New developments in the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase.
McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.
Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.
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 Techniques, 2(4), 289-292.
A procedure for the measurement of fungal and bacterial α-amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end) p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and α-glucosidase, and is absolutely specific for α-amylase. The assay procedure is simple, reliable and accurate.
An improved enzymic method for the measurement of starch damage in wheat flour.
Gibson, T. S., Al Qalla, H. & McCleary, B. V. (1992). Journal of Cereal Science, 15(1), 15-27.
An improved enzymic method for the determination of starch damage in wheat flour has been developed and characterized. The proposed method is simple and reliable, and enables up to 20 samples to be measured in duplicate in 2 h. A single assay takes approximately 40 min. The assay protocol is in two phases. In the first, the flour sample is incubated with purified fungal alpha-amylase to liberate damaged starch granules as soluble oligosaccharides. After centrifugation, the oligosaccharides in the supernatant are hydrolysed by amyloglucosidase to glucose in phase 2. The glucose is then quantified with a glucose oxidase/peroxidase reagent. The proposed method therefore avoids potential errors associated with existing standard assays, which employ unpurified amylase preparations and non-specific reducing group methods to quantify the hydrolytic products. Despite the use of purified assay components, the proposed starch damage method did not exhibit an absolute end-point to the action of alpha-amylase in phase 1. This was due to a low rate of hydrolysis of undamaged granules, and is a feature of enzymic methods for starch damage determination. Other amylolytic enzymes, including beta-amylase, isoamylase and pullulanase, and combinations of these enzymes, were evaluated as alternatives to alpha-amylase in the proposed method. These enzymes, when used alone, gave no benefits over the use of alpha-amylase. When used in combination with alpha-amylase, there was a synergistic action on undamaged granules. A test kit based on the assay format described in this paper is the subject of an international interlaboratory evaluation.
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%.
The physicochemical properties and in vitro digestibility of selected cereals, tubers and legumes grown in China.
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.
Determination of “Net Carbohydrates” using high-performance anion exchange chromatography.
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%.
Physical, microscopic and chemical characterisation of industrial rye and wheat brans from the Nordic countries.
Kamal-Eldin, A., Lærke, H. N., Knudsen, K. E. B., Lampi, A. M., Piironen, V., Adlercreutz, H., Katina, K., Poutanen, K. & Ɨman, P. A. (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.
A simplified modification of the AOAC official method for determination of total dietary fiber using newly developed enzymes: preliminary interlaboratory study.
Kanaya, K., Tada, S., Mori, B., Takahashi, R., Ikegami, S., Kurasawa, S., Okuzaki, M., Mori, Y., Innami, S. & Negishi, Y. (2007). Journal of AOAC International, 90(1), 225-237.
A preliminary interlaboratory study was conducted to evaluate the validity of the modified AOAC method for determination of total dietary fiber by Tada and Innami, in which the 3-step enzymatic digestion process in AOAC Method 991.43 is modified to a 2-step process without pH adjustment. Total dietary fiber contents in 8 representative foodstuffs were measured using both the original AOAC Method 991.43 and the modified method in 6 research facilities in Japan. Repeatability relative standard deviations, reproducibility relative standard deviations, and Horwitz ratio values from the modified method were equivalent to those from AOAC Method 991.43, except in the rice sample. However, this exceptional case shown in the modified method was entirely dissolved by the addition of -amylase stabilizing agents. The modified method, which shortens the process of enzymatic digestion from 3 to 2 steps and in which only reaction temperature is adjusted under the same pH, was found not only to give accurate values comparable to the original method, but also to substantially reduce the labor required by the laboratory staff in the process of routine analysis. This study revealed that the validity of the modified method was further ensured by adding -amylase stabilizing agents to the reaction system.
Treatment of cereal products with a tailored preparation of Trichoderma enzymes increases the amount of soluble dietary fiber.
Napolitano, A., Lanzuise, S., Ruocco, M., Arlotti, G., Ranieri, R., Knutsen, S. H., Lorito, M. & Fogliano, V. (2006). Journal of Agricultural and Food Chemistry, 54(20), 7863-7869.
