Resistant Starch Assay Kit (Rapid)

Play Training Video

00:08   Introduction
01:09    Principle
02:35    Reagent Preparation
06:03    Weighing of samples
06:34    Hydrolysis & Solubilization of Digestible Starch
07:53    Waterbath Alignment
08:22    Ethanol washing & centrifugation
10:26    Procedure
14:26     Measurement of Digestible, Non-Resistant Starch
16:57     Calculations

Resistant Starch Assay Kit Rapid K-RAPRS Scheme
   
Reference code: K-RAPRS
SKU: 700004333

100 assays per kit

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: Resistant Starch
Assay Format: Spectrophotometer
Detection Method: Absorbance
Wavelength (nm): 510
Signal Response: Increase
Linear Range: 4 to 100 mg of D-glucose per assay
Limit of Detection: 0.036 g/100 g
Reaction Time (min): ~ 5 h
Application examples: Plant materials, starch samples and other materials.
Method recognition: Novel method

The Resistant Starch Assay Kit (Rapid) method is suitable for the analysis of resistant starch in pure starch, cereal and legume seeds and food samples.

This method is an update of the method of McCleary et al1 (AOAC Method 2002.02, AACC Method 32-40.01) employing incubation conditions similar to those used in AOAC Method 2017.16 for dietary fiber. The enzyme mixture employed [pancreatic α-amylase (PAA) and amyloglucosidase (AMG)] are those used by Englyst et al.2 except that both enzymes have been purified, standardised and stabilised. Digestion is performed using saturating levels of PAA and AMG with stirring at pH 6 and 37oC for 4 h, to simulate in vivo conditions in the human small intestine. Recent studies on the hydrolysis of “newer” resistant starch materials such as phosphate crosslinked starch (RS4) indicated that these incubation conditions are an essential requirement to obtain meaningful physiologically relevant values for RS.

The incubation conditions parallel those used in AOAC Method 2017.16, a new, rapid integrated procedure for the measurement of total dietary fiber (Megazyme method K-RINTDF). This method is physiologically based and designed to service the definition of DF announced by Codex Alimentarius in 2009.

1. McCleary, B. V., McNally, M. & Rossiter, P. (2002).  Measurement of Resistant Starch by Enzymic Digestion in Starch and Selected Plant Materials - Collaborative Study.  J. AOAC Int., 85, 1103-1111.

2. Englyst, H. N., Kingman, S. M. & Cummings, J. H. (1992).  Classification and measurement of nutritionally important starch fractions.  Eur. J. Clin. Nutr., 46 (Suppl. 2), S33-S50.

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Validation of Methods
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Safety Data Sheet
FAQs Assay Protocol Data Calculator Product Performance Validation Report
Publications
Megazyme publication

Measurement of available carbohydrates, digestible, and resistant starch in food ingredients and products.

McCleary, B. V., McLoughlin, C., Charmier, L. M. J. & McGeough, P. (2019). Cereal Chemistry, 97(1), 114-137.

Background and objectives: The importance of selectively measuring available and unavailable carbohydrates in the human diet has been recognized for over 100 years. The levels of available carbohydrates in diets can be directly linked to major diseases of the Western world, namely Type II diabetes and obesity. Methodology for measurement of total carbohydrates by difference was introduced in the 1880s, and this forms the basis of carbohydrate determination in the United States. In the United Kingdom, a method to directly measure available carbohydrates was introduced in the 1920s to assist diabetic patients with food selection. The aim of the current work was to develop simple, specific, and reliable methods for available carbohydrates and digestible starch (and resistant starch). The major component of available carbohydrates in most foods is digestible starch. Findings: Simple methods for the measurement of rapidly digested starch, slowly digested starch, total digestible starch, resistant starch, and available carbohydrates have been developed, and the digestibility of phosphate cross‐linked starch has been studied in detail. The resistant starch procedure developed is an update of current procedures and incorporates incubation conditions with pancreatic α‐amylase (PAA) and amyloglucosidase (AMG) that parallel those used AOAC Method 2017.16 for total dietary fiber. Available carbohydrates are measured as glucose, fructose, and galactose, following complete and selective hydrolysis of digestible starch, maltodextrins, maltose, sucrose, and lactose to glucose, fructose, and galactose. Sucrose is hydrolyzed with a specific sucrase enzyme that has no action on fructo‐oligosaccharides (FOS). Conclusions: The currently described “available carbohydrates” method together with the total dietary fiber method (AOAC Method 2017.16) allows the measurement of all carbohydrates in food products, including digestible starch. Significance and novelty: This paper describes a simple and specific method for measurement of available carbohydrates in cereal, food, and feed products. This is the first method that provides the correct measurement of digestible starch and sucrose in the presence of FOS. Such methodology is essential for accurate labeling of food products, allowing consumers to make informed decisions in food selection.

