Resistant Starch Assay Kit

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.

A method is described for the measurement of dietary fibre, including resistant starch (RS), non-digestible oligosaccharides (NDO) and available carbohydrates. Basically, the sample is incubated with pancreatic α-amylase and amyloglucosidase under conditions very similar to those described in AOAC Official Method 2002.02 (RS). Reaction is terminated and high molecular weight resistant polysaccharides are precipitated from solution with alcohol and recovered by filtration. Recovery of RS (for most RS sources) is in line with published data from ileostomy studies. The aqueous ethanol extract is concentrated, desalted and analysed for NDO by high-performance liquid chromatography by a method similar to that described by Okuma (AOAC Method 2001.03), except that for logistical reasons, D-sorbitol is used as the internal standard in place of glycerol. Available carbohydrates, defined as D-glucose, D-fructose, sucrose, the D-glucose component of lactose, maltodextrins and non-resistant starch, are measured as D-glucose plus D-fructose in the sample after hydrolysis of oligosaccharides with a mixture of sucrase/maltase plus β-galactosidase.

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.

A robust and reliable method was developed to measure resistant starch (RS), i.e., starch that enters the large intestine. In vivo conditions were reflected as much as possible while a user-friendly format was maintained. Parameters investigated included α-amylase concentration, pH of incubation, maltose inhibition of α-amylase, the need for amyloglucosidase inclusion, the effect of shaking and stirring on determined values, and problems in recovering and analyzing the RS-containing pellet. The RS values obtained were in good agreement with published in vivo data. An interlaboratory evaluation of the method has been completed (First Action Method 2002.02).

Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSD_r) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSD_R) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSD_r and RSD_R values are unacceptably high.

Enzyme activity and purity of these topics, the easiest to deal with is the importance of enzyme purity and activity. As a scientist actively involved in polysaccharide research over the past 25 years, I have come to appreciate the importance of enzyme purity and specificity in polysaccharide modification and measurement (7). These factors translate directly to dietary fiber (DF) methodology, because the major components of DF are carbohydrate polymers and oligomers. The committee report published in the March issue of Cereal FOODS WORLD refers only to the methodology for measuring enzyme purity and activity (8) that led up the AOAC method 985.29 (2). In this work enzyme purity was gauged by the lack of hydrolysis (i.e., complete recovery) of a particular DF component (e.g. β-glucan, larch galactan or citrus pectin). Enzyme activity was measured by the ability to completely hydrolyze representative starch and protein (namely wheat starch and casein). These requirements and restrictions on enzyme purity and activity were adequate at the time the method was initially developed and served as a useful working guide. However, it was recognized that there was a need for more stringent quality definitions and assay procedures for enzymes used in DF measurements.

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%.

Growing demand for environmentally sustainable protein sources is shifting dietary preferences toward plant-derived alternatives such as legumes. Pea (Pisum sativum L.) seeds offer great potential for expanded human consumption, but sensory quality is key for consumer acceptance and cultivar development. In this study, a diversity panel of 15 pea accessions was evaluated for nutrients and phytochemicals (protein, resistant and non-resistant starch, fatty acids, choline, phytate, saponins, and sucrose) and their sensory attributes (taste, aroma, mouthfeel, and aftertaste). Among the sensory attributes, mouthfeel and aroma contributed most to the variation. Principal component analysis revealed two large, distinct clusters, primarily separated by seed coat (testa) colour. Accessions with a dark-coloured testa were generally perceived more odour intense and with more texture, while accessions with light-coloured testa were sweeter and juicier. Accessions with wrinkled seeds stood out in their content of non-resistant starch, sucrose, total choline, and phytate, when compared to smooth and dimpled seeds. Shorter cooking times were positively correlated to the perception of higher bitterness. This study highlights the potential in combining seed compositional analysis and sensory evaluations for screening pea accessions suitable for the development of future food products.