Nutritionists recommend increasing the intake of soluble dietary fiber (SDF), which is very low in most cereal-based products. Conversion of insoluble DF (IDF) into SDF can be achieved by chemical treatments, but this affects the sensorial properties of the products. In this study, the possibility of getting a substantial increase of SDF from cereal products using a tailored preparation of Trichoderma enzymes is reported. Enzymes were produced cultivating Trichoderma using durum wheat fiber (DWF) and barley spent grain (BSG) as unique carbon sources. Many Trichoderma strains were screened, and the hydrolysis conditions able to increase by enzymatic treatment the amount of SDF in DWF and BSG were determined. Results demonstrate in both products that it is possible to triple the amount of SDF without a marked decrease of total DF. The enzymatic treatment also causes the release of hydroxycinnamic acids, mainly ferulic acid, that are linked to the polysaccharides chains. This increases the free phenolic concentration, the water-soluble antioxidant activity, and, in turn, the phenol compounds bioavailability.
Method for the direct determination of available carbohydrates in low-carbohydrate products using high-performance anion exchange chromatography.
Ellingson, D., Potts, B., Anderson, P., Burkhardt, G., Ellefson, W., Sullivan, D., Jacobs, W. & Ragan, R. (2010). Journal of AOAC International, 93(6), 1897-1904.
An improved method for direct determination of available carbohydrates in low-level products has been developed and validated for a low-carbohydrate soy infant formula. The method involves modification of an existing direct determination method to improve specificity, accuracy, detection levels, and run times through a more extensive enzymatic digestion to capture all available (or potentially available) carbohydrates. The digestion hydrolyzes all common sugars, starch, and starch derivatives down to their monosaccharide components, glucose, fructose, and galactose, which are then quantitated by high-performance anion-exchange chromatography with photodiode array detection. Method validation consisted of specificity testing and 10 days of analyzing various spike levels of mixed sugars, maltodextrin, and corn starch. The overall RSD was 4.0 across all sample types, which contained within-day and day-to-day components of 3.6 and 3.4, respectively. Overall average recovery was 99.4 (n = 10). Average recovery for individual spiked samples ranged from 94.1 to 106 (n = 10). It is expected that the method could be applied to a variety of low-carbohydrate foods and beverages.
Low folate content in gluten-free cereal products and their main ingredients.
Yazynina, E., Johansson, M., Jägerstad, M. & Jastrebova, J. (2008). Food Chemistry, 111(1), 236-242.
Folate content in some gluten-free cereal products and their main ingredients was determined using a validated method based on reversed-phase high performance liquid chromatography (HPLC) with fluorescence and diode array detection. The main folate forms found in gluten-free products were 5-methyl-tetrahydrofolate and tetrahydrofolate. Starches and low protein flours commonly used as main components in gluten-free products appeared to be poor folate sources with folate content ≤ 6 µg/100 g fresh weight. Folate content in gluten-free breads was higher (15.1–35.9 µg folate/100 g fresh weight) due to use of bakery yeast which is a rich folate source. Overall, folate content in gluten-free products was lower than in their gluten-containing counterparts. Therefore, fortification of gluten-free products with folic acid or enrichment of these products with nutrient-dense fractions of cereals naturally free from gluten (such as buckwheat, quinoa, amaranth or millet) can be of interest.
Starch transformation in bran-enriched extruded wheat flour.
Robin, F., Théoduloz, C., Gianfrancesco, A., Pineau, N., Schuchmann, H. P. & Palzer, S. (2011). Carbohydrate Polymers, 85(1), 65-74.
