably related to its moderate β-amylase activity, and thus the low maltose content of its dough. Colour was correlated with heat damage, as einkorn breads were lighter than the others. The results show that einkorn bread undergoes lower heat damage than analogous products from durum and bread wheat, thus probably better preserving its nutritional value.
Effect of unmalted oats (Avena sativa L.) on the quality of high-gravity mashes and worts without or with exogenous enzyme addition.
Schnitzenbaumer, B. & Arendt, E. K. (2014). European Food Research and Technology, 238(2), 225-235.
Barley malt is the preferred brewing material these days because of its high extract content and high enzyme activities. However, when substituting malted barley with oats to create a unique beer flavor and aroma, endogenous malt enzymes become the limiting factor. Therefore, the objectives of this study were to evaluate the effect of 10–40% unmalted oats on the quality of high-gravity mashes/worts and to investigate the limitations of endogenous malt enzymes as well as the benefits of the application of industrial enzymes. The enzyme mix Ondea® Pro was found to be particularly suitable for mashing with unmalted oats and was therefore used in the present rheological tests and laboratory-scale mashing trials. In order to gain detailed information about the biochemical processes occurring during mashing, the quality of mashes was comprehensively analyzed after each mash rest using standard methods described by Mitteleuropäische Brautechnische Analysenkommission and Lab-on-a-Chip capillary electrophoresis. Mashing with up to 40% oats resulted in increased mash consistencies, color/pH (20°C) values, β-glucan concentrations, wort viscosities 12.0%, and filtration times as well as decreased FAN and extract contents. The application of Ondea® Pro enormously increased the color of worts despite lower pH values but considerably improved the quality and processability of 30 or 40% oat-containing mashes/worts. However, the substitution of up to 20% barley malt with unmalted oats can easily be realized without the addition of exogenous enzymes.
Implementation of commercial oat and sorghum flours in brewing.
Schnitzenbaumer, B., Kaspar, J., Titze, J. & Arendt, E. K. (2014). European Food Research and Technology, 238(3), 515-525.
Brewing with commercial flours has the potential to reduce mashing times and improve brewhouse efficiency. At present, however, no studies are available assessing the application of commercial oat and sorghum flours as brewing adjuncts. Therefore, the objectives of this study were to evaluate the quality and processability of mashes/worts produced with 10–90% oat or sorghum flour as well as to reveal the advantages and limitations of their use as a substitute for barley malt. For these purposes, both flour types were fully analyzed in terms of brewing-relevant characteristics using standard methods, Lab-on-a-Chip capillary electrophoresis, and scanning electron microscopy. Laboratory-scale mashing trials were performed to assess the effect of up to 90% flour adjunct on mash/wort quality. Equivalent factors were introduced to determine the performance efficiency of different oat/sorghum flour concentrations. Commercial oat flour sourced in Ireland exhibited significantly more protein, β-glucan, and fat, less starch, ash, and polyphenols, as well as a lower starch gelatinization temperature than commercial sorghum flour obtained from the USA. Worts produced with 10–90% oat or sorghum flour had lighter colors, higher pH values, and lower concentrations of foam-positive proteins as well as free amino nitrogen compared to 100% barley malt worts. In terms of extract yields, the use of up to 70% oat flour and 50% sorghum flour, respectively, has proven economically beneficial. Worts containing up to 70% oat flour showed a very good or good fermentability, those containing 30–50% sorghum flour resulted, however, in a lower alcohol production.
Oat malt as a baking ingredient – A comparative study of the impact of oat, barley and wheat malts on bread and dough properties.
Mäkinen, O. E. & Arendt, E. K. (2012). Journal of Cereal Science, 56(3), 747-753.
