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|Content:||10 mL or 40 mL or 100 mL or 100 mL (ANKOM)|
|Formulation:||In 50% (v/v) glycerol|
|Stability:||> 4 years at 4oC|
|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.|
~ 36 U/mg (40oC, pH 4.5 on soluble starch);
~ 200 U/mL (40oC, pH 4.5 on p-nitrophenyl β-maltoside);
~ 3,260 U/mL (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:||Applications for the measurement of starch and dietary fiber in the food and feeds industry.|
|Method recognition:||AOAC Method 2000.11 and GB Standard 5009.245-2016|
High purity Amyloglucosidase (Aspergillus niger) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
For use in Megazyme Total Starch and Dietary Fiber methods. Amyloglucosidase, Fructanase (E-FRMXPD) and Isoamylase (E-ISAMY) are used in the enzyme hydrolysis step of two validated methods for the determination of polydextrose (a low molar mass dietary fiber) in foods: AOAC method 2000.11 and Chinese GB Standard 5009.245-2016.
E-AMGDF-A-100ML specifically to be used with ANKOMTDF Dietary Fiber Analyzer.
Data booklets for each pack size are located in the Documents tab.
Validation of Methods
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
McCleary, B. V. (2014). Journal of AOAC International, 97(3), 896-901.
AOAC Official Methods 2009.01 and 2011.25 have been modified to allow removal of resistant maltodextrins produced on hydrolysis of various starches by the combination of pancreatic α-amylase and amyloglucosidase (AMG) used in these assay procedures. The major resistant maltodextrin, 63,65-di-α-D-glucosyl maltopentaose, is highly resistant to hydrolysis by microbial α-glucosidases, isoamylase, pullulanase, pancreatic, bacterial and fungal α-amylase and AMG. However, this oligosaccharide is hydrolyzed by the mucosal α-glucosidase complex of the pig small intestine (which is similar to the human small intestine), and thus must be removed in the analytical procedure. Hydrolysis of these oligosaccharides has been by incubation with a high concentration of a purified AMG at 60°C. This incubation results in no hydrolysis or loss of other resistant oligosaccharides such as FOS, GOS, XOS, resistant maltodextrins (e.g., Fibersol 2) or polydextrose. The effect of this additional incubation with AMG on the measured level of low molecular weight soluble dietary fiber (SDFS) and of total dietary fiber in a broad range of samples is reported. Results from this study demonstrate that the proposed modification can be used with confidence in the measurement of dietary fiber.Hide Abstract
Measurement of total dietary fiber using AOAC method 2009.01 (AACC International approved method 32-45.01): Evaluation and updates.
McCleary, B. V., Sloane, N., Draga, A. & Lazewska, I. (2013). Cereal Chemistry, 90(4), 396-414.
The Codex Committee on Methods of Analysis and Sampling recently recommended 14 methods for measurement of dietary fiber, eight of these being type I methods. Of these type I methods, AACC International Approved Method 32-45.01 (AOAC method 2009.01) is the only procedure that measures all of the dietary fiber components as defined by Codex Alimentarius. Other methods such as the Prosky method (AACCI Approved Method 32-05.01) give similar analytical data for the high-molecular-weight dietary fiber contents of food and vegetable products low in resistant starch. In the current work, AACCI Approved Method 32-45.01 has been modified to allow accurate measurement of samples high in particular fructooligosaccharides: for example, fructotriose, which, in the HPLC system used, chromatographs at the same point as disaccharides, meaning that it is currently not included in the measurement. Incubation of the resistant oligosaccharides fraction with sucrase/β-galactosidase removes disaccharides that interfere with the quantitation of this fraction. The dietary fiber value for resistant starch type 4 (RS4), varies significantly with different analytical methods, with much lower values being obtained with AACCI Approved Method 32-45.01 than with 32-05.01. This difference results from the greater susceptibility of RS4 to hydrolysis by pancreatic α-amylase than by bacterial α-amylase, and also a greater susceptibility to hydrolysis at lower temperatures. On hydrolysis of samples high in starch in the assay format of AACCI Approved Method 32-45.01 (AOAC method 2009.01), resistant maltodextrins are produced. The major component is a heptasaccharide that is highly resistant to hydrolysis by most of the starch-degrading enzymes studied. However, it is hydrolyzed by the maltase/amyloglucosidase/isomaltase enzyme complex present in the brush border lining of the small intestine. As a consequence, AOAC methods 2009.01 and 2011.25 (AACCI Approved Methods 32-45.01 and 32-50.01, respectively) must be updated to include an additional incubation with amyloglucosidase to remove these oligosaccharides.Hide Abstract
McCleary, B. V., DeVries, J. W., Rader, J. I., Cohen, G., Prosky, P., Mugford, D. C., Champ, M. & Okuma, K. (2012). Journal of AOAC International, 95(3), 824-844.
