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%.

Seventeen pigmented maize cultivated in different locations of India were collected and evaluated for their nutritional, anti-nutritional and starch profile. A wide variation was observed in all the parameters evaluated. Protein and total dietary fiber content varied between 7.18 to 10.85 g/100g and 8.76 to 14.04 g/100g, respectively. Among the water-soluble vitamins, B₂ varied from 0.071 to 0.134; B_3, 1.50 to 2.79; B_5, 0.17 to 0.70 and B₆, 0.141 to 0.245 mg/100g; while total tocopherol and tocotrienol varied 1417 to 2246 µg/100g and 18.57 to 30.01 µg/100g, respectively. The resistant starch, total starch and amylose varied significantly and observed between 0.49 to 4.78 %, 61.33 to 69.65 % and 10.91 to 32.01 %, respectively. Among the minerals, Fe and Zn were observed between 1.51 to 5.01 and 2.25 to 5.51 mg/100g, respectively. Total polyunsaturated and total monounsaturated fatty acids were observed up to 45.72 and 55.54 %, respectively. Amongst the anti-nutrients, phytate was detected the lowest in Laal makki, whereas total and soluble oxalates were the lowest in Kashmiri red maize.

Rice flour is useful as a substitute for wheat flour, however, to obtain fine flour, millers need special milling facilities, which increase the cost of milling. To reduce the milling cost, we developed a floury mutant line by irradiating gamma-rays to dry seeds of the japonica cultivar ‘Hoshinoyume’. The line was registered as a new cultivar, ‘Hoshinoko’. Genetical analysis of the floury trait was conducted using an F2 population derived from a cross between ‘Hoshinoko’ and ‘Corbetti’ (a japonica rice cultivar with normal endosperm), which indicated the involvement of a single recessive gene located near the RM163 marker on the long arm of rice chromosome 5, flanking flo4 identified by Kang et al. (2005). Sequence analysis of flo4 showed a two-bp (CA) insertion in the eighth exon of in ‘Hoshinoko’ compared to that of ‘Hoshinoyume’, which led to a frameshift mutation. The CAPS-based genotype of flo4 gene completely correlated to the phenotype of endosperm in two populations. This CAPS marker could be helpful for rice breeders to develop new cultivars harboring floury endosperm of the flo4-303 gene.

This study aimed to investigate the effects of different physicochemical modifications of Riceberry rice (Oryza Sativa L.) starch on its morphology, pasting behavior, and functional properties. The methods include HCl lintnerization (1 N, 1.5 N, 2 N), autoclaving (135°C for 14 min), HCl lintnerization combined with autoclaving, and freeze-thawing (-20°C for 30 min). The result demonstrates that all modifications applied to the native starch increased the resistant starch (RS) content. The autoclaved Riceberry rice starch showed the highest RS content (49.66 ± 0.14%) and lowest estimated glycemic index (eGI) (50.03 ± 0.01%), indicating a healthy, low glycemic food. All treatments improved the solubility and water holding capacity (WHC) of native starch without destroying granule structure, whereas swelling power decreased with increased acid temperature-dependent concentration. Autoclave treatment increased WHC significantly of the modified starches with the damaged starch granules. The color values of lintnerized starch were reduced due to the removal of color pigment anthocyanin. Therefore, the result of this study provides a better understanding of the various modification methods for the functional properties of Riceberry rice starches for potential applications in a wide range of food applications.

Autoclaving (15 min, 121°C, 1 atm overpressure) and high-intensity ultrasound (3 pulses of 1 min, 750 W, 20 kHz, 80% amplitude) were evaluated individually or combined in cassava pulp to reduce the digestible starch and increase the fibre content. Ultrasound alone produced minimal modifications, and the subsequent autoclaving increased the oil-binding capacity and swelling. Autoclaving alone significantly increased total dietary fibre (TDF) from 14.7% to 21.4%, and the ratio of hemicellulose to lignocellulose. It also reduced the extractable starch from 42.7% to 7.5% and the resistant starch from 7.4% to 3.1%. Despite the higher digestibility, the starch digestion rate with pancreatic α-amylase was slower. Starch solubility increased at 25, 68, and 90°C. Ultrasonication after autoclaving reduced the hemicellulose percentage, but the sorption properties remained unchanged. Resistant starch substantially increased to 39.9%, mainly composed of RS1 (inaccessible starch), and starch solubility decreased at the three temperatures assayed.

