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).
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.
A new procedure for the measurement of fungal and bacterial α-amylase.
Sheehan, H. & McCleary, B. V. (1988). Biotechnology Technniques, 2, 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.
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.
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%.
Thickeners for dysphagic patients: comparison of a new amylase resistant product with four standard starch-based products - in vitro study.
Oudhuis, A. A. C. M., Helmens, H. J. & Bos, M. A. ESPEN 2: e83. 2007.
Patients with dysphagia are commonly prescribed thickened drinks to promote safe swallowing. Standard starch-based thickeners are sensitive to α-amylase, and may thin during consumption resulting in patients not receiving their prescribed consistency. This study compares the effect of human saliva on the consistency of drinks (water, full fat milk and black coffee) thickened with a newly developed thickener with α-amylase resistant features and four standard starch-based thickeners.
Pharmacometrics of 3-Methoxypterostilbene: A Component of Traditional Chinese Medicinal Plants.
Martinez, S. E., Sayre, C. L. & Davies, N. M. (2013). Evidence-Based Complementary and Alternative Medicine, Article ID 261468.
3-Methoxypterostilbene is a naturally occurring stilbene with potential in the treatment of diabetes. The preclinical pharmacokinetics and pharmacodynamics of 3-methoxypterostilbene were evaluated in the present study. The right jugular veins of male Sprague-Dawley rats were cannulated. The rats were dosed 10 mg/kg or 100 mg/kg of 3-methoxypterostilbene intravenously (IV) or orally (PO), respectively. Serum and urine samples were analyzed using a previously validated reversed-phase HPLC method. Serum AUC, serum t1/2, urine t1/2, Cltotal, and Vd for IV dosing were 48.1 ± 23.8 µg/h/mL, 18.1 ± 10.9 h, 9.54 ± 1.51 h, 47.8 ± 23.7 L/h/kg, and 5.11 ± 0.38 L/kg, respectively (mean ± SEM, n = 4). Serum AUC, serum t1/2, urine t1/2, Cltotal, and Vd for PO dosing were 229.8 ± 44.6 µg/h/mL, 73.3 ± 8.91 h, 20.6 ± 3.01 h, 0.48 ± 0.008 L/h/kg, and 52.0 ± 10.5 L/kg, respectively (mean ± SEM, n = 4). Bioavailability of the stilbene was determined to be 50.6% ± 10.0%. A 3-methoxypterostilbene glucuronidated metabolite was detected in both serum and urine. 3-Methoxypterostilbene exhibited antidiabetic activity including α-glucosidase and α-amylase inhibition as well as concentration-dependent antioxidant capacity similar to resveratrol. 3-Methoxypterostilbene also exhibited anti-inflammatory activity. 3-Methoxypterostilbene appears to be a bioactive compound and may be useful in reducing postprandial hyperglycemia.
Dose-and tissue-specific interaction of monoterpenes with the gibberellin-mediated release of potato tuber bud dormancy, sprout growth and induction of α-amylases and β-amylases.
Rentzsch, S., Podzimska, D., Voegele, A., Imbeck, M., Müller, K., Linkies, A. & Leubner-Metzger, G. (2012). Planta, 235(1), 137-151.
Gibberellins (GA) are involved in bud dormancy release in several species. We show here that GA-treatment released bud dormancy, initiated bud sprouting and promoted sprout growth of excised potato tuber bud discs (‘eyes’). Monoterpenes from peppermint oil (PMO) and S-(+)-carvone (CAR) interact with the GA-mediated bud dormancy release in a hormesis-type response: low monoterpene concentrations enhance dormancy release and the initiation of bud sprouting, whereas high concentrations inhibit it. PMO and CAR did, however, not affect sprout growth rate after its onset. We further show that GA-induced dormancy release is associated with tissue-specific regulation of α- and β-amylases. Molecular phylogenetic analysis shows that potato α-amylases cluster into two distinct groups: α-AMY1 and α-AMY2. GA-treatment induced transcript accumulation of members of both α-amylase groups, as well as α- and β-amylase enzyme activity in sprout and ‘sub-eye’ tissues. In sprouts, CAR interacts with the GA-mediated accumulation of α-amylase transcripts in an α-AMY2-specific and dose-dependent manner. Low CAR concentrations enhance the accumulation of α-AMY2-type α-amylase transcripts, but do not affect the α-AMY1-type transcripts. Low CAR concentrations also enhance the accumulation of α- and β-amylase enzyme activity in sprouts, but not in ‘sub-eye’ tissues. In contrast, high CAR concentrations have no appreciable effect in sprouts on the enzyme activities and the α-amylase transcript abundances of either group. The dose-dependent effects on the enzyme activities and the α-AMY2-type α-amylase transcripts in sprouts are specific for CAR but not for PMO. Different monoterpenes therefore may have specific targets for their interaction with hormone signalling pathways.