A method for the determination of insoluble (IDF), soluble (SDF), and total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official MethodsSM 985.29, 991.43, 2001.03, and 2002.02, the method quantitates water-insoluble and water-soluble dietary fiber. This method extends the capabilities of the previously adopted AOAC Official Method 2009.01, Total Dietary Fiber in Foods, Enzymatic-Gravimetric-Liquid Chromatographic Method, applicable to plant material, foods, and food ingredients consistent with CODEX Definition 2009, including naturally occurring, isolated, modified, and synthetic polymers meeting that definition. The method was evaluated through an AOAC/AACC collaborative study. Twenty-two laboratories participated, with 19 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 10.45 to 29.90%. Digestion of samples under the conditions of AOAC 2002.02 followed by the isolation, fractionation, and gravimetric procedures of AOAC 985.29 (and its extensions 991.42 and 993.19) and 991.43 results in quantitation of IDF and soluble dietary fiber that precipitates (SDFP). The filtrate from the quantitation of water-alcohol-insoluble dietary fiber is concentrated, deionized, concentrated again, and analyzed by LC to determine the SDF that remains soluble (SDFS), i.e., all dietary fiber polymers of degree of polymerization = 3 and higher, consisting primarily, but not exclusively, of oligosaccharides. SDF is calculated as the sum of SDFP and SDFS. TDF is calculated as the sum of IDF and SDF. The within-laboratory variability, repeatability SD (Sr), for IDF ranged from 0.13 to 0.71, and the between-laboratory variability, reproducibility SD (sR), for IDF ranged from 0.42 to 2.24. The within-laboratory variability sr for SDF ranged from 0.28 to 1.03, and the between-laboratory variability sR for SDF ranged from 0.85 to 1.66. The within-laboratory variability sr for TDF ranged from 0.47 to 1.41, and the between-laboratory variability sR for TDF ranged from 0.95 to 3.14. This is comparable to other official and approved dietary fiber methods, and the method is recommended for adoption as Official First Action.Hide Abstract
McCleary, B. V., DeVries, J. W., Rader, J. I., Cohen, G., Prosky, L., Mugford, D. C., Champ, M. & Okuma, K. (2010). Journal of AOAC International, 93(1), 221-233.
A method for the determination of total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official MethodsSM 985.29, 991.43, 2001.03, and 2002.02, the method quantitates high- and low-molecular-weight dietary fiber (HMWDF and LMWDF, respectively). In 2007, McCleary described a method of extended enzymatic digestion at 37°C to simulate human intestinal digestion followed by gravimetric isolation and quantitation of HMWDF and the use of LC to quantitate low-molecular-weight soluble dietary fiber (LMWSDF). The method thus quantitates the complete range of dietary fiber components from resistant starch (by utilizing the digestion conditions of AOAC Method 2002.02) to digestion resistant oligosaccharides (by incorporating the deionization and LC procedures of AOAC Method 2001.03). The method was evaluated through an AOAC collaborative study. Eighteen laboratories participated with 16 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 11.57 to 47.83. Digestion of samples under the conditions of AOAC Method 2002.02 followed by the isolation and gravimetric procedures of AOAC Methods 985.29 and 991.43 results in quantitation of HMWDF. The filtrate from the quantitation of HMWDF is concentrated, deionized, concentrated again, and analyzed by LC to determine the LMWSDF, i.e., all nondigestible oligosaccharides of degree of polymerization 3. TDF is calculated as the sum of HMWDF and LMWSDF. Repeatability standard deviations (Sr) ranged from 0.41 to 1.43, and reproducibility standard deviations (SR) ranged from 1.18 to 5.44. These results are comparable to other official dietary fiber methods, and the method is recommended for adoption as Official First Action.Hide Abstract
McCleary, B. V., Mills, C. & Draga, A. (2009). Quality Assurance and Safety of Crops & Foods, 1(4), 213–224.
An integrated total dietary fibre (TDF) method, consistent with the recently accepted CODEX definition of dietary fibre, has been developed. The CODEX Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) has been deliberating for the past 8 years on a definition for dietary fibre that correctly reflects the current consensus thinking on what should be included in this definition. As this definition was evolving, it became evident to us that neither of the currently available methods for TDF (AOAC Official Methods 985.29 and 991.43), nor a combination of these and other methods, could meet these requirements. Consequently, we developed an integrated TDF procedure, based on the principals of AOAC Official Methods 2002.02, 991.43 and 2001.03, that is compliant with the new CODEX definition. This procedure quantitates high- and low-molecular weight dietary fibres as defined, giving an accurate estimate of resistant starch and non-digestible oligosaccharides also referred to as low-molecular weight soluble dietary fibre. In this paper, the method is discussed, modifications to the method to improve simplicity and reproducibility are described, and the results of the first rounds of interlaboratory evaluation are reported.Hide Abstract
An integrated procedure for the measurement of total dietary fibre (including resistant starch), non-digestible oligosaccharides and available carbohydrates.
McCleary, B. V. (2007). Analytical and Bioanalytical Chemistry, 389(1), 291-308.