Oat malt is a nutritionally rich ingredient mainly used in a small number of speciality products. The aim of this study was to evaluate the suitability of oat malt in wheat baking. The effect of oat malt on bread and dough properties at levels ranging from 0.5% to 5% was studied and compared with barley and wheat malts. The addition of all malts increased loaf specific volumes. Barley and wheat malts at levels above 2.5% led to a sticky and coarse crumb, but the effect of oat malt on the crumb grain was negligible. Rheological characterisation could not explain the superior baking performance of oat malt, as it increased extensibility and decreased resistance extensively indicating weakening of the extensional properties of the gluten network. The high lipolytic activity may have compensated for the loss of dough strength by improving the surface properties of gas cells. The results show that oat malt can be used in wheat baking to improve the loaf volume and nutritional quality without the detrimental effects associated with the excess amylolytic activity of barley and wheat malts.
Heat damage of water biscuits from einkorn, durum and bread wheat flours.
Hidalgo, A. & Brandolini, A. (2011). Food Chemistry, 128(2), 471-478.
To limit heat damage and improve the nutritional properties of bakery products, furosine, glucosylisomaltol, hydroxymethylfurfural, furfural, sugars, α-amylase, β-amylase and colour were assessed during the production of water biscuits from three einkorn, three bread and one durum wheat flours. Heat damage indices, colour and aw development during baking (from 25 to 75 min duration) of water biscuits from one bread wheat were also studied. Furosine was more abundant in durum (86.0 ± 6.29 mg/100 g protein) and bread wheat (42.5 ± 6.93) than in einkorn (15.7 ± 3.92) water biscuits, while GLI was detected only in durum (10.0 ± 2.02 mg/kg DM) and bread wheat (5.2 ± 1.52) products; hydroxymethylfurfural and furfural were always absent. The limited heat damage of Triticum monococcum products was probably due to the moderate β-amylase activity of einkorn, and hence to the low maltose content of its mixes. The colour was correlated to heat damage, as einkorn water biscuits were lighter than those from other wheats.
Effect of drying temperature and time on alpha-amylase, beta-amylase, limit dextrinase activities and dimethyl sulphide level of teff (Eragrostis tef) malt.
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.
Carbohydrate metabolism and tissue differentiation during potato tuber initiation, growth and dormancy induction.
Akoumianakis, K. A., Alexopoulos, A. A., Karapanos, I. C., Kalatzopoulos, K., Aivalakis, G. & Passam, H. C. (2016). Australian Journal of Crop Science, 10(2), 185.
The duration of potato tuber dormancy has economic importance for both ware potatoes and seed tubers. The aim of this study was to shed light on the time at which tuber dormancy is induced. Potato tubers were selected at different stages of tuberisation: initial swelling of the stolon tip and early stages of tuber growth (tuber diameter 3, 7 and 14 mm). At each stage of tuberisation, the diameter of the pith and the cortex was measured, the activity of the enzymes beta-amylase, glucose-6-phosphate dehydrogenase and succinate dehydrogenase was determined, and starch and RNA levels recorded. It was observed that during tuber initiation the pith and perimedullary zone showed the greatest increase in size, whereas the cortical parenchyma increased mainly when the tuber diameter was 7-14 mm. Moreover, during stolon swelling and initial tuber development (3 mm diameter) total RNA accumulation was observed. Starch accumulation varied with the stages of development. Glucose-6-phosphate dehydrogenase and succinate dehydrogenase exhibited their highest activity during stolon swelling whereas beta-amylase activity was highest both during stolon swelling and at the 3 mm diameter stage. From the changes in tuber anatomy/morphology and the assays of enzyme activity, it is clear that dormancy is not induced in all the tissues of the tuber at the time of tuber initiation, but is imposed on the individual tissues as they are formed. Consequently, we may refer to 'tuber dormancy' only when the last bud has completed its differentiation.
Modulation of steeping conditions influence the diastatic enzymes and protein profile in pearl millet malt.
Kolawole, A. N. & Ebiloma, I. B. (2017). Biokemistri, 29(1).