A kinetic model to explain the maximum in α-amylase activity measurements in the presence of small carbohydrates.
Baks, T., Janssen, A. E. & Boom, R. M. (2006). Biotechnology and Bioengineering, 94(3), 431-440.
The effect of the presence of several small carbohydrates on the measurement of the α-amylase activity was determined over a broad concentration range. At low carbohydrate concentrations, a distinct maximum in the α-amylase activity versus concentration curves was observed in several cases. At higher concentrations, all carbohydrates show a decreasing α-amylase activity at increasing carbohydrate concentrations. A general kinetic model has been developed that can be used to describe and explain these phenomena. This model is based on the formation of a carbohydrate–enzyme complex that remains active. It is assumed that this complex is formed when a carbohydrate binds to α-amylase without blocking the catalytic site and its surrounding subsites. Furthermore, the kinetic model incorporates substrate inhibition and substrate competition. Depending on the carbohydrate type and concentration, the measured α-amylase activity can be 75% lower than the actual α-amylase activity. The model that has been developed can be used to correct for these effects in order to obtain the actual amount of active enzyme.
Toward a Microfluidic‐Based Rapid Amylase Assay System.
Holmes, R. J., Summersgil, P., Ryan, T., Brown, B. J. T., Mockbil, A., Grieve, B. D. & Fielden, P. R. (2009). Journal of Food Science, 74(6), N37-N43.
This article describes work into a prototype system for the assay of amylase, using microfludic technologies. The new system has a significantly shorter cycle time than the current laboratory methods, which generally use microtitre plates, yet is capable of generating significantly superior results. As such, we have shown that sensitivity is enhanced by a factor of 10 in the standard assay trials, and by a factor of 2 in the real-sample lab trials. In both assays, the use of a microreactor system reduced the reaction time by a factor of 6.2, from 20 min incubation to 3.2 min. Basing the conclusion on the Megazyme Cerealpha Standard Method, and using the Cerealpha units as a measure of assay efficiency, the typical response for the microfluidic assay was shown to be 1.0 × 10-3 CU/mL (standard deviation [SD] 2.5 × 10-4 CU/mL), compared to 2.56 × 10-4 CU/mL (SD 5.94 × 10-5 CU/mL) for the standard macroassay. It is believed that this improvement in the reaction schematics is due to the inherent advantages of microfluidic devices such as superior mixing, higher thermal efficiency, and enhanced reaction kinetics.
Effect of Gelatinization and Hydrolysis Conditions on the Selectivity of Starch Hydrolysis with α-Amylase from Bacillus licheniformis.
Baks, T., Bruins, M. E., Matser, A. M., Janssen, A. E. M. & Boom, R. M. (2008). Journal of Agricultural and Food Chemistry, 56(2), 488-495.
Enzymatic hydrolysis of starch can be used to obtain various valuable hydrolyzates with different compositions. The effects of starch pretreatment, enzyme addition point, and hydrolysis conditions on the hydrolyzate composition and reaction rate during wheat starch hydrolysis with α-amylase from Bacillus licheniformis were compared. Suspensions of native starch or starch gelatinized at different conditions either with or without enzyme were hydrolyzed. During hydrolysis, the oligosaccharide concentration, the dextrose equivalent, and the enzyme activity were determined. We found that the hydrolyzate composition was affected by the type of starch pretreatment and the enzyme addition point but that it was just minimally affected by the pressure applied during hydrolysis, as long as gelatinization was complete. The differences between hydrolysis of thermally gelatinized, high-pressure gelatinized, and native starch were explained by considering the granule structure and the specific surface area of the granules. These results show that the hydrolyzate composition can be influenced by choosing different process sequences and conditions.