A method is described for the measurement of dietary fibre, including resistant starch (RS), non-digestible oligosaccharides (NDO) and available carbohydrates. Basically, the sample is incubated with pancreatic α-amylase and amyloglucosidase under conditions very similar to those described in AOAC Official Method 2002.02 (RS). Reaction is terminated and high molecular weight resistant polysaccharides are precipitated from solution with alcohol and recovered by filtration. Recovery of RS (for most RS sources) is in line with published data from ileostomy studies. The aqueous ethanol extract is concentrated, desalted and analysed for NDO by high-performance liquid chromatography by a method similar to that described by Okuma (AOAC Method 2001.03), except that for logistical reasons, D-sorbitol is used as the internal standard in place of glycerol. Available carbohydrates, defined as D-glucose, D-fructose, sucrose, the D-glucose component of lactose, maltodextrins and non-resistant starch, are measured as D-glucose plus D-fructose in the sample after hydrolysis of oligosaccharides with a mixture of sucrase/maltase plus β-galactosidase.Hide Abstract
Dietary fiber and available carbohydrates.
McCleary, B. V. & Rossiter, P. C. (2007). “Dietary Fiber: An International Perspective for Harmonization of Health Benefits and Energy Values”, (Dennis T. Gordon and Toshinao Goda, Eds.), AACC International, Inc., pp. 31-59.
Debate continues on the definition of dietary fiber (DF), methods for measurement of DF, and methods for measurement of the carbohydrates that are readily hydrolyzed and absorbed in the human small intestine. Henneberg and Stahmann developed the 'Wende' proximate system for analysis of foods in 1860, and a set of values obtained using this method were published by Atwater and Bryant in 1900. This method is still in use in the USA for the measurement of total carbohydrate. In this procedure, total carbohydrate is measured by difference after deducting the moisture, protein, fat and ash from the total weight. Carbohydrate calculated in this way contains not only sugar and starch, but also the 'unavailable carbohydrate' of DF. However, there are a number of problems with this approach, as the 'by difference' figure includes a number of non-carbohydrate components such as lignin, organic acids, tannins, waxes and some Maillard products. In addition to this error, it combines all of the analytical errors from the other analyses (FAO 1997). A need for information on the carbohydrate composition of foods for diabetics prompted McCance and Lawrence (1929) to attempt to measure carbohydrate composition to gain results that would be of biological significance. They divided the carbohydrates in foods into two broad groups, 'available' and 'unavailable'. The available carbohydrates, that is, sugar plus starch, were defined as those that are digested and absorbed by man and are glucogenic. The unavailable carbohydrates were defined as those that are not digested by the endogenous secretions of the human digestive tract. In the mid 1920s, McCance obtained a grant of £30 per year from the Medical Research Council to analyse raw and cooked fruits and vegetables for total "available carbohydrate"; values needed for calculating diabetic diets.Hide Abstract
McCleary, B. V. & Rossiter, P. (2004). Journal of AOAC International, 87(3), 707-717.
With the recognition that resistant starch (RS) and nondigestible oligosaccharides (NDO) act physiologically as dietary fiber (DF), a need has developed for specific and reliable assay procedures for these components. The ability of AOAC DF methods to accurately measure RS is dependent on the nature of the RS being analyzed. In general, NDO are not measured at all by AOAC DF Methods 985.29 or 991.43, the one exception being the high molecular weight fraction of fructo-oligosaccharides. Values obtained for RS, in general, are not in good agreement with values obtained by in vitro procedures that more closely imitate the in vivo situation in the human digestive tract. Consequently, specific methods for the accurate measurement of RS and NDO have been developed and validated through interlaboratory studies. In this paper, modifications to AOAC fructan Method 999.03 to allow accurate measurement of enzymically produced fructo-oligosaccharides are described. Suggested modifications to AOAC DF methods to ensure complete removal of fructan and RS, and to simplify pH adjustment before amyloglucosidase addition, are also described.Hide Abstract
McCleary, B. V. (2003). Proceedings of the Nutrition Society, 62, 3-9.
The 'gold standard' method for the measurement of total dietary fibre is that of the Association of Official Analytical Chemists (2000; method 985.29). This procedure has been modified to allow measurement of soluble and insoluble dietary fibre, and buffers employed have been improved. However, the recognition of the fact that non-digestible oligosaccharides and resistant starch also behave physiologically as dietary fibre has necessitated a re-examination of the definition of dietary fibre, and in turn, a re-evaluation of the dietary fibre methods of the Association of Official Analytical Chemists. With this realisation, the American Association of Cereal Chemists appointed a scientific review committee and charged it with the task of reviewing and, if necessary, updating the definition of dietary fibre. It organised various workshops and accepted comments from interested parties worldwide through an interactive website. More recently, the (US) Food and Nutrition Board of the Institute of Health, National Academy of Sciences, under the oversight of the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, assembled a panel to develop a proposed definition(s) of dietary fibre. Various elements of these definitions were in agreement, but not all. What was clear from both reviews is that there is an immediate need to re-evaluate the methods that are used for dietary fibre measurement and to make appropriate changes where required, and to find new methods to fill gaps. In this presentation, the 'state of the art' in measurement of total dietary fibre and dietary fibre components will be described and discussed, together with suggestions for future research.Hide Abstract
Two issues in dietary fiber measurement.
