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Amylose/Amylopectin Assay Kit

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00:01   Introduction
01:16    Theory of the Analytical Procedure
02:33   Kit Content & Reagent Preparation
06:15    Weighing of Samples
06:44    A. Starch Pretreatment
12:04    B. Precipitation of Amylopectin with Con A & the Determination of Amylose
16:11      C. Determination of Total Starch
18:35     Determination of D-Glucose in Amylose & Total Starch Fractions
20:37    Calculation of Amylose Content

Amylose Amylopectin Assay Kit K-AMYL Scheme
Product code: K-AMYL

100 assays per kit

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Content: 100 assays per kit
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: Amylopectin, Amylose
Assay Format: Spectrophotometer
Detection Method: Absorbance
Wavelength (nm): 510
Signal Response: Increase
Limit of Detection: Amylose 5-95% of total starch content
Total Assay Time: ~ 120 min
Application examples: Cereal starches, flours, pure starches and foods.
Method recognition: Novel method

The Amylose/Amylopectin test kit is suitable for the measurement and analysis of amylose/amylopectin ratio and content in cereal starches and flours. Based on a Con A precipitation procedure.

See our full list of polysaccharide test kits.

Scheme-K-AMYL K-AMYL Megazyme

  • Very cost effective (cost per test) 
  • All reagents stable for > 12 months after preparation 
  • Only enzymatic kit available 
  • Accurate and reliable amylose / amylopectin ratio determination  
  • Simple format 
  • Standard included
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator Product Performance Validation Report
Megazyme publication
Measurement of carbohydrates in grain, feed and food.

McCleary, B. V., Charnock, S. J., Rossiter, P. C., O’Shea, M. F., Power, A. M. & Lloyd, R. M. (2006). Journal of the Science of Food and Agriculture, 86(11), 1648-1661.

Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successfully subjected to interlaboratory evaluation. All methods are based on the use of enzymes either purified by conventional chromatography or produced using molecular biology techniques. Such methods allow specific, accurate and reliable quantification of a particular component. Problems in calculating the actual weight of galactosyl-sucrose oligosaccharides in test samples are discussed in detail.

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Megazyme publication
Measurement of total starch in cereal products by amyloglucosidase-alpha-amylase method: collaborative study.

McCleary, B. V., Gibson, T. S. & Mugford, D. C. (1997). Journal of AOAC International, 80, 571-579.

An American Association of Cereal Chemists/AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measurement of total starch in a range of cereal grains and products. The flour sample is incubated at 95 degrees C with thermostable alpha-amylase to catalyze the hydrolysis of starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze the dextrins to glucose. Glucose is measured with glucose oxidase-peroxidase reagent. Thirty-two collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included chicken feed pellets, white bread, green peas, high-amylose maize starch, white wheat flour, wheat starch, oat bran, and spaghetti. All samples were analyzed by the standard procedure as detailed above; 4 samples (high-amylose maize starch and wheat starch) were also analyzed by a method that requires the samples to be cooked first in dimethyl sulfoxide (DMSO). Relative standard deviations for repeatability (RSD(r)) ranged from 2.1 to 3.9%, and relative standard deviations for reproducibility (RSD(R)) ranged from 2.9 to 5.7%. The RSD(R) value for high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%; the value was reduced to 2.9% when the DMSO procedure was used, and the determined starch values increased from 86.9 to 97.2%.

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Megazyme publication
A procedure to measure amylose in cereal starches and flours with concanavalin A.

Gibson, T. S., Solah, V. A. & McCleary, B. V. (1997). Journal of Cereal Science, 25(2), 111-119.

A modified procedure for the determination of amylose in cereal starches and flours based on complex formation between the lectin concanavalin A (Con A) and amylopectin has been developed and characterised. The assay format is suitable for multi-sample analysis, allowing the analysis of up to 20 samples per day. In the procedure, the amylopectin in a solubilised, lipid-free starch sample is precipitated by reaction with Con A and removed by centrifugation. The amylose remaining in the supernatant is then determined after amylolytic hydrolysis to glucose and expressed as a proportion (%) of the glucose derived from amylolytic hydrolysis of the total starch in a separate aliquot of the solubilised sample (i.e. prior to Con A treatment). The Con A procedure correlates well (r>0•993) with existing Con A-based and iodine-based procedures and yields a linear standard curve for starch samples containing from ∼0 to ∼100% amylose. Advantages of this modified Con A procedure for amylose determination include its applicability to flour samples without the need for prior starch purification; it allows the simultaneous estimation of total starch and does not require a calibration curve. Repeated analyses of a set of samples yielded repeatability (within laboratory) relative standard deviations of <5% for pure starches and ∼10% for flours. A commercially available assay kit has been developed to facilitate the use of the Con A procedure in routine amylose determinations.

