|Content:||100 mg or 500 mg|
|Storage Temperature:||Below -10oC|
|Formulation:||Supplied as a lyophilised powder|
|Stability:||Minimum 1 year at < -10oC. Check vial for details.|
|Synonyms:||glucan 1,4-alpha-glucosidase; 4-alpha-D-glucan glucohydrolase; glucoamylase|
|Expression:||From Aspergillus niger|
|Specificity:||Hydrolysis of terminal α-1,4 and α-1,6 D-glucose residues successively from non-reducing ends of maltodextrins.|
|Specific Activity:||~ 35 U/mg (40oC, pH 4.5 on soluble starch)|
|Unit Definition:||One Unit of amyloglucosidase activity is defined as the amount of enzyme required to release one µmole of D-glucose reducing-sugar equivalents per minute from soluble starch at pH 4.5 and 40oC.|
|Application examples:||Recommended for use in AOAC Method 997.08 (fructan).|
|Method recognition:||AOAC Method 997.08|
High purity Amyloglucosidase (Aspergillus niger) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
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Validation of Methods
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
McCleary, B. V., Sloane, N. & Draga, A. (2015). Starch/Stärke, 67(9-10), 860–883.
The new definition of dietary fibre introduced by Codex Alimentarius in 2008 includes resistant starch and the option to include non-digestible oligosaccharides. Implementation of this definition required new methodology. An integrated total dietary fibre method was evaluated and accepted by AOAC International and AACC International (AOAC Methods 2009.01 and 2011.25; AACC Method 32–45.01 and 32–50.01, and recently adopted by Codex Alimentarius as a Type I Method. However, in application of the method to a diverse range of food samples and particularly food ingredients, some limitations have been identified. One of the ongoing criticisms of this method was that the time of incubation with pancreatic α-amylase/amyloglucosidase mixture was 16 h, whereas the time for food to transit through the human small intestine was likely to be approximately 4 h. In the current work, we use an incubation time of 4 h, and have evaluated incubation conditions that yield resistant starch and dietary values in line with ileostomy results within this time frame. Problems associated with production, hydrolysis and chromatography of various oligosaccharides have been addressed resulting in a more rapid procedure that is directly applicable to all foods and food ingredients currently available.Hide Abstract
McCleary, B. V. (2000). Journal of AOAC International, 83(4), 997-1005.
A study was made of the effect of the activity and purity of enzymes in the assay of total dietary fiber (AOAC Method 985.29) and specific dietary fiber components: resistant starch, fructan, and β-glucan. In the measurement of total dietary fiber content of resistant starch samples, the concentration of α-amylase is critical; however, variations in the level of amyloglucosidase have little effect. Contamination of amyloglucosidase preparations with cellulase can result in significant underestimation of dietary fiber values for samples containing β-glucan. Pure β-glucan and cellulase purified from Aspergillus niger amyloglucosidase preparations were used to determine acceptable critical levels of contamination. Sucrose, which interferes with the measurement of inulin and fructooligosaccharides in plant materials and food products, must be removed by hydrolysis of the sucrose to glucose and fructose with a specific enzyme (sucrase) followed by borohydride reduction of the free sugars. Unlike invertase, sucrase has no action on low degree of polymerization (DP) fructooligosaccharides, such as kestose or kestotetraose. Fructan is hydrolyzed to fructose and glucose by the combined action of highly purified exo- and endo-inulinases, and these sugars are measured by the p-hydroxybenzoic acid hydrazide reducing sugar method. Specific measurement of β-glucan in cereal flour and food extracts requires the use of highly purified endo-1,3:1,4 β-glucanase and A. niger β-glucosidase. β-glucosidase from almonds does not completely hydrolyze mixed linkage β-glucooligosaccharides from barley or oat β-glucan. Contamination of these enzymes with starch, maltosaccharide, or sucrose-hydrolyzing enzymes results in production of free glucose from a source other than β-glucan, and thus an overestimation of β-glucan content. The glucose oxidase and peroxidase used in the glucose determination reagent must be essentially devoid of catalase and α- and β-glucosidase.Hide Abstract
Enzyme purity and activity in fibre determinations.
McCleary, B. V. (1999). Cereal Foods World, 44(8), 590-596.