McCleary, B. V. (2001). Cereal Foods World, 46, 164-165.
Enzyme activity and purity of these topics, the easiest to deal with is the importance of enzyme purity and activity. As a scientist actively involved in polysaccharide research over the past 25 years, I have come to appreciate the importance of enzyme purity and specificity in polysaccharide modification and measurement (7). These factors translate directly to dietary fiber (DF) methodology, because the major components of DF are carbohydrate polymers and oligomers. The committee report published in the March issue of Cereal FOODS WORLD refers only to the methodology for measuring enzyme purity and activity (8) that led up the AOAC method 985.29 (2). In this work enzyme purity was gauged by the lack of hydrolysis (i.e., complete recovery) of a particular DF component (e.g. β-glucan, larch galactan or citrus pectin). Enzyme activity was measured by the ability to completely hydrolyze representative starch and protein (namely wheat starch and casein). These requirements and restrictions on enzyme purity and activity were adequate at the time the method was initially developed and served as a useful working guide. However, it was recognized that there was a need for more stringent quality definitions and assay procedures for enzymes used in DF measurements.Hide Abstract
Measurement of dietary fibre components: the importance of enzyme purity, activity and specificity.
McCleary, B. V. (2001), “Advanced Dietary Fibre Technology”, (B. V. McCleary and L. Prosky, Eds.), Blackwell Science, Oxford, U.K., pp. 89-105.
Interest in dietary fibre is undergoing a dramatic revival, thanks in part to the introduction of new carbohydrates as dietary fibre components. Much emphasis is being placed on determining how much fibre is present in a food. Linking a particular amount of fibre to a specific health benefit is now an important area of research. The term 'dietary fibre' first appeared in 1953, and referred to hemicelluloses, celluloses and lignin (Theandere/tf/.1995). Trowell (1974) recommended this term as a replacement for the no longer acceptable term 'crude fibre'. Burkitt (1995) has likened the interest in dietary fibre to the growth of a river from its first trickle to a mighty torrent He observes that dietary fibre 'was first viewed as merely the less digestible constituent of food which exerts a laxative action by irritating the gut', thus acquiring the designation 'roughage' - a term later replaced by 'crude fibre' and ultimately by 'dietary fibre'. Various definitions of dietary fibre have appeared over the years, partly due to the various concepts used in deriving the term (i.e. origin of material, resistance to digestion, fermentation in the colon, etc.), and partly to the difficulties associated with its measurement and labelling (Mongeau et al. 1999). The principal components of dietary fibre, as traditionally understood, are non-starch polysaccharides (which in plant fibre are principally hemicelluloses and celluloses), and the non-carbohydrate phenolic components, cutin, suberin and waxes, with which they are associated in nature. In 1976, the definition of dietary fibre was modified to include gums and some pectic substances, based on the resistance to digestion of these components in the upper intestinal tract. For the purposes of labelling, Englyst et al. (1987) proposed that dietary fibre be defined as 'non-starch polysaccharides (NSP) in the diet that are not digested by the endogenous secretions of the human digestive tract'. Methods were concurrently developed to specifically measure NSP (Englyst et al. 1994).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
Measuring dietary fibre.
McCleary, B. V. (1999). The World of Ingredients, 50-53.
Interest in dietary fibre is undergoing a dramatic revival thanks in part to the introduction of new carbohydrates as dietary fibre components. Much emphasis is being placed on determining how much fibre is present in a food. Linking a particular amount of fibre to a specific health benefit is now an important area of research. Total Dietary Fibre. The term “dietary fibre” first appeared in 1953 and referred to hemicelluloses, celluloses and lignin (1). In 1974, Trowell (2) recommended this term as a replacement for the no longer acceptable term “crude fibre” Burkitt (3) has likened the interest in dietary fibre to the growth of a river from its first trickle to a mighty torrent. He observes that dietary fibre “was viewed as merely the less digestible constituent of food which exerts a laxative action by irritating the gut “thus acquiring the designation “roughage” a term which was later replaced by “crude fibre” and ultimately by “dietary fibre” Various definitions of dietary fibre have appeared over the years, partly due the various concepts used in deriving the term (i.e. origin of material, resistance to digestion, fermentation in the colon etc.), and partly to the difficulties associated with its measurement and labelling (4). The principle components of dietary fibre, as traditionally understood, are non-starch polysaccharides, which in plant fibre are principally hemicelluloses and celluloses, and the non-carbohydrate phenolic components, cutin, suberin and waxes with which they are associated in Nature.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
Berger, K., Falck, P., Linninge, C.,Nilsson, U., Axling, U., Grey, C., Stålbrand, H., Karlsson, E. N., Nyman, M., Holm, C. & Adlercreutz, P. (2014). Journal of Agricultural and Food Chemistry, 62(32), 8169-8178.