Water caltrop starch (WCS) isolated from the dehydrated water caltrop (DWCS) showed higher content of protein and ash as compared to starch isolated from fresh water caltrop (FWCS), leading to slightly lower starch purity. However, the pasting profiles of DWCS and FWCS are similar under a constant solid content of 4% (w/w). WCS/gum mixed systems contain 0.3%–0.9% gum generally showed higher pasting temperature, peak viscosity, and final viscosity than the starch alone system. The increase in peak and final viscosity during rapid-viscosity analysis was most pronounced for WCS/gum mixed systems with konjac glucomannan (KG), followed by guar gum (GG) and xanthan gum (XG). All WCS/gum mixed systems showed shear thinning and thixotropic characteristics. The extent of pseudoplasticity and shear stability, as evidenced by the smaller flow behavior index (n) and area of the hysteresis loop, was most pronounced for WCS/gum mixed system with XG, followed by mixed systems with GG and KG. Dynamic viscoelastic analysis revealed that the addition of gum increased both the storage and loss moduli of WCS, and lowered the frequency dependence of the loss factor significantly. However, the loss factor of WCS/gum mixed systems was generally higher than that of the starch alone system, implying the addition of gums modified the rheological characteristic of WCS to more viscous dominant behavior.

Resistant starch (RS) has caught much attention for its potential to exert a beneficial impact on intestine and certain members of its resident microbiota. In this study, we examined how dietary RS promotes intestinal barrier in meat ducks by microbiome-metabolomics analysis. Ducklings were fed corn-soybean basal diet or RS diet. Dietary RS improved intestinal morphology and enhanced barrier function in ileum, evidenced by lower permeability and upregulated tight junction proteins and Mucin-2 gene expression. Microbiome analysis showed that RS administration elevated the proportion of Firmicutes and butyrate-producing bacteria, and increased butyrate contents in cecum. Furthermore, significant alterations in metabolic profiles were observed, with most of these were associated with the amino acid metabolism (especially tryptophan), lipid metabolism, and intestinal inflammation. Together, diet with RS improved gut integrity and caused corresponding alterations in gut metabolome and microbiome, yielding better insights of the mechanism by RS improved the gut system of ducks.

Mainstreaming the low glycemic index (GI) trait in breeding programs is constrained by low-throughput and high-cost clinical GI phenotyping. This study aimed to evaluate the potential of starch fine structure components and simulated digestion parameters in predicting GI in rice. Amylose (AM1 and AM2; r = −0.94 and r = −0.80, respectively, p < .05) and amylopectin fine structure (MCAP, SCAP, and SCAP1; r = 0.78-0.86, p < .05) measured through size-exclusion chromatography along with resistant starch (r = −0.81, p < .05) in seven (7) rice accessions showed high correlation with in vivo GI. Meanwhile, starch hydrolysis extent (SH) and the corresponding area under the digestion curve (AUC) obtained through in vitro digestion were found to be of higher correlation with GI, even within shorter digestion periods of 5 min or 30 min (r = 0.96, p < .01). These results highlight the potential use of these parameters as predictors of GI, with improved predictive capacity through a multiple regression model. Higher correlations of simulated digestion AUC with GI may be due to its ability to account for the overall food matrix native macro- and micro-structures, gaining an added advantage over SEC method as a predictive tool in studying rice GI variability. Validation in a larger population is an inevitable next step.

Green banana flour (GBF) is considered a functional ingredient that could improve banana world's production sustainability. The banana drying method might influence the physicochemical and nutritional properties of GBF, affecting its performance in bread-making. The study aims to determine the impact of freeze-drying and oven-drying on gluten-free banana bread's quality. Freeze-dried banana flour (FDBF) bread had higher specific volume, resilience, and less brown coloration than oven-dried banana flour (ODBF) bread. Also, FDBF bread presented higher resistant starch (RS). The slowly digested starch (SDS) was similar in both types of bread. In contrast, rapidly digested starch (RDS) was significantly higher in FDBF bread, which led to higher expected glycemic index. Even though the nutritional fractions (RS and SDS) of both gluten-free banana bread exceeded 20 g/100 g bread (db), with no significant difference concerning the drying type, the FDBF bread presented improved characteristic.