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Comparative analysis of malt quality and starch characteristics of three South Korean barley cultivars.

Park, J., Chung, H. J., Park, H. Y., Park, H. J. & Oh, S. K. (2023). Food Science and Biotechnology, 1-11.

In this study, malt was produced in pilot-scale facilities and conditioned using three barley (Hordeum vulgare L.) cultivars in South Korea (Heugho, Hopum, and Kwangmaeg). Quality and starch characteristics were compared. The starch content was considerably reduced in all malts. Coleoptile elongation was higher in Heugho (HHM; 85.7% ± 12.6%) and Hopum (HPM; 83.9% ± 10.7%) than in Kwangmaeg (KMM; 78.1% ± 9.9%) malt. Malt yield ranged from 81.8 to 84.9%, with no significant difference. All samples presented type A crystallinity, and granules showed discoid shapes. After malting, the mono- and di-saccharide contents (not including sucrose) were increased. The fermentable sugar level was the highest in HHM, whereas non-fermentable sugar was the highest in KMM. These results suggest that HPM enables efficient scarification based on the rapid degradation of starch, while Heugho barley and HHM have a high potential for beer and malt production, respectively.

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High-throughput phenotyping of nutritional quality components in sweet potato roots by near-infrared spectroscopy and chemometrics methods.

Tang, C., Jiang, B., Ejaz, I., Ameen, A., Zhang, R., Mo, X. & Wang, Z. (2023). Food Chemistry: X, 20, 100916.

The lack of an efficient approach for quality evaluation of sweet potatoes significantly hinders progress in quality breeding. Therefore, this study aimed to establish a near-infrared spectroscopy (NIRS) assay for high-throughput analysis of sweet potato root quality, including total starch, amylose, amylopectin, the ratio of amylopectin to amylose, soluble sugar, crude protein, total flavonoid content, and total phenolic content. A total of 125 representative samples were utilized and a dual-optimized strategy (optimization of sample subset partitioning and variable selection) was applied to NIRS modeling. Eight optimal equations were developed with an excellent coefficient of determination for the calibration (R2C) at 0.95-0.99, cross-validation (R2CV) at 0.93-0.98, external validation (R2V) at 0.89–0.96, and the ratio of prediction to deviation (RPD) at 6.33-11.35. Overall, these NIRS models provide a feasible approach for high-throughput analysis of root quality and permit large-scale screening of elite germplasm in future sweet potato breeding.

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In-mouth mechanisms leading to physicochemical changes and textural perception of cooked rice prepared with different heating rates.

Yin, X., Chen, X., Hu, J., Zhu, L., Zhang, H., Hong, Y. & Zhang, Z. (2023). Food Bioscience, 56, 103282.

Oral textural perception is a complex process that involves tooth fragmentation and salivary lubrication. To reveal the associated in-mouth mechanisms, three stages of testing were conducted in this study, including the initial cooked rice stage (0%), the deformation stage (50%), and the swallowing stage (100%). The total chewing time of high/low heating rate rice (HHRR/LHRR) was 25.46 ± 6.22 s and 22.11 ± 5.02 s, respectively. However, no significant difference in the chewing frequency. The salivary flow rate in LHRR (28.18 mg/s) was higher than in HHRR (22.57 mg/s). At the 0% stage, LHRR had a higher moisture content and expansion rate. At the 50% stage, rice boluses exhibited a significant difference in texture properties measured by instrumental analysis. At the 100% stage, two rice boluses reached similar hardness and chewiness, with smaller particles for swallowing. These results indicated that the deformation stage (50%) played a critical role in differentiating the textural perception. The initial properties of cooked rice influenced chewing manner and salivary secretion, contributing to differences in the reduction of particle size, water migration, and the inner microstructure evolution of corresponding rice boluses. These differences will feed back as different texture perceptions during oral processing.

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Effects of explosion puffing on the native structural organization and oil adsorption properties of starch.