Barley husks, rye bran, and a fiber residue from oat milk production were processed by heat pretreatment, various separation steps, and treatment with an endoxylanase in order to improve the prebiotic potential of these cereal byproducts. Metabolic functions were intended to improve along with improved microbial activity. The products obtained were included in a high-fat mouse diet so that all diets contained 5% dietary fiber. In addition, high-fat and low-fat controls as well as partially hydrolyzed guar gum were included in the study. The soluble fiber product obtained from rye bran caused a significant increase in the bifidobacteria (log copies of 16S rRNA genes; median (25–75 percentile): 6.38 (6.04–6.66) and 7.47 (7.30–7.74), respectively; p < 0.001) in parallel with a tendency of increased production of propionic acid and indications of improved metabolic function compared with high-fat fed control mice. The oat-derived product caused an increase in the pool of cecal propionic (from 0.62 ± 0.12 to 0.94 ± 0.08) and butyric acid (from 0.38 ± 0.04 to 0.60 ± 0.04) compared with the high-fat control, and it caused a significant increase in lactobacilli (log copies of 16S rRNA genes; median (25–75 percentile): 6.83 (6.65–7.53) and 8.04 (7.86–8.33), respectively; p < 0.01) in the cecal mucosa. However, no changes in measured metabolic parameters were observed by either oat or barley products.Hide Abstract
Nemeth, C., Andersson, A. A. M., Andersson, R., Mangelsen, E., Sun, C. & Åman, P. (2014). Food and Nutrition Sciences, 5, 581-589.
Fructans are important in the survival of plants and also valuable for humans as potentially health promoting food ingredients. In this study fructan content and composition were determined in grains of 20 barley breeding lines and cultivars with a wide variation in chemical composition, morphology and country of origin, grown at one site in Chile. There was significant genotypic variation in grain fructan content ranging from 0.9% to 4.2% of grain dry weight. Fructan degree of polymerisation (DP) was analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Changes in the distribution of different chain lengths and the pattern of structures of fructan were detected with increasing amount of fructan in the different barleys. A positive correlation was found between fructan content and the relative amount of long chain fructan (DP > 9) (r = 0.54, p = 0.021). Our results provide a basis for selecting promising barley lines and cultivars for further research on fructan in barley breeding with the aim to produce healthy food products.Hide Abstract
Doti, S., Suárez-Belloch, J., Latorre, M. A., Guada, J. A. & Fondevila, M. (2014). Livestock Science, 164, 119-127.
Barley, rice, maize and peas as starch sources were tested to study if differences in glycemic index may affect feed utilization, productive performances and quality traits for growing pigs. Four experimental diets were formulated to include 420 g starch/kg, provided by barley, barley/broken rice, barley/maize or barley/peas combinations (diets coded B, R, M and P, respectively). A 45% of total starch from diets R, M and P was provided by broken rice, maize or peas. The in vitro characterization of feeds showed that the rapidly digestible starch fraction was highest for R (P<0.001) and no differences were recorded among the other diets, whereas the slowly digestible fraction was higher for B and M than for R (P<0.01) and that higher than for P (P<0.01). Then the resistant starch fraction was higher for P than for B and M (P<0.001) and those higher than for R (P<0.001). The rate of glucose release was fastest with R (P<0.001), and estimations of glycemic index were highest for R and lowest for P (P<0.001). For the in vivo trial, a total of 72 Duroc×(Landrace×Large White) crossbred gilts, with 63.0±4.55 kg body weight (116±3 days of age) was used. Statistical trends (P model<0.10) were observed for ADG and FCR; gilts fed diet P grew faster (P<0.05) and had a lower FCR (P<0.05) than those fed diet R. The apparent organic matter digestibility for diets R and P was lower than for diet B (P<0.001) but higher than for diet M (P<0.01). Similarly, the apparent crude protein digestibility was higher with diet R than with diet M (P<0.01). Gilts consumed from 32% to 40% of available feed within the first 2 h after offering, then the rate dropped from 2 to 4 h (P<0.05) and was virtually nil from 10 to 12 h for all diets. There was a limited influence of diet on carcass and meat characteristics but the intramuscular fat from pigs fed B showed higher (P<0.001) total saturated fatty acids and lower (P<0.001) total monounsaturated fatty acid contents than that from pigs fed M. It can be concluded that the pattern of gut digestion of the starch source may affect backfat thickness and growth performance of gilts, with scant effect on meat quality.Hide Abstract
Doehlert, D. C., Simsek, S. & McMullen, M. S. (2012). Cereal Chemistry, 89(5), 230-236.