This study aimed to determine the effects of different irrigation levels (50%, 75%, and 100% of ETo values calculated using evaporation from Class-A pan) and nitrogen doses (0, 90, 180, and 270 kg ha ⁻ ¹) on yield, yield components, and the nutritional properties of sorghum grains. According to the research results, increasing irrigation and nitrogen fertilization levels enhanced plant height, thousand-grain weight, grain number per panicle, grain weight per panicle, and grain yield. The highest grain yield (7120 kg ha ⁻ ¹) was obtained with 100% irrigation and 180 kg ha ⁻ ¹ N application. While increasing irrigation levels increased oil content, higher nitrogen doses caused a decrease for it. The highest oil content (6.64%) was recorded with 100% irrigation and 0 kg ha ⁻ ¹ N application. Protein content increased with irrigation and nitrogen applications, reaching the highest level (11.85%) with 100% irrigation and 270 kg ha ⁻ ¹ N application. Higher irrigation levels also increased total starch and phytic acid content. Among nitrogen applications, the dose of 270 kg ha ⁻ ¹ resulted in the maximum total starch (77.29%) and phytic acid content (1.83%). The ratio of resistant starch (RS) was found to be high at 50% irrigation with low nitrogen doses, indicating an inverse relationship with the total starch content. Both irrigation and nitrogen applications significantly affected the ratios of oleic and linoleic acids. Specifically, increased irrigation raised the linoleic acid content, while nitrogen applications enhanced the oleic acid content. Additionally, as irrigation levels increased, the contents of potassium (K), magnesium (Mg), iron (Fe), phosphorus (P), and zinc (Zn) also increased. Conversely, the levels of calcium (Ca) and manganese (Mn) decreased. Generally, higher nitrogen doses resulted in increased mineral content, with the highest levels of magnesium, iron, and zinc observed at nitrogen doses between 180 and 270 kg ha ⁻ ¹. According to the research results, the most suitable irrigation level for optimizing high yield and grain nutritional properties was determined to be 100%, with a nitrogen dose of 180–270 kg ha ⁻ ¹. These findings will contribute to future studies on different sorghum varieties under varying climate and soil conditions.

Euryale ferox (EF), a highly nutritious food, is an excellent source of resistant starch (RS). This study compared the structure, physicochemical properties, and probiotic activities of RS from North (NEFRS) and South EF (SEFRS). NEFRS exhibited a higher RS content (~10%) than SEFRS (~4%) and demonstrated superior crystallinity (21.66%), thermal stability (ΔH = 21.85 J/g), and molecular order, whereas SEFRS contained more double helices (ΔH = 4.17 J/g). Both displayed type A crystalline structures, with RS5 amylose-lipid complexes being more abundant in NEFRS during growth. Gas chromatography-mass spectrometry identified bound fatty acids, including palmitic, linoleic, trans-oleic, and stearic acids, confirmed through in vitro synthesis. Probiotic assays revealed EFRS enhanced the growth of Bifidobacterium and Lactobacillus acidophilus, while NEFRS exhibited stronger inhibition against Escherichia coli and Staphylococcus aureus. Overall, this study systematically elucidated the EFRS differences between two species, providing valuable insights into functional product development and EF deep processing.

Limited information is available on the starch digestion properties in high-amylose japonica rice. This study compared starch digestion properties and physiochemical characteristics-including texture profiles, starch and protein composition, and pasting properties—between the high-amylose japonica rice variety Youtangdao 3 (Y3) and the high-amylose indica variety Guangluai 4 (G4). Y3 exhibited a 36% shorter active digestion duration, 24% faster glucose production rate, and 21% lower glucose production than G4. The faster starch digestion rate in Y3 was attributed to its lower cohesiveness, which was associated with its higher resistant starch content-driven by a higher amylose-to-amylopectin ratio and higher protein content, particularly albumin and glutelin. The lower glucose production in Y3 resulted from its lower total starch content-linked to higher protein content-and higher resistant starch content. This study clarifies varietal differences in starch digestion and related physiochemical traits between high-amylose japonica and indica rice. Additionally, this study highlights that a higher resistant starch content does not always correspond to slower starch digestion in rice.

Oat bran addition to rice bread results in quality deterioration especially during storage, but its superfine particle size probably relieves this side effect. This study focuses on the gluten-free and rice starch-based bread. The effects of different particle sizes at medium (MOB, 359.67 μm), fine (FOB, 153.67 μm) and superfine (SOB, 35.77 μm) on the short-term storage quality of rice bread was confirmed. Oat bran addition affected pasting properties of rice flour, leading to a significant decrease in values of final viscosity and setback, which was intensified much more by the smaller particle sizes. The decrease in particle size of oat bran also led to an increase in brightness and whiteness values of rice bread. Gas-holding capacity of rice bread was reduced by MOB and FOB, including specific volume, gas cell diameter and volume, but SOB reversed this reduction and recovered to the same level as control bread. SOB mitigated the increase in bread firmness during storage and led to the highest springiness while the lowest baking loss. The water-binding capacity of rice bread with SOB was much higher than that with MOB and FOB, according to the moisture migration analysis. The inhibition of water evaporation by SOB during storage further delayed the amylopectin retrogradation and starch recrystallization. Additionally, SOB prevented the rice starch hydrolysis, exhibited an increase in RS (35.39 %), which was much higher than those of MOB (23.30%) and FOB (25.30%). These findings supported the efficient inhibition of SOB on rice bread staling.