Malting is targeted at getting the optimum point of enzymatic induction without losing much energy during the embryo metabolism and growth. Successful production of malt includes production of various hydrolytic enzymes and controlled degradation of the grain endosperm structure. Attention is at the centre stage of using Pearl millet as a substitute for barley, wheat and sorghum due to the cost of importation of barley and wheat to tropical countries. This study seeks to understand the effect of different steeping conditions with respect to varying pH, temperature and time on key enzymes associated with malting processes. Activities of α-amylase, β-amylase, β-glucanase, β-glucan content, protein profiles were monitored with respect to the varying steeping conditons. There was a steady increase (from 0 to 96 h) in the α-amylase activity at 30°C under all the pH stress conditions with the exception of acidic pH malted pearl millet where the enzyme activity decreased from 191.04 ± 1.5 U/g to 142.50 ± 2.20 U/g between the 72nd and 96th hour. Optimal activity (248.04 ± 0.20 U/g) was observed at 96 h for alkaline pH steeped pearl grains germinated at 30°C. However activity decreases as germination days prolong. Optimal activity was recorded at the 96th hour for malted pearl millet grains subjected to alkaline stress (2.73 ± 0.20 U/g) as compared with the control. β-glucanase activities of the malted pearl millet grains were high especially under the 30°C heat stress. Peak activity was observed at the 96th hour for the pearl millet grains subjected to alkaline pH stress (892.34 ± 0.20 U/kg). β-glucan content under the alkaline pH stress, acidic pH stress and control conditions at 30°C were within the same range of approximately 4-8 % w/w malt flour.
The interaction between starch hydrolysis and acidification kinetic determines the quality of a malted and fermented sorghum beverage.
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.
Effect of cold shock on the enhancement of β‐amylase activity during malting and malt processability for a red sorghum intended for brewing use.
Bwanganga, J. C. T. (2015). Journal of the Institute of Brewing, 121(2), 219-223.
The low β-amylase activity of sorghum malt is a major concern when malts are intended for use in brewing. Several studies have shown that the germination temperature plays an important role in β-amylase synthesis. In this study, the cold shock treatment was envisioned as a means of improving β-amylase synthesis during red sorghum malting. The results show that, when a high-frequency decrease in the germination temperature is used, the obtained malt exhibits a significantly increased β-amylase activity. This study shows that this increase is not sufficient to consider cold shock as a means of improving β-amylase activity for red sorghum brewing use, as the processabilty of the malts is unsatisfactory.
Chemical composition of wild and feral diploid wheats and their bearing on domesticated wheats.
Brandolini, A., Hidalgo, A., Gabriele, S. & Heun, M. (2015). Journal of Cereal Science, 63, 122-127.
The diploid wild wheats Triticum monococcum ssp. thaoudar and Triticum urartu are the direct ancestors of all domesticated wheats, but almost nothing is known about the chemical composition of their kernels. Aim of this research was to assess their content in several compositional traits and to compare it with that of domesticated wheats. To this end, fifteen diploid, tetraploid and hexaploid accessions belonging to different Triticum species were tested for 1000 kernel weight, protein, ash and starch content, β-amylase activity, carotenoid, tocol, anthocyanin and polyphenol concentration. The wild einkorns had high protein (21.7 ± 0.74 g/100 g), ash (3.0 ± 0.06 g/100 g), tocol (75.1 ± 3.95 mg/kg), carotenoid (8.0 ± 0.91 mg/kg) and anthocyanin (43.0 ± 4.66 mg/kg) content, and low β-amylase activity (20.2 ± 0.84 B3U/g). T. urartu instead coupled high protein (28.0 ± 0.07 g/100 g), ash (3.3 ± 0.03 g/100 g) and tocol (63.9 ± 2.91 g/100 g) content with low carotenoid (2.7 ± 0.02 g/100 g) and high β-amylase (57.7 ± 0.11 g/100 g) levels. These results fit well with those observed in the derived wheats, i.e. domesticated and feral einkorn on one side and emmer, durum, spelt and bread wheat on the other. Several positive nutritional traits present in the diploid wild species were not lost during the transition from wild to domesticated forms.