Extraction protocols for β-glucan from oat flour were tested to determine optimal conditions for β-glucan quality testing, which included extractability and molecular weight. We found mass yields of β-glucan were constant at all temperatures, pH values, and flour-to-water ratios, as long as sufficient time and enough repeat extractions were performed and no hydrolytic enzymes were present. Extracts contained about 30–60% β-glucan, with lower proportions associated with higher extraction temperatures in which more starch and protein were extracted. All commercial starch hydrolytic enzymes tested, even those that are considered homogenous, degraded β-glucan apparent molecular weight as evaluated by size-exclusion chromatography. Higher concentration β-glucan solutions could be prepared by controlling the flour-to-water ratio in extractions. Eight grams of flour per 50 mL of water generated the highest native β-glucan concentrations. Routine extractions contained 2 g of enzyme-inactivated flour in 50 mL of water with 5mM sodium azide (as an antimicrobial), which were stirred overnight, centrifuged, and the supernatant boiled for 10 min. The polymer extracted had a molecular weight of about 2 million and was stable at room temperature for at least a month.Hide Abstract
Guzar, I., Ragaee, S. & Seetharaman, K. (2012). Journal of Food Science, 77(11), C1192-C1196.
A series of experiments were conducted to highlight the mechanism of inhibition of hydrolysis and differences in hydrolysis among starches from different sources in the presence of green or black tea extract. The first experiment showed that black tea extract was more effective at reducing final viscosity for all starches. The second experiment showed that black tea was more effective at inhibiting starch hydrolysis compared to green tea when starch, tea extract, and pancreatin were added at the beginning of pasting. The third experiment, when starches were pretreated with tea extracts, showed that both treatments reduced starch hydrolysis. Analysis of supernatant free phenolic content and of soluble dextrins showed that amyloglucosidase activity was affected, with exceptions for potato starch. These observations suggest that starch hydrolysis is affected by interactions and also by the impact on specific enzymes based on starch structure.Hide Abstract
Lin, Q., Huang, B., Zhang, M., Zhang, X., Rivenbark, J., Lappe, R. L., James, M. G., Myers, A. M. & Hennen-Bierwagen, T. A. (2012). Plant Physiology, 158(2), 679-692.
This study characterized genetic interactions between the maize (Zea mays) genes dull1 (du1), encoding starch synthase III (SSIII), and isa2, encoding a noncatalytic subunit of heteromeric isoamylase-type starch-debranching enzyme (ISA1/ISA2 heteromer). Mutants lacking ISA2 still possess the ISA1 homomeric enzyme. Eight du1- mutations were characterized, and structural changes in amylopectin resulting from each were measured. In every instance, the same complex pattern of alterations in discontinuous spans of chain lengths was observed, which cannot be explained solely by a discrete range of substrates preferred by SSIII. Homozygous double mutants were constructed containing the null mutation isa2-339 and either du1-Ref, encoding a truncated SSIII protein lacking the catalytic domain, or the null allele du1-R4059. In contrast to the single mutant parents, double mutant endosperms affected in both SSIII and ISA2 were starch deficient and accumulated phytoglycogen. This phenotype was previously observed only in maize sugary1 mutants impaired for the catalytic subunit ISA1. ISA1 homomeric enzyme complexes assembled in both double mutants and were enzymatically active in vitro. Thus, SSIII is required for normal starch crystallization and the prevention of phytoglycogen accumulation when the only isoamylase-type debranching activity present is ISA1 homomer, but not in the wild-type condition, when both ISA1 homomer and ISA1/ISA2 heteromer are present. Previous genetic and biochemical analyses showed that SSIII also is required for normal glucan accumulation when the only isoamylase-type debranching enzyme activity present is ISA1/ISA heteromer. These data indicate that isoamylase-type debranching enzyme and SSIII work in a coordinated fashion to repress phytoglycogen accumulation.Hide Abstract
Rakha, A., Åman, P. & Andersson, R. (2011). International journal of molecular sciences, 12(5), 3381-3393.
Extractable dietary fiber (DF) plays an important role in nutrition. This study on porridge making with whole grain rye investigated the effect of rest time of flour slurries at room temperature before cooking and amount of flour and salt in the recipe on the content of DF components and molecular weight distribution of extractable fructan, mixed linkage (1→3)(1→4)-β-D-glucan (β-glucan) and arabinoxylan (AX) in the porridge. The content of total DF was increased (from about 20% to 23% of dry matter) during porridge making due to formation of insoluble resistant starch. A small but significant increase in the extractability of β-glucan (P = 0.016) and AX (P = 0.002) due to rest time was also noted. The molecular weight of extractable fructan and AX remained stable during porridge making. However, incubation of the rye flour slurries at increased temperature resulted in a significant decrease in extractable AX molecular weight. The molecular weight of extractable β-glucan decreased greatly during a rest time before cooking, most likely by the action of endogenous enzymes. The amount of salt and flour used in the recipe had small but significant effects on the molecular weight of β-glucan. These results show that whole grain rye porridge made without a rest time before cooking contains extractable DF components maintaining high molecular weights. High molecular weight is most likely of nutritional importance.Hide Abstract
Monro, J. A., Mishra, S. & Venn, B. (2010). British Journal of Nutrition, 103(2), 295-305.