Prebiotics are nondigestible polysaccharides that feed gut microbiota, maintaining host-gut microbiota symbiosis and mitigating chronic illnesses. This study extracted and purified resistant starch (RS) from finger millet starch (FMS) by physical treatments, ultrasonication (P-US-FMS), and annealing (P-A-FMS) to characterize their prebiotic potential. When RS samples were subjected to in vitro digestion, treated samples, especially P-US-FMS, showed higher resistance to enzymatic hydrolysis. RS samples supported the growth of the probiotic strain Lactobacillus rhamnosus MTCC 1423 in the range of 7.59-8.41 log₁₀ CFU/mL. Applying ultrasonication and annealing has improved RS content to 7.2% and 6.5%, respectively. Physical treatments have modified the crystallinity from A-type to B-type. FT-IR data revealed the presence of high-intensity peaks at 990 cm⁻¹ (C-O-C linkage) and 2925-2930 cm⁻¹ (C-H linkage). Thermal properties of resistant starch were enhanced due to the remodeling of starch granules during retrogradation. The most desirable qualities, such as water solubility index, swelling power, and water absorption capacity, were high in the ultrasonicated sample. These findings underscore the potentiality of finger millet-resistant starch as a prebiotic, offering insights for developing functional foods to prevent gastrointestinal diseases.

High-resistant starch rice is a valuable food for human health, especially for individuals with type 2 diabetes, as it supports effective blood sugar control and provides cardiovascular and intestinal benefits. However, developing rice varieties with high resistant starch content remains a major challenge. In this study, we identified a mutant, chalk2, with increased chalkiness from the mutant library of indica rice ZJ100. The chalk2 mutants exhibited significantly higher amylose and protein contents, while total starch and lipid contents were reduced. Analysis of resistant starch in chalk2 revealed substantial increases in two resistant starch (RS) types RS2 and RS3. Electron microscopy revealed abnormal starch granule development in the endosperm. The chalk2 mutant also showed reduced grain length, width, and thickness, as well as a decreased seed setting rate, which ultimately led to a significant reduction in grain yield. Through physical localization, Mut-Map analysis, and transgene complementation, we found that SBEIIb was responsible for the chalk2 phynotypes, a member of the starch branching enzyme (SBE) family, specifically expressed in the endosperm. Furthermore, the expression levels, enzyme activity, and protein abundance of SBEIIb were significantly reduced in chalk2 mutants. These findings suggest that SBEIIb plays a crucial role in regulating the composition of starch and resistant starch formation in indica rice.

Background: The physical properties of resistant starch (RS) are similar to those of dietary fiber; thus, RS is often added to food products to provide the same health benefits as dietary fiber. Methods: In this study, four types of RS were mixed with wheat flour, and gels were prepared. RS-2, high amylose corn starch (HACS), was used alongside three types of RS-4: phosphate cross-linked tapioca starch (XLTS) and low- and high-hydroxypropylated phosphate tapioca starch (LHTS and HHTS, respectively). The flour suspension (16.7 w/w %) consisted of a mixture of medium wheat flour (1:1 mixture of low- and high-gluten wheat flour) and RS, combined in a 95:5 ratio. The suspension was heated at either 90 or 120°C. The control sample consisted of wheat flour only. Compressive analysis, texture analysis, microscopic observations, RS measurements, and thermal properties analysis were performed. Results: The gel made with HACS was soft after heating at 90°C, and this gel showed the highest RS content. Additionally, the control and HACS gels had increased RS content when heated at 120°C. In contrast, while the physical properties of the RS-4 mixed gels (XLTS, LHTS, and HHTS) changed upon heating, the RS content did not increase in the gels heated at 120°C. Therefore, the RS-4 mixed gels may inhibit wheat starch aging during retort cooking. Conclusions: These results indicate that mixing HACS into flour is the most effective way to increase the RS content in a water-dispersed flour system with high-moisture content, with higher heating temperatures facilitating this process.