The genotypic difference in the effect of water stress after anthesis on the malt quality parameters in barley.
Wu, X. J., Chen, X., Zeng, F. R. & Zhang, G. P. (2015). Journal of Cereal Science, 65, 209-214.
It is well known that water stress is a major abiotic factor causing barley yield loss and unstable malt quality. However it is not quite clear about the altered pattern of the main malt quality traits for the barley genotypes differing in water stress tolerance. In this study, the effect of water stress during grain filling stage on grain weight, β-glucan content, β-amylase and limit dextrinase (LD) activities of three Tibetan wild and two cultivated barley genotypes were investigated. The results showed that water stress reduced biomass accumulation and grain weight of all genotypes, and increased protein content, but the reduced or increased extent differed significantly among genotypes, with XZ147 being less affected. In general, water stress increased β-amylase activity, reduced β-glucan content, had little effect on LD activity, which are attributed to increased protein and hordein contents, reduced grain weight, and its ineffective form in grains, respectively. Tibetan wild barley XZ147 is characterized by little change in grain weight and β-glucan content, dramatically increase of β-amylase activity under water stress, indicating its great potential for use in developing barley cultivars with high tolerance to water stress and high malt quality.
Diastatic enzymes malting process optimisation of African finger millet for biotechnological uses.
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.
I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.
Ouedraogo, J. P., Arentshorst, M., Nikolaev, I., Barends, S. & Ram, A. F. (2015). Applied Microbiology and Biotechnology, 99(23), 10083-10095.
Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae ISceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.
Gene transcript accumulation and enzyme activity of β-amylases suggest involvement in the starch depletion during the ripening of cherry tomatoes.
Maria, T., Tsaniklidis, G., Delis, C., Nikolopoulou, A. E., Nikoloudakis, N., Karapanos, I. & Aivalakis, G. (2016). Plant Gene, 5, 8-12.
The flavor of tomato fruits is mostly influenced by the accumulation of sugars and organic acids. During fruit ripening a conversion of starch to sugars occurs, which modulates significantly the taste and consequently the quality of the ripe tomato fruits. β-Amylases, a group of major starch hydrolytic enzymes involved in starch degradation were examined in developing cherry tomatoes. Our results suggest that the enzyme activity and the gene transcript accumulation of plastidial β-amylase isoenzymes were elevated during the late stages of fruit development indicating a participation of the enzyme in starch depletion and in the increase of total soluble sugar levels in ripe tomatoes.
Electromagnetic heating for industrial kilning of malt: a feasibility study.
Ferrari-John, R. S., Katrib, J., Zerva, E., Davies, N., Cook, D. J., Dodds, C. & Kingman, S. (2016). Food and Bioprocess Technology, 10(4), 687-698.
Industrial malting operations use ~ 800 kWh/t of energy to produce the heat required to kiln malt. Electromagnetic heating technologies are suggested as a way to potentially improve the energy efficiency of the kilning processing. In this work, the potential for using electromagnetic heating to dry malt to commercially acceptable moisture levels whilst preserving the activity of enzymes critical for downstream brewing processes is investigated. The 2450 MHz bulk dielectric properties of malt at moisture contents consistent with those occurring at different points in the kilning process are evaluated; 12% is shown to be a critical moisture level below which drying becomes more energy intensive. Calculated penetration depths of electromagnetic energy in malt at radio frequency are 100-fold higher than at microwave frequencies, showing a significant advantage for commercial-scale batch processing. The moisture contents and alpha and beta amylase activity of malt subjected to RF heating at different temperatures, treatment times and RF energy inputs in the intermediate and bound water drying regions were determined. It is shown for the first time that whilst significantly reduced process times are attainable, significant energy efficiency improvements compared to conventional kilning can only be achieved at higher product temperatures and thus at the expense of enzyme survival. It is suggested that RF heating may be feasible where higher bulk temperatures are not critical for downstream use of the material or when used in hybrid systems.