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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
Product code: K-RAPRS

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: 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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FAQs Assay Protocol Data Calculator Product Performance Validation Report
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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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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Fundamental study of the application of brewers spent grain and fermented brewers spent grain on the quality of pasta.

Neylon, E., Arendt, E. K., Zannini, E. & Sahin, A. W. (2021). Food Structure, 100225.

Upcycling and repurposing of side streams from food processing have become a necessity to merge our world into a more sustainable future. Brewers spent grain (BSG) is a highly abundant and nutrient rich by-product of the brewing industry. The aim of this study was to investigate the effect of fermentation on BSG (FBSG) while also examining the effects of including fibre rich BSG and FBSG ingredients on techno-functional and nutritional properties of semolina-based pasta. The gluten network formation, starch gelatinisation, texture, cooking loss, optimal cooking time, in vitro starch digestibility and ultrastructure of the pasta was investigated. BSG and FBSG inclusion weakened gluten network properties versus the semolina control but was more favourable than the wholemeal control. Addition of BSG and FBSG produced pasta with a greater nutritional profile, having a higher fibre content and lower predicted glycaemic index compared to semolina pasta. BSG and FBSG addition enhanced tensile strength and pasta firmness versus wholemeal pasta. An increased reduction in the predicted glycaemic index was noted with FBSG inclusion at the higher level of addition compared to BSG, suggesting fermentation of BSG may further enhance nutritional properties of the BSG ingredient.

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Rejuvenated Brewer's Spent Grain: The impact of two BSG-derived ingredients on techno-functional and nutritional characteristics of fibre-enriched pasta.

Sahin, A. W., Hardiman, K., Atzler, J., Vogelsang-O'Dwyer, M., Valdeperez, D., Münch, S., Cattaneo, G., O’Riordan, P. & Arendt, E. K. (2021). Innovative Food Science & Emerging Technologies, 68, 102633.

Brewer's Spent Grain (BSG), rich in fibre and protein is mostly used for animal feed but has great potential to be used as an ingredient for cereal based products. Originated from BSG, the two ingredients EverVita Fibra (EVF) high in fibre; and EverVita Pro (EVP) high in protein, were used to produce fibre-enriched pasta and compared to semolina, wholemeal flour and a commercial fibre-rich pasta. Analysis of gluten network development and pasting properties revealed the formation of a stronger network by the incorporation of EVP resulting in a compact pasta structure which led to a higher pasta firmness and tensile strength and a decrease in predicted glycaemic index compared to the controls. EVF resulted in an inferior product compared to EVP but was comparable to the semolina control. Hence, EVF and EVP have the potential to increase nutritional value of pasta while maintaining or even improving pasta quality and encouraging the recycling of by-streams for food production.

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Roles of Waxy and Soluble Starch Synthase Iia Alleles in Determining Different Type Resistant Starch Contents of Rice.

You, H., Zhang, O., Liang, X., Liang, C., Chen, Y., & Xiang, X. (2020). In Press.

Background: Resistant Starch (RS) is a functional starch that has functions of regulating diabetes, hypertension and obesity. The effects of most starch synthesis-related genes (SSRGs) on RS content and their relationships are largely unknown. Result: In current study, ninety-nine lines from a recombinant inbred line were selected to investigate the effects of SSRGs on the RS content in different process status. Results revealed that RS content decreased dramatically after cooking, but it did not increase significantly after cooling for 7 days. And RS was closely related to many indexes of physicochemical properties, but was not correlated with granule size. Waxy ( Wx ) played an important role in controlling RS content and Wx a could elevate RS content in raw milled rice, cooked rice and retrograded rice. Soluble starch synthase IIa ( SSIIa ) had an impact on RS2, and RS2 content of indica SSIIa were significantly higher than that of japonica SSIIa ( SSIIaj ). Moreover, interaction of Wx and SSIIa was responsible for variations of RS content in three sample types, RS2 and volume proportion of different size starch granules. Conclusions: Wx and SSIIa together significantly regulate different types content of RS in rice, but SSIIa only affects RS2. Wx a - SSIIaj is favorable to forming large-diameter starch granules.