Wheat flour was extruded at different conditions of barrel temperature (120°C and 180°C), water content (18% and 22%) and screw speed (400 rpm and 800 rpm) with an increasing concentration of wheat bran fibers (2.8%, 12.6% and 24.4%). In the tested extrusion conditions, starch crystallites were fully dissociated. The estimated starch solubility was influenced by the process conditions and ranged from 24.1% to 63.1%. At same process conditions, the starch solubility was increased only at the highest bran level. The bran concentration influenced the glass transition temperature, melting temperature and sorption isotherm of the unprocessed wheat flour. At the extrusion conditions, it showed that higher bran levels led to a higher amount of free water and a decrease in starch glass transition temperature of up to 13 K. The differences in starch transformation, induced by the concentration of bran, might contribute to the modulation of the expansion properties of bran-containing starchy foams.
Potato genotype differences in nutritionally distinct starch fractions after cooking, and cooking plus storing cool.
Monro, J., Mishra, S., Blandford, E., Anderson, J. & Genet, R. (2009). Journal of Food Composition and Analysis, 22(6), 539-545.
Rapidly digestible (RDS), slowly digestible (SDS) and resistant starch (RS) were measured in 9 New Zealand supermarket potatoes and in 37 lines from a potato breeding program by in vitro digestion immediately after cooking, and after storing at 4°C for 44 h post-cooking. The aim was to measure the range in the tendency to form SDS and RS in the potato gene pool in New Zealand. Immediately after cooking, the potatoes contained (mean and across-cultivar range, dry matter basis) 68% RDS (range 62–73%), 3% SDS (range 0–8.5%), and 3.9% RS (range 3–6.4%). Cool storage after cooking altered the distribution and ranges to 44% RDS (range 33–53%), 23% SDS (range 15–34%) and 7% RS (range 4.7–15.8%). There was no significant relationship between RS and SDS in the cooked-cooled potatoes. In the 37 potato lines, SDS ranged from 7 to 37% of total starch, RS from 12 to 27% of total starch after the post-cooking cool treatment. The results suggest that the glycaemic impact of some potatoes may be substantially reduced by cool-storing after cooking, and that the differences between cultivars in the tendency to form cold-induced SDS and RS are sufficient for these traits to be used in conventional plant breeding.
Molecular, mesoscopic and microscopic structure evolution during amylase digestion of maize starch granules.
Shrestha, A. K., Blazek, J., Flanagan, B. M., Dhital, S., Larroque, O., Morell, M. K., Gilbert, E. P. & Gilbert, M. J. (2012). Carbohydrate Polymers, 90(1), 23-33.
Cereal starch granules with high (>50%) amylose content are a promising source of nutritionally desirable resistant starch, i.e. starch that escapes digestion in the small intestine, but the structural features responsible are not fully understood. We report the effects of partial enzyme digestion of maize starch granules on amylopectin branch length profiles, double and single helix contents, gelatinisation properties, crystallinity and lamellar periodicity. Comparing results for three maize starches (27, 57, and 84% amylose) that differ in both structural features and amylase-sensitivity allows conclusions to be drawn concerning the rate-determining features operating under the digestion conditions used. All starches are found to be digested by a side-by-side mechanism in which there is no major preference during enzyme attack for amylopectin branch lengths, helix form, crystallinity or lamellar organisation. We conclude that the major factor controlling enzyme susceptibility is granule architecture, with shorter length scales not playing a major role as inferred from the largely invariant nature of numerous structural measures during the digestion process (XRD, NMR, SAXS, DSC, FACE). Results are consistent with digestion rates being controlled by restricted diffusion of enzymes within densely packed granular structures, with an effective surface area for enzyme attack determined by external dimensions (57 or 84% amylose – relatively slow) or internal channels and pores (27% amylose – relatively fast). Although the process of granule digestion is to a first approximation non-discriminatory with respect to structure at molecular and mesoscopic length scales, secondary effects noted include (i) partial crystallisation of V-type helices during digestion of 27% amylose starch, (ii) preferential hydrolysis of long amylopectin branches during the early stage hydrolysis of 27% and 57% but not 84% amylose starches, linked with disruption of lamellar repeating structure and (iii) partial B-type recrystallisation after prolonged enzyme incubation for 57% and 84% amylose starches but not 27% amylose starch.