Glycaemic responses to foods reflect the balance between glucose loading into, and its clearance from, the blood. Current in vitro methods for glycaemic analysis do not take into account the key role of glucose disposal. The present study aimed to develop a food intake-sensitive method for measuring the glycaemic impact of food quantities usually consumed, as the difference between release of glucose equivalents (GGE) from food during in vitro digestion and a corresponding estimate of clearance of them from the blood. Five foods – white bread, fruit bread, muesli bar, mashed potato and chickpeas – were consumed on three occasions by twenty volunteers to provide blood glucose response (BGR) curves. GGE release during in vitro digestion of the foods was also plotted. Glucose disposal rates estimated from downward slopes of the BGR curves allowed GGE dose-dependent cumulative glucose disposal to be calculated. By subtracting cumulative glucose disposal from cumulative in vitro GGE release, accuracy in predicting the in vitro glycaemic effect from in vitro GGE values was greatly improved. GGEin vivo = 0·99GGEin vitro+0·75 (R2 0·88). Furthermore, the difference between the curves of cumulative GGE release and disposal closely mimicked in vivo incremental BGR curves. We conclude that valid measurement of the glycaemic impact of foods may be obtained in vitro, and expressed as grams of glucose equivalents per food quantity, by taking account not only of GGE release from food during in vitro digestion, but also of blood glucose clearance in response to the food quantity.Hide Abstract
Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S. & Walton, J. D. (2010). Biotechnology for Biofuels, 3(1), 22.
Background: Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers’ grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings. Results: When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h. Conclusion: The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.Hide Abstract
Kamal-Eldin, A., Lærke, H. N., Knudsen, K. E. B., Lampi, A. M., Piironen, V., Adlercreutz, H., Katina, K., Poutanen, K. & Aman, P. (2009). Food & Nutrition Research, 53.
Background: Epidemiological studies show inverse relationship between intake of wholegrain cereals and several chronic diseases. Components and mechanisms behind possible protective effects of wholegrain cereals are poorly understood. Objective: To characterise commercial rye bran preparations, compared to wheat bran, regarding structure and content of nutrients as well as a number of presumably bioactive compounds. Design: Six different rye brans from Sweden, Denmark and Finland were analysed and compared with two wheat brans regarding colour, particle size distribution, microscopic structures and chemical composition including proximal components, vitamins, minerals and bioactive compounds. Results: Rye brans were generally greener in colour and smaller in particle size than wheat brans. The rye brans varied considerably in their starch content (13.2–28.3%), which reflected variable inclusion of the starchy endosperm. Although rye and wheat brans contained comparable levels of total dietary fibre, they differed in the relative proportions of fibre components (i.e. arabinoxylan, β-glucan, cellulose, fructan and Klason lignin). Generally, rye brans contained less cellulose and more β-glucan and fructan than wheat brans. Within small variations, the rye and wheat brans were comparable regarding the contents of tocopherols/tocotrienols, total folate, sterols/stanols, phenolic acids and lignans. Rye bran had less glycine betaine and more alkylresorcinols than wheat brans. Conclusions: The observed variation in the chemical composition of industrially produced rye brans calls for the need of standardisation of this commodity, especially when used as a functional ingredient in foods.Hide Abstract
Mishra, S., Monro, J. & Hedderley, D. (2008). Starch‐Stärke, 60(9), 500-507.
The effect of a number of laboratory-scale pretreatments on the proportions of rapidly digested (RDS), slowly digested (SDS) and resistant starch (RS) in raw and cooked potato has been examined using an in vitro digestion procedure. Potatoes of the variety Frisia were prepared in three states: raw, cooked, and cooked followed by a cold treatment (4°C, two days). Each preparation was then subjected in triplicate to freeze-drying, coarsely mincing, pasting, freezing, dry-milling after freeze-drying, in 22 different combinations, before digesting. In raw potato, very little RDS and SDS (<5% total starch (TS)) were present, and the mechanical treatments of the potato did not affect the amounts of RDS and SDS. Cooking resulted in an almost complete conversion to RDS (>95% TS) in freshly-cooked potato, but after post-cooking cold treatment much of the RDS transformed to SDS, which reached a maximum of about 45% TS. SDS formation was independent of the degree of tissue disruption after cooking, and was generally associated with formation of RS, however, freezing after cooking allowed SDS formation without prolonged cold treatment and with very little associated RS (SDS 35% and RS 4% of TS). Freeze-drying caused an increase in RS in most treatments of the cooked potatoes. The observed effects provided guidance for sample handling in potato research, but also suggested several approaches to the enrichment of SDS and/or RS, with a concurrent reduction in RDS, that could be used to improve the nutritional profile of potato products by decreasing RDS (lowered glycaemic impact), and increasing SDS (more sustained energy availability) and RS (prebiotic benefits).Hide Abstract
Liu, Q., Gu, Z., Donner, E., Tetlow, I. & Emes, M. (2007). Cereal Chemistry, 84(1), 15-21.