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Unveiling the nutritional spectrum: A comprehensive analysis of protein quality and antinutritional factors in three varieties of quinoa (Chenopodium quinoa Wild).

Manzanilla-Valdez, M. L., Boesch, C., Orfila, C., Montaño, S. & Hernández-Álvarez, A. J. (2024). Food Chemistry: X, 24, 101814.

Quinoa (Chenopodium quinoa) is renowned for its high protein content and balanced amino acid profile. Despite promising protein characteristics, plant-based sources usually possess antinutritional factors (ANFs). This study aimed to analyze the nutritional and ANFs composition of three quinoa varieties (Black, Yellow, and Red), and assessed the protein quality. Among these varieties, Black quinoa showed the highest protein content (20.90 g/100 g) and total dietary fiber (TDF) (22.97 g/100 g). In contrast, Red quinoa exhibited the highest concentration of phenolic compounds (338.9 mg/100 g). The predominant ANFs identified included oxalates (ranging from 396.9 to 715.2 mg/100 g), saponins (83.27–96.82 g/100 g), and trypsin inhibitors (0.35–0.46 TUI/100 g). All three varieties showed similar in vitro protein digestibility (IVPD) (> 76.9 %), while Black quinoa exhibited the highest protein quality. In conclusion to ensure reduction of ANFs, processing methods are necessary in order to fully benefit from the high protein and nutritional value of quinoa.

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Cell wall polysaccharides determine cooking quality in cassava roots.

Sathitnaitham, S., Ceballos, H., Wonnapinij, P., Kraichak, E., Utthiya, S., Suttangkakul, A., Gomez, L. D., Kittipadakul, P., Siriwong, N., Kongsil, P. & Vuttipongchaikij, S. (2024). Plants, People, Planet, 6(6), 1534-1551.

Cassava is a vital food source for millions worldwide, crucial for food security and economic stability. This study analyzed cell wall polysaccharides in cassava roots to understand their impact on cooking properties. We found these polysaccharides influence the textural attributes of cassava roots, essential for both cooking and consumption. The research highlights the need to further identify and analyze cassava cell wall components. By improving our understanding of these components, we can improve food security, affordability, and resilience in diverse regions, ultimately contributing to global food security and better aligning with consumer preferences.

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Solid-State Fermentation of Quinoa Flour: An In-Depth Analysis of Ingredient Characteristics.

Gautheron, O., Nyhan, L., Ressa, A., Torreiro, M. G., Tlais, A. Z. A., Cappello, C., Gobbetti, M., Hammer, A. K., Zannini, E., Arendt, E. K. & Sahin, A. W. (2024). Fermentation, 10(7), 360.

Plant protein ingredients are gaining attention for human nutrition, yet they differ significantly from animal proteins in functionality and nutrition. Fungal solid-state fermentation (SSF) can modulate the composition and functionality, increasing their applicability in foods. Quinoa flour (QF) served as a substrate for Aspergillus oryzae and Rhizopus oligosporus, resulting in two fermented ingredients (QFA and QFR) with different nutritional, functional, and aroma characteristics. A higher increase in protein (+35%) and nitrogen (+24%) was observed in the QFA, while fat was predominantly increased in the QFR (+78%). Fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) decreased in the QFR but increased in the QFA due to polyol production. Metabolomic analysis revealed higher lactic acid concentrations in the QFA, and higher citric, malic, and fumaric acid contents in the QFR. The SSF reduced most antinutrients, while R. oligosporus produced saponins. Olfactometry showed the development of fruity ester compounds and a decrease in metallic and cardboard aromas. Both ingredients showed an enhanced water-holding capacity, with the QFA also demonstrating an increased oil-holding capacity. Complex formation increased the particle size, reduced the solubility, and decreased the foaming properties. Mycelium production darkened the ingredients, with the QFR having a higher differential colour index. This study highlights the potential of SSF to produce ingredients with improved nutritional, sensory, and functional properties.