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Leuconostoc citreum TR116 as a Microbial Cell Factory to Functionalise High-Protein Faba Bean Ingredients for Bakery Applications.

Hoehnel, A., Bez, J., Sahin, A. W., Coffey, A., Arendt, E. K. & Zannini, E. (2020). Foods, 9(11), 1706.

Grain legumes, such as faba beans, have been investigated as promising ingredients to enhance the nutritional value of wheat bread. However, a detrimental effect on technological bread quality was often reported. Furthermore, considerable amounts of antinutritional compounds present in faba beans are a subject of concern. Sourdough-like fermentation can positively affect baking performance and nutritional attributes of faba bean flours. The multifunctional lactic acid bacteria strain Leuconostoc citreum TR116 was employed to ferment two faba bean flours with different protein contents (dehulled flour (DF); high-protein flour (PR)). The strain’s fermentation profile (growth, acidification, carbohydrate metabolism and antifungal phenolic acids) was monitored in both substrates. The fermentates were applied in regular wheat bread by replacing 15% of wheat flour. Water absorption, gluten aggregation behaviour, bread quality characteristics and in vitro starch digestibility were compared to formulations containing unfermented DF and PR and to a control wheat bread. Similar microbial growth, carbohydrate consumption as well as production of lactic and acetic acid were observed in both faba bean ingredients. A less pronounced pH drop as well as a slightly higher amount of antifungal phenolic acids were measured in the PR fermentate. Fermentation caused a striking improvement of the ingredients’ baking performance. GlutoPeak measurements allowed for an association of this observation with an improved gluten aggregation. Given its higher potential to improve protein quality in cereal products, the PR fermentate seemed generally more promising as functional ingredient due to its positive impact on bread quality and only moderately increased starch digestibility in bread.

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Physico-chemical properties of flour, dough and bread from wheat and hydrothermally-treated finger millet.

Onyango, C., Luvitaa, S. K., Unbehend, G. & Haase, N. (2020). Journal of Cereal Science, 102954.

Hydrothermally-treated (HTT) finger millet was prepared by tempering the grains twice with water (10:1) followed by incubation at about 25-30°C in a woven polypropylene sack for 10 days. Hydrothermally-treated finger millet was darker and had higher α-amylase activity and lower starch digestibility than native (NAT) grains. The HTT finger millet was composited with wheat flour and used to prepare bread. Composite dough had higher dough stability, dough development time and degree of softening but lower dough energy, extensibility and resistance to extension than WHE dough. The higher specific volume and lower crumb firmness and chewiness of WHE-HTT compared to WHE-NAT bread was attributed to the high α-amylase activity and water absorption capacity of HTT finger millet. Wheat-HTT bread had higher dietary fibre, phytate and phenolic acid content but the same starch and protein digestibility as WHE bread.

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Design and synthesis of modified and resistant starch-based oil-in-water emulsions.

Jain, S., Winuprasith, T. & Suphantharika, M. (2019). Food Hydrocolloids, 89, 153-162.

In this study, rice starch was modified by different modifications that included debranching (DB), esterification with octenyl succinic anhydride (OSA), debranching followed by OSA modification (DBOS) and OSA modification followed by debranching (OSDB). Following modification, the resistant starch content and emulsifying properties of the modified starches markedly increased in comparison with the native starch with the DBOS starch having the highest resistant starch content and the best emulsifying properties. This can be attributed to its higher degree of OSA substitution resulting in an enhanced amphiphilic character and better emulsion stability. Microstructure, physical stability and rheological properties of oil-in-water emulsions using these modified rice starches were also investigated and it was observed that emulsions stabilized by OSA and DBOS starches exhibited smaller oil droplets, were more stable to creaming and possessed stronger three-dimensional structure when compared with the other modified starch-based emulsions. Since the DBOS starch was found to demonstrate the highest resistant starch content and better emulsifying properties than the other starches, it was then chosen for optimization of its concentration to give the best emulsion characteristics and stability. The emulsions stabilized by the DBOS starch at a concentration of 4% or 5% (w/w) were found to be more stable and very little phase separation was observed after a storage period of 30 days.

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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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