In this study, the functional properties of A- and B-type wheat starch granules from two commercial wheat flours were investigated for digestibility in vitro, chemical composition (e.g., amylose, protein, and ash content), gelatinization, retrogradation, and pasting properties. The branch chain length and chain length distribution of these A- and B-type wheat starch granules were also determined using high-performance anion exchange chromatography (HPAEC). Wheat starches with different granular sizes not only had different degrees of enzymatic hydrolysis and thermal and pasting properties, but also different molecular characteristics. Different amylose content, protein content, and branch chain length of amylopectin in A- and B-type wheat starch granules could also be the major factors besides granular size for different digestibility and other functional properties of starch. The data indicate that different wheat cultivars with different proportion of A- and B-type granular starch could result in different digestibility in wheat products.Hide Abstract
Liu, Q., Donner, E., Yin, Y., Huang, R. L. & Fan, M. Z. (2006). Food Chemistry, 99(3), 470-477.
Digestibility, gelatinization, retrogradation and pasting properties of starch in various cereal, tuber and legume flours were determined. Rapidly and slowly digestible starch and resistant starch were present in 11 selected flours. In general, cereal starches were more digestible than legume starches and tuber starches contained a high amount of resistant starch. Thermal and rheological properties of flours were different depending on the crop source.Hide Abstract
Lilla, Z., Sullivan, D., Ellefson, W., Welton, K. & Crowley, R. (2005). Journal of AOAC International, 88(3), 714-719.
For labeling purposes, the carbohydrate content of foods has traditionally been determined by difference. This value includes sugars, starches, fiber, dextrins, sugar alcohols, polydextrose, and various other organic compounds. In some cases, the current method may lack sufficient specificity, precision, and accuracy. These are subsequently quantitated by high performance anion exchange chromatography with pulsed amperometric detection and expressed as total nonfiber saccharides or percent “net carbohydrates.” In this research, a new method was developed to address this need. The method consists of enzyme digestions to convert starches, dextrins, sugars, and polysaccharides to their respective monosaccharide components. These are subsequently quantified by high-performance anion exchange chromatography with pulsed amperometric detector and expressed as total nonfiber saccharides or percent “net carbohydrates.” Hydrolyzed end products of various novel fibers and similar carbohydrates have been evaluated to ensure that they do not register as false positives in the new test method. The data generated using the “net carbohydrate” method were, in many cases, significantly different than the values produced using the traditional methodology. The recoveries obtained in a fortified drink matrix ranged from 94.9 to 105%. The coefficient of variation was 3.3%.Hide Abstract
Xie, X. S. & Liu, Q. (2004). Starch‐Stärke, 56(8), 364-370.
Resistant starch has drawn broad interest for both potential health benefits and functional properties. In this study, a technology was developed to increase resistant starch content of corn starch using esterification with citric acid at elevated temperature. Waxy corn, normal corn and high-amylose corn starches were used as model starches. Citric acid (40% of starch dry weight) was reacted with corn starch at different temperatures (120–150°C) for different reaction times (3–9 h). The effect of reaction conditions on resistant starch content in the citrate corn starch was investigated. When conducting the reaction at 140°C for 7 h, the highest resistant starch content was found in waxy corn citrate starch (87.5%) with the highest degree of substitution (DS, 0.16) of all starches. High-amylose corn starch had 86.4% resistant starch content and 0.14 DS, and normal corn starch had 78.8% resistant starch and 0.12 DS. The physicochemical properties of these citrate starches were characterized using various analytical techniques. In the presence of excess water upon heating, citrate starch made from waxy corn starch had no peak in the DSC thermogram, and small peaks were found for normal corn starch (0.4 J/g) and Hylon VII starch (3.0 J/g) in the thermograms. This indicates that citrate substitution changes granule properties. There are no retrogradation peaks in the thermograms when starch was reheated after 2 weeks storage at 5°C. All the citrate starches showed no peaks in RVA pasting curves, indicating citrate substitution changes the pasting properties of corn starch as well. Moreover, citrate starch from waxy corn is more thermally stable than the other citrate starches.Hide Abstract
Quigley, M. E., Hudson, G. J. & Englyst, H. N. (1999). Food Chemistry, 65(3), 381-390.
We have proposed the term short-chain carbohydrates (SCC) for those species, other than the free sugars, that are soluble in 80% ethanol under well-defined conditions. We describe a technique for the measurement of resistant SCC (RSCC), which are not susceptible to pancreatic amylase or the brush border enzymes and therefore sometimes termed non-digestible oligosaccharides. In the procedure, alpha-glucans (starch and maltodextrins) are hydrolysed enzymatically to glucose and the non-starch polysaccharides (NSP) are precipitated in ethanol. Fructans are hydrolysed enzymatically and the monosaccharide constituents are reduced to acid-stable alditol derivatives before the remaining RSCC are hydrolysed with sulphuric acid. All the constituent sugars are measured as alditol acetate derivatives by gas–liquid chromatography. The protocol allows both the measurement of total RSCC and a separate, specific measurement of fructans.Hide Abstract