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Investigating the impact of boiling and pressure cooking on resistant starch levels in food.

Karunarathna, S., Wickramasinghe, I., Brennan, C., Truong, T., Navaratne, S. & Chandrapala, J. (2024). International Journal of Food Science & Technology, 59(6), 3907-3917.

Resistant starch (RS) has been shown to manipulate food digestion and nutrient metabolism as well as being important in promoting gut health. However, the presence of RS varies across diverse food systems depending upon process conditions. To investigate this, chickpeas, potatoes, arrowroots, unripe bananas and oats were subjected to boiling and pressure cooking at various cooking durations and the RS% was determined. After 45 min of boiling and 15 min of pressure cooking, RS% of chickpeas increased by ~29% and ~41%, respectively. Increasing the cooking time, together with boiling and pressure cooking, led to higher RS% in chickpeas, mainly due to the retrogradation of starch after gelatinisation. In contrast, boiling and pressure cooking led to a reduction in RS% for potatoes, arrowroots, unripe bananas and oats, while a further decrease was encountered with the increase in processing time. After 45 min of boiling, RS% of potatoes, arrowroots, unripe bananas, and oats were reduced by 58%, 22%, 80% and 70%, respectively. After 15 min of pressure cooking, RS% of potatoes, arrowroots and unripe bananas were reduced by 41%, 22% and 83%, respectively. This was mainly due to the disruption of the ordered structure of starch molecules during thermal processing in the presence of water. This study clearly elucidates the impact of boiling and pressure cooking on RS% as a function of processing time and type of RS source.

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Effect of baking conditions on resistant starch: Model systems and cake formulations.

Gutiérrez-Luna, K., Ansorena, D. & Astiasaran, I. (2024). Food Chemistry, 449, 139174.

Five ingredients rich in RS (resistant starch) were assessed in a model system simulating baking process (water addition 30-50%, 180°C/35 min) and in reformulated cakes (50% replacement of wheat flour). Moreover, two enzymatic methods used for RS determination (official and rapid) were compared. The combined effect of heating and water addition (50%) significantly decreased the RS content in all ingredients. Reformulated vegan cakes presented significantly lower RS values than those theoretically expected, according to the RS value of raw ingredients. The highest RS amount was observed for Hi Maize, which kept 84% and 72% of the initial RS content in the model systems and cake, respectively. Only the cakes made with Hi Maize reached the criteria for the health claim related to the reduction of post-prandial glycemic response (European Union regulation). Finally, differences between the official and rapid methods were less significant in the cakes than in the model systems.

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Dynamic in-vitro system indicates good digestibility characteristics for novel upcycled plant protein; correlation to techno-functional properties.

Jaeger, A., Ahern, N., Sahin, A. W., Nyhan, L., Mes, J. J., van der Aa, C., Vrasidas, T. & Arendt, E. K. (2024). Innovative Food Science & Emerging Technologies, 92, 103571.

An increasing global population demands the broadening of the plant-based protein portfolio. The high volume of by-products produced across the food industry presents the opportunity to reduce food waste while reclaiming valuable nutrition. The current study examines a novel protein, BRP, extracted from brewers spent grains regarding its techno-functionality and digestibility, in comparison to a variety of proteins including whey, soy, rice and two pea protein sources. Compositional, foaming, emulsifying, structural and rheological properties were examined, while digestion kinetics were determined using the dynamic tiny-TIM in vitro digestion model. BRP performed similarly to whey protein in many respects, demonstrating a high solubility, high nitrogen bioavailability (>90%) and comparable N digestion kinetics, however BRP exhibited no rheological changes over heating. The digestible Indispensable amino acid score for BRP was 67% with lysine as the sole limiting amino acid, a higher value than soy, rice and one of the pea proteins investigated.