Shao, M., Junejo, S. A., Zhang, B. & Huang, Q. (2024). Carbohydrate Polymers, 324, 121518.

The effects of explosion puffing (EP) on the native structural organization (i.e., thermal properties, crystalline structure, short-range order, granule morphology and powder properties) and oil adsorption properties of puffed starch (PS) were investigated. The results showed that EP treatment could decrease the melting enthalpy of starch double helices and increase the V-type crystallinity. The highest V-type crystallinity (24.7 %) was obtained when the puffing pressure was 0.4 MPa and the starch:ethanol:water ratio was 1:2:1 (w/w). By controlling the puffing conditions, EP treatment can alter the morphology, and increase the particle size, flowability and specific surface area of PS. The high amorphous proportion and porous sheet structure of PS resulted in the highest oil adsorption capacity when the starch:ethanol:water ratio was 1:1:1 (w/w). Pearson correlation analysis showed that oil adsorption capacity was significantly and positively correlated with the 1022/995 cm−1 value and V-type crystallinity, but negatively correlated with bulk density and angle of repose. Furthermore, oil retention capacity was strongly dependent on V-type crystallinity. These findings demonstrated that EP is an innovative technology with the potential to enhance the V-type crystallinity and adsorption performance of starch.

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Effects of single and dual modifications with debranching and heat-moisture treatments on physicochemical, rheological, and digestibility properties of proso millet starch.

Kumar, S. R., Tangsrianugul, N., Sriprablom, J., Winuprasith, T., Wansuksri, R. & Suphantharika, M. (2023). Carbohydrate Polymer Technologies and Applications, 6, 100399.

The effects of single and dual modifications with heat-moisture treatment (HMT) and enzymatic debranching on the structural, physicochemical, rheological, and in-vitro digestibility properties of proso millet starch (PMS) were comparatively studied. Amylose content increased after gelatinization and all modification treatments. Debranching and dual modification (debranching followed by HMT) decreased the average chain length and swelling power but increased the solubility of PMS. HMT did not change the average chain length and chain length distribution in native and debranched starches but decreased the swelling power and solubility of native starch. The A-type X-ray diffraction pattern of PMS was converted to the A + V type by HMT and to the B + V type by debranching and dual modification. FTIR investigation showed that the short-range molecular order of all modified starches was disrupted. The viscoelasticity and viscosity of all starch pastes decreased in the following order: PMS > HMT starch > debranched starch > dual modified starch. In-vitro starch digestibility showed that HMT increased slowly digestible starch (SDS) to 32.88 %, while debranching increased resistant starch (RS) to 77.14 %. HMT increased RS to 86.17 % in the dual modified starch. Debranching therefore predominates in the dual modification of starch compared to HMT.

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Extrusion as pretreatment for complexation of high-amylose starch with glycerin monostearin: Dependence on the guest molecule.

Zhuang, P., Wu, X., Li, Q., Su, X. & Chen, L. (2024). International Journal of Biological Macromolecules, 256, 128439.

Low-moisture extrusion (LME) can modify starch structures and enrich their functionality. These LME-made starches may efficiently form inclusion complexes (ICs) with hydrophobic guest molecules, which is profoundly impacted by the guest molecule concentration. In this work, the influence of glycerin monostearin (GMS) concentration on the structure and in vitro digestibility of pre-extruded starch-GMS complexes was investigated. The results showed that LME pretreatment increased the complex index of high-amylose starch with GMS by 13 %. The appropriate GMS concentrations produced ICs with high crystallinity and excellent thermostability. The presence of IC retarded amylose retrogradation and dominated bound water in starches. In addition, highly crystallized ICs were resistant to enzymolysis and had a higher proportion of resistant starch. The acquired knowledge would provide a better understanding of the LME-modified starch and GMS concentration-regulated IC formation.

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Effect of static magnetic field pretreatment on the structure and oil absorption properties of normal maize starch.

Cui, Y., Wang, W., Gao, W., Shi, M., Liu, S., Liu, C., Zheng, M., Liu, M., Liu, H. & Liu, J. (2024). Food Hydrocolloids, 149, 109589.

In this work, we report the effect of static magnetic field (SMF) on normal maize starch (NMS) structure and oil absorption properties. The results showed that the untreated NMS had the total oil absorption of 83.90%, solubility of 19.00%, swelling power of 20.32 g/g, and amylose content of 26.95%. Besides, compared with untreated NMS, pretreatment with different SMF strengths significantly reduced the total oil absorption (61.78-78.12%), solubility (16.99–18.61%), swelling power (17.62–19.02 g/g), and amylose content (19.77–24.73%) of NMS. Besides, the SMF-pretreated NMS had a rough surface with more pits and pores, a denser particle structure, and a smaller specific surface area and particle size than untreated NMS. However, the high SMF intensity (50 mT) made NMS form a porous structure, leading to the highest oil absorption of NMS (78.12%), but still less than untreated NMS (83.90%). FT-IR, XRD, and DSC studies further showed that SMF pretreatment improved the structural ordering and thermal stability of NMS particles, inhibiting the oil absorption of NMS. To investigate whether SMF pretreatment could reduce starch-based fried food oil absorption, LF-NMR was used to determine the oil content of fried NMS treated with different SMF strengths. Results indicated the oil content of NMS after frying was significantly reduced by SMF pretreatment than untreated NMS, and the lowest was 0.2751 g/g for SMF-30. Therefore, SMF pretreatment can effectively reduce the oil absorption capacity of NMS before and after frying and serve as a prospective technology to reduce the oil content of starch-based fried foods.

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Seasonal Changes in Amylose and Starch Compositions in ‘Ambrosia’Apples Associated with Rootstocks and Orchard Climatic Conditions.

Lu, C., Xu, H., Lannard, B. & Yang, X. (2023). Agronomy, 13(12), 2923.

The dynamics of amylose (AM) and total starch content (TSC) have been studied in several apple varieties; however, their responses to environmental variables and rootstocks are less understood in new low-climacteric varieties. In addition, the pertinence of AM and TSC to fruit dry matter content (DMC), fruit quality at harvest, and quality retention after storage is little understood. In this study, we investigated the seasonal changes in AM and TSC of ‘Ambrosia’ apples grafted onto dwarfing rootstocks Malling 9 (M.9) and Budagovsky 9 (B.9) in two consecutive years, 2016 and 2017, in the Okanagan-Similkameen Valley, British Columbia, Canada. Enzymatic methods with Megazyme® kits were used to analyze the fruit samples collected from four orchards in the growing season. In 2016, the orchard microclimate exhibited cooler nights and greater diurnal temperature ranges (DTRs) compared to 2017. As a result, the AM content and AM portion in TSC were significantly higher in 2016, with levels peaking over 40% compared to the values observed in 2017. However, there were no significant differences in peak values of TSC between the two years. Additionally, the apples grown on the B.9 rootstock consistently showed higher levels of AM and AM percentage compared to those grown on the M.9 rootstock. The study also used Felix 750 Produce Quality Meter, a handheld infrared spectrometer, to assess the dry matter content (DMC) of growing fruit, which showed a strong correlation with AM content (r > 0.8). After being harvested at commercial maturity and stored in controlled atmospheric chambers (CA) for 8 months, fruit quality analysis suggested that the AM level and its percentage in TSC played an important role in determining ‘Ambrosia’ apple quality after long-term storage.

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Insight into wheat starch characteristics and fat absorption of breaded fish nuggets during frying.

Feng, J., Chen, J., Chen, C., Peng, L., Zhang, P. & Liu, G. (2024). Journal of Food Engineering, 366, 111855.

Understanding the effects of frying temperature and time on fat absorption is crucial for the manufacturing of low-fat fried products. The crystalline structure and pasting properties of the starch were analyzed for fried batter-breaded fish nuggets. Also, the amylose content, calorimetric properties, thermogravimetric properties, oil distribution of the crust were measured. The results showed that the amylose content of the starch was significantly increased with the increasing frying temperature and time, while enthalpy change, relative crystallinity, peak viscosity and final viscosity exhibited an opposite trend. Changes in crystalline structure, calorimetric and thermogravimetric properties demonstrated that starch was bound to lipids to form starch-lipid complexes and the type II starch-lipid complexes were disintegrated during continuously frying, which led to a large-scale oil distribution of the crust. These results suggested that the frying temperature and time significantly affected the wheat starch characteristics and oil distribution, thereby leading to the fat absorption.

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Characterization of temperate and tropical popcorn populations and GWAS for zeins and starch contents.

Gotardi, L. F., Viana, J. M. S., Ribeiro, M. P., de Castro, R. B., de Oliveira Ramos, H. J. & Fietto, J. L. R. (2023). Plant Breeding, In Press.

