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%.

Starch, used in various applications; offers restricted utilities due to the limited functionalities in its native form. Several methods have been explored to enhance its functionality. Among these, non-thermal processes such as ultrasound (US) and freeze-thaw (FT) cycles impact starch properties, making them promising processes for emerging applications. In this study, starch (50%, w/v in distilled water) was processed (either individually or combined) using US at different amplitudes (70, 80, and 90%) for 30 min at room temperature, and multiple FT (1, 3, and 5) at −40 °C for 3 h per cycle. Amylose content (AC), water holding capacity (WHC), scanning electron microscopy (SEM), and X-ray diffraction (XRD) were measured. The suitability of modified starches for 3D printing was also evaluated. The treatments resulted in modified starches with a decrease in AC varying from 19.5% to 17.1%, and an increase in WHC ranging from 53.7% to 66.1% (p < 0.05). The modified starches exhibited enhanced pasting properties, indicating improved thermal stability and gelation ability. SEM showed increased surface deformation in modified starch granules compared to the smooth surfaces of those unmodified. XRD patterns showed typical A-type peaks; however, starches subjected to combined treatments exhibited a reduction in relative crystallinity. The use of the starches for 3D printing showed that the modified starches had a more defined surface compared to native starch. Therefore, the combined US and FT treatments can be applied to enhance functionality and 3D printability of cassava starch, opening new avenues for its use as a functional material for food development.

This study aimed to evaluate in vitro and in vivo iron bioavailability of biofortified cowpeas (Vigna unguiculata (L) Walp) (Aracê, Xiquexique and Tumucumaque), compared to a conventional cultivar (Pajeú). In vitro bioavailability was evaluated in Caco-2 cells. For