Dietary fiber is mainly composed of plant cell wall polysaccharides such as cellulose, hemicellulose, and pectic substances, but it also includes lignin and other minor components (1). Basically, it covers the polysaccharides that are not hydrolyzed by the endogenous secretions of the human digestive tract (2,3). This definition has served as the target for those developing analytical procedures for the measurement of dietary fiber for quality control and regulatory considerations (4). Most procedures for the measurement of total dietary fiber (TDF), or specific polysaccharide components, either involve some enzyme treatment steps or are mainly enzyme-based. In the development of TDF procedures such as the Prosky method (AOAC International 985.29, AACC 32—05) (5), the Uppsala method (AACC32-25) (6), and the Englyst method (7), the aim was to remove starch and protein through enzyme treatment, and to measure the residue as dietary fiber (after allowing for residual, undigested protein and ash). Dietary fiber was measured either gravimetrically or by chemical or instrumental procedures. Many of the enzyme treatment steps in each of the methods, particularly the prosky (5) and the Uppsala (6) methods are very similar. As a new range of carbohydrates is being introduced as potential dietary fiber components, the original assay procedures will need to be reexamined, and in some cases slightly modified, to ensure accurate and quantitative measurement of these components and of TDF. These “new” dietary fiber components include resistant nondigestible oligosaccharides; native and chemically modified polysaccharides of plant and algal origin (galactomannan, chemically modified celluloses, and agars and carrageenans); and resistant starch. To measure these components accurately, the purity, activity, and specificity of the enzymes employed will become much more important. A particular example of this is the mesurement of fructan. This carbohydrate consists of a fraction with a high degree of polymerization (DP) that is precipitated in the standard Prosky method (5,8) and a low DP fraction consequently is not measured (9). Resistant starch poses a particular problem. This component is only partially resistant to degradation by α-amylase, so the level of enzyme used and the incubation conditions (time and temperature) are critical.Hide Abstract
McCleary, B. V., Bouhet, F. & Driguez, H. (1991). Biotechnology Techniques, 5(4), 255-258.
An enzyme-linked assay for the measurement of amyloglucosidase in commercial enzyme mixtures and crude culture filtrates is described. A method for the synthesis of the substrate employed, p-nitrophenyl β-D-maltoside, is also described. The substrate is used in the presence of saturating levels of β-glucosidase. With a range of Aspergillus sp. culture filtrates, an excellent correlation was found for values obtained with this assay and a conventional assay employing maltose as substrate with measurement of released glucose.Hide Abstract
McCleary, B. V. & Anderson, M. A. (1980). Carbohydrate Research, 86(1), 77-96.
Culture filtrates of Cladosporium resinae ATCC 20495 contain a mixture of enzymes able to convert starch and pullulan efficiently into D-glucose. Culture conditions for optimal production of the pullulan-degrading activity have been established. The amylolytic enzyme preparation was fractionated by ion-exchange and molecular-sieve chromatography, and shown to contain α-D-glucosidase, α-amylase, and two glucoamylases. The glucoamylases have been purified to homogeneity and their substrate specificities investigated. One of the glucoamylases (termed P) readily hydrolyses the (1→6)-α-D linkages in pullulan, amylopectin, isomaltose, panose, and 63-α-D-glucosylmaltotriose. Each of the glucoamylases cleaves the (1→6)-α-D linkage in panose much more readily than that in isomaltose.Hide Abstract
Relationship between the digestion rate and internal fatty acid content of starch rendered resistant by addition of fatty acids and heat treatment.
Nakashima, K., Oki, S., Toyoshima, R., Tozawa, S., Nakayama, M., Ishikawa, K., Zhang, H., Chen, J., Mori, D. & Akiyama, Y. (2018). Japan Journal of Food Engineering, 19(3), 185-189.
Potato starch was rendered digestion resistant by the addition of fatty acids (lauric, myristic, palmitic, stearic, oleic, or linoleic) and heat treatment. The effect of different fatty acids on starch digestibility was investigated, as well as the relationship between digestibility and the quantity of fatty acids able to form starch complexes. Although myristic acid reduced digestibility by the greatest degree in samples adjusted to 15% moisture content, no significant difference (p<0.05) among fatty acid types was observed in samples adjusted to 20% moisture content. Digestibility tended to decrease with increasing internal free fatty acid (IFFA) content up to 4 mg per 1 g of starch (dry basis) but did not change substantially for IFFA content greater than 4 mg. This result suggests that starch-fatty acid complex formation reaches saturation when IFFA content is approximately 4 mg and that further addition of fatty acid has no remarkable effect on digestibility.Hide Abstract
Shrestha, A. K., Ng, C. S., Lopez-Rubio, A., Blazek, J., Gilbert, E. P. & Gidley, M. J. (2010). Carbohydrate Polymers, 80(3), 699-710.
Gelose 80, a high amylose maize starch, was extruded in a twin screw extruder at different feed moistures, cooled, stored for 12 days at 4°C, and cryo-milled. The raw and extruded starches were analysed for in vitro enzyme-resistant starch content (ERS), morphology, in vitro digestibility, long range (X-ray diffraction) and short range (FTIR) molecular order. Extrusion markedly increased the rate of starch digestion and reduced the ERS content, irrespective of whether B-type or B- and V-type polymorphs were present. Increasing feed moisture and storage resulted in only slight increases in ERS content, with X-ray diffraction and FTIR also showing small changes in long and short range molecular order, respectively. Analysis of residues from in vitro digestion showed the mechanism of enzyme resistance of granular and extruded high amylose starch to be markedly different, both qualitatively and quantitatively. Enzyme digestion of granular high amylose maize starch showed initial disorganization in structure followed by slow reorganization at later stages of digestion. In contrast, molecular reorganization took place throughout the enzyme digestion of extruded high amylose maize starch. Higher levels of crystallinity were found in digested extrudates compared with digested granules, showing that there is no direct correlation between starch crystallinity and enzyme digestion rates.Hide Abstract