Because measuring expansion volume (EV) is simple and inexpensive, popcorn breeders have developed high-quality single crosses ignoring the contents of zeins, starch, lipids, and cellular wall components in selection. However, some methods of quantification of these quality-related traits can be applied to popcorn breeding, increasing the selection efficacy for quality. The objectives of this study were to assess methods of zeins and starch quantification that can be used in popcorn breeding, characterize a temperate and a tropical populations for zeins and starch contents and identify candidate genes for these quality-related traits. We genotyped and phenotyped 286 plants. For quantification of total zeins and zein subunits we choose the ‘lab-on-a-chip’ microfluidic electrophoresis. For quantification of starch and amylose/amylopectin, we choose the Megazyme's Amylose/Amylopectin kit assay. The temperate population has superior EV (36.0%), a higher level of the 19 kDa zein subunit (32.0%), lower levels of the 21, 22 and 27 kDa subunits (-1543.0%, -40.0% and −47.0%, respectively) and no statistical difference for the 10 kDa zein content, relative to the tropical population. Although there are statistical differences between the two populations regarding starch, amylose, and amylose/amylopectin ratio, the differences are not significant (−2.0% to 8.0%). Thirteen candidate genes were identified for the 19 and 22 kDa zeins, two for amylose and one for starch, with emphasis on the genes coding for the 19 and 22 kDa alpha-zeins, located on chromosome 4. The evaluated quantification methods can be used in popcorn breeding but for a limited number of samples, mainly because costs.

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Unravelling the anthocyanin-binding capacity of native starches from different botanical origins.

Li, Q., Liu, Y., Li, Y., Rao, L., Zhao, L., Wang, Y. & Liao, X. (2024). Food Chemistry, 434, 137390.

In this study, the cyanidin-3-O-glucoside (C3G)-binding capacities of three native starches were investigated. While potato starch had the largest binding capacity of 0.34 mg/100 mg, corn and pea starch had binding capacities of 0.17 and 0.06 mg/100 mg. Confocal microscopy confirmed the binding results and revealed close associations between the surface properties and binding capacities. These findings were further substantiated with wettability and gelatinization results. The morphological observations showed that corn starch had advantageous particle sizes and more surface gullies, providing more opportunities to bind C3G. The zeta potential results, however, indicated that potato starch had the highest negative surface charges (−24 mV). These favorable electronic characteristics were believed to be responsible for the strongest electrostatic interactions. Hydrogen bonds, however, had a negligible effect on the formation of complexes. Overall, the negative surface charges and specific surface areas of the native starches were the most important factors determining C3G-binding capacities.

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Barley SS2a single base mutation at the splicing site led to obvious change in starch.

Bang, Wang., Jing, Liu., Chen, X. L., Qiang, XU., ZHANG, Y. Z., Dong, H. X., Tang, H. P., Qi, P. F., Deng, M., Ma, J., Wnag, J. R., Chen, G. Y., Wei, Y. M., Zheng, Y. L. & Jiang, Q. T. (2023). Journal of Integrative Agriculture, In Press.

Starch biosynthesis is a complex process that relies on the coordinated action of multiple enzymes. Resistant starch is not digested in the small intestine, thus preventing the rapid rise of the glycemic index. Starch synthase 2a (SS2a), a key enzyme in amylopectin biosynthesis, has significant effects on starch structure and properties. In this study, we identified an ss2a null mutant (M3-1413) with a single base mutation from an ethyl methane sulfonate (EMS)-mutagenized population of barley. The mutation was located at the 3´ end of the first intron of the RNA splicing receptor (AG) site, resulting in abnormal RNA splicing and two abnormal transcripts of ss2a, which caused the inactivation of the SS2a gene. The starch structure and properties were significantly altered in the mutant, with M3-1413 containing decreased total starch and increased amylose and resistant starch levels. This study sheds light the effect of barley ss2a null mutations on starch properties and helps to guide new applications of barley starch to develop nutritious food products.

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Safety Information
Symbol : GHS05, GHS08
Signal Word : Danger
Hazard Statements : H314, H315, H317, H319, H334, H361
Precautionary Statements : P201, P202, P260, P261, P264, P272, P280, P284, P301+P330+P331, P302+P352, P303+P361+P353, P304+P340, P342+P311, P501
Safety Data Sheet
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