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Effect of fibre fortification of low FODMAP pasta.

Atzler, J. J., Crofton, E. C., Sahin, A. W., Ispiryan, L., Gallagher, E., Zannini, E. & Arendt, E. K. (2024). International Journal of Food Sciences and Nutrition, 75(3), 293-305.

Irritable bowel syndrome (IBS) is a condition affecting the digestive system and can be triggered by several different factors, including diet. To ease symptoms of IBS, a diet low in fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) is often recommended. Pasta, as a staple food in the Western World, is naturally high in FODMAPs. This study investigates the impact of insoluble and soluble dietary fibre ingredients in low-FODMAPs pasta. The assessment included physicochemical, sensory, and nutritional quality. Soluble fibre strengthened gluten network, which caused a lower cooking loss and a lower release of sugars during in vitro starch digestion. Insoluble fibre interfered with the gluten network development to a higher extent causing a higher sugar release during digestion. This study reveals the most suitable fibre ingredients for the development of pasta with elevated nutritional value and sensory characteristics compared to commercial products on the market. This type of pasta has a high potential of being suitable for IBS patients.

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Impact of different fibre ingredients on a low-FODMAP biscuit model system.

Sahin, A. W., Atzler, J. J., Crofton, E., Gallagher, E., Zannini, E., Walter, J. & Arendt, E. K. (2023). Food & Function, 14(15), 7082-7095.

Fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) are carbohydrates which can cause symptoms of irritable bowel syndrome (IBS). Cereal-based products are high in FODMAPs, as they are part of the carbohydrate fraction in flour. Low-FODMAP products are starch-based which leads to a low dietary fibre content. Hence, the fortification with dietary fibre ingredients low in FODMAPs is essential. This study reveals the impact of three different fibre ingredients, resistant starch, cellulose, and arabinoxylan, and their interactions with each other in a low-FODMAP biscuit model system using response surface methodology. All fibre ingredients have an affinity to water which was further increased by their coexistence in the model system. Fibersym RW affected the biscuit hardness by its morphology and potential to recrystallise leading to a maximum inclusion level of 40%. VITACEL L 600-30 also increased biscuit hardness due to its plasticising character leading to a maximum inclusion of 20%. AgriFiber BFG mainly impacted the colour of the product restricting its inclusion to 2.3%. Additionally, it reduced the degree of starch digestibility of the biscuit by the formation of a film imbedding the starch granules and reducing enzyme attack. This research provides an in-depth insight into the integration potential of these fibre ingredients into a low-FODMAP biscuit, their interactions within the system and inclusion levels which allow their coexistence.

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Immunomodulatory and antioxidant properties of Ipomoea batatas flour and extracts obtained by green extraction.

Boukhers, I., Morel, S., Kongolo, J., Domingo, R., Servent, A., Ollier, L., Kodja, H., Petit, T. & Poucheret, P. (2023). Current Issues in Molecular Biology, 45(9), 6967-6985.

 

Sweet potato (SP), Ipomoea batatas Lam, belongs to the Convolvulaceae family. It produces edible storage roots. Currently, orange varieties contribute to improving food systems and managing vitamin A deficiency. Processing of this food crop into flour allows better conservation. However, nutrition health data regarding SP flour obtained by green extraction remains scarce. In this study, we therefore explored its phytochemistry and its associated bioactivity potential for human health. We analyzed the nutritional composition of orange flesh sweet potato (OFSP) flour and assessed the antioxidant (free radical scavenging) and immunomodulatory (on inflammatory murine macrophages) properties of the extract. More specifically, we measured the impact of OFSP flour extract on mediators such as Nitric Oxide (NO) and the production of pro-inflammatory cytokines such as Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-alpha), Monocyte Chemoattractant Protein-1 (MCP-1), and Prostaglandin-E2 (PGE-2). Our results indicated significant fiber, mineral, beta-carotene, and polyphenols content in the extracts, and antioxidant and immunomodulatory bioactivities were also demonstrated with a concentration-dependent inhibition of cytokine production. Taken together, our results suggest that Ipomoea batatas flour could, in addition to being a good source of energy and beta-carotene provitamin A, constitute a food of interest for the prophylaxis of metabolic diseases associated with an underlying low-grade inflammatory state.

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Associating Compositional, Nutritional and Techno-Functional Characteristics of Faba Bean (Vicia faba L.) Protein Isolates and Their Production Side-Streams with Potential Food Applications.

Krause, M., Sørensen, J. C., Petersen, I. L., Duque-Estrada, P., Cappello, C., Tlais, A. Z. A., Di Cagno, R., Ispiryan, L., Sahin, A. W., Arendt, E. K. & Zannini, E. (2023). Foods, 12(5), 919.

Faba beans (Vicia faba L.) show exciting prospects as a sustainable source of protein and fibre, with the potential to transition to a more sustainable food production. This study reveals the compositional, nutritional and techno-functional characteristics of two protein isolates from faba beans (Vicia faba L.), a high-starch fraction and a high-fibre side-stream. During the analysis of those four ingredients, particular attention was paid to the isolates’ protein profile and the side-streams’ carbohydrate composition. The isoelectric precipitated protein isolate 1 showed a protein content of 72.64 ± 0.31% DM. It exhibited low solubility but superior digestibility and high foam stability. High foaming capacity and low protein digestibility were observed for protein isolate 2, with a protein content of 71.37 ± 0.93% DM. This fraction was highly soluble and consisted primarily of low molecular weight proteins. The high-starch fraction contained 83.87 ± 3.07% DM starch, of which about 66% was resistant starch. Over 65% of the high-fibre fraction was insoluble dietary fibre. The findings of this study provide a detailed understanding of different production fractions of faba beans, which is of great value for future product development.

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Functional Properties of Brewer’s Spent Grain Protein Isolate: The Missing Piece in the Plant Protein Portfolio.

Jaeger, A., Sahin, A. W., Nyhan, L., Zannini, E. & Arendt, E. K. (2023). Foods, 12(4), 798.

Plant protein sources, as a part of developing sustainable food systems, are currently of interest globally. Brewer’s spent grain (BSG) is the most plentiful by-product of the brewing industry, representing ~85% of the total side streams produced. Although nutritionally dense, there are very few methods of upcycling these materials. High in protein, BSG can serve as an ideal raw material for protein isolate production. This study details the nutritional and functional characteristics of BSG protein isolate, EverPro, and compares these with the technological performance of the current gold standard plant protein isolates, pea and soy. The compositional characteristics are determined, including amino acid analysis, protein solubility, and protein profile among others. Related physical properties are determined, including foaming characteristics, emulsifying properties, zeta potential, surface hydrophobicity, and rheological properties. Regarding nutrition, EverPro meets or exceeds the requirement of each essential amino acid per g protein, with the exception of lysine, while pea and soy are deficient in methionine and cysteine. EverPro has a similar protein content to the pea and soy isolates, but far exceeds them in terms of protein solubility, with a protein solubility of ~100% compared to 22% and 52% for pea and soy isolates, respectively. This increased solubility, in turn, affects other functional properties; EverPro displays the highest foaming capacity and exhibits low sedimentation activity, while also possessing minimal gelation properties and low emulsion stabilising activity when compared to pea and soy isolates. This study outlines the functional and nutritional properties of EverPro, a brewer’s spent grain protein, in comparison to commercial plant protein isolates, indicating the potential for the inclusion of new, sustainable plant-based protein sources in human nutrition, in particular dairy alternative applications.

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Effect of mild heat-moisture treatment for harvested raw paddy rice on physicochemical properties and in vitro starch digestibility of cooked rice.

Thuengtung, S., Ketnawa, S., Ding, Y., Cai, Y. & Ogawa, Y. (2023). Food Hydrocolloids for Health, 3, 100133.

In this study, heat-moisture treatment (HMT) at 60°C and 65°C for 12 h and 18 h was applied to harvested raw paddy rice (Oryza sativa L. cv. Toyomeki), and changes in the quality characteristics and starch hydrolysis during simulated gastrointestinal digestion were investigated. The surface color of the treated rice grains was mainly caused by the period of HMT rather than the heating temperature. HMT at mild temperatures caused a minor change with no significant effect in the total starch content of the treated rice, as well as the moisture content and firmness of the cooked rice (P > 0.05). However, the resistant starch content increased with increasing HMT temperature and time, and a significant difference was found between the control and the treated rice subjected at 65°C. The mild HMT also promoted the reduction trend of starch hydrolysis for the intact cooked rice grain, which could have health benefits. Thus, the mild HMT of raw paddy rice could be regarded as a profitable technique modifying the digestibility of cooked rice but maintaining rice sensory characteristics.

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Acute postprandial gut hormone, leptin, glucose and insulin responses to resistant starch in obese children: a single blind crossover study.

Suntharesan, J., Atapattu, N., Jasinghe, E., Ekanayake, S., de Silva, D. A. G. H., Dunseath, G., Luzio, S. & Premawardhana, L. (2022). Archives of Disease in Childhood, In Press.

Introduction Resistant starch (RS) has beneficial effects on postprandial glucose metabolism in both animals and adults. Hitherto, there have been no studies in children of the acute metabolic and hormonal effects of RS-containing meals. Objectives We aimed to compare serial plasma glucose, insulin, gut hormone, leptin profiles and satiety scores in obese children after meals containing variable amounts of RS. Methods This was a single blind, non-randomised, crossover study of 20 obese children aged 10–14 years old without comorbidities. Three test meals containing rice (M1), rice cooked with coconut oil (M2), rice cooked in coconut oil with lentils (M3) were given in sequence after a 12-hour fast . Blood samples were analysed for glucose (PG), insulin, leptin, glucagon-like polypeptide (GLP) 1, ghrelin and peptide YY (PYY) at appropriate times between 0 and 180 min. Results Meal M2 resulted in significantly lower postprandial glucose values compared with meal M1 (maximal incremental glucose, ∆Cmax, p<0.05; area under the curve, ∆AUC0–3, p<0.01) and meal M3 (maximal concentration, Cmax, p<0.01; ∆Cmax, p<0.001, and ∆AUC0–3p<0.01). M2 also produced lower insulin values compared with M1 (p<0.05). Postprandial ghrelin was significantly higher after M1 compared with M3 (p<0.05). PYY, GLP1 and median satiety scores were not significantly different between the three meals. Conclusion This study shows that M2, the meal containing RS alone, induced beneficial effects on acute postprandial glucose, insulin and ghrelin concentrations in obese children without diabetes. Acute postprandial satiety scores were not significantly affected by the three meals.

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Utilisation of Amaranth and Finger Millet as Ingredients in Wheat Dough and Bread for Increased Agro-Food Biodiversity.

Onyango, C., Luvitaa, S. K., Lagat, K., Hüsken, A., Smit, I. & Schmidt, M. (2022). Foods, 11(7), 911.

Amaranth and finger millet are important food security crops in Africa but show poor bread making ability, even in composite wheat breads. Malting and steaming are promising approaches to improve composite bread quality, which have not been fully explored yet. Therefore, in this study, wheat was blended with native, steamed or malted finger millet or amaranth in the ratio of 70:30. Wheat/native amaranth (WHE-NAM) and wheat/malted amaranth (WHE-MAM) had longer dough development times and higher dough stabilities, water absorption capacities and farinograph quality numbers than wheat/steamed amaranth (WHE-SAM), wheat/native finger millet (WHE-NFM), wheat/steamed finger millet (WHE-SFM) or wheat/malted finger millet (WHE-MFM). The WHE-NAM and WHE-MAM breads had lower crumb firmness and chewiness, higher resilience and cohesiveness and lighter colours than WHE-NFM, WHE-SFM and WHE-MFM. Starch and protein digestibility of composite breads were not different (p > 0.05) from each other and ranged between 95-98% and 83-91%, respectively. Composite breads had higher ash (1.9-2.5 g/100 g), dietary fibre (5.7-7.1 g/100 g), phenolic acid (60-122 mg/100 g) and phytate contents (551-669 mg/100 g) than wheat bread (ash 1.6 g/100 g; dietary fibre 4.5 g/100 g; phenolic acids 59 mg/100 g; phytate 170 mg/100 g). The WHE-NAM and WHE-MAM breads possessed the best crumb texture and nutritional profile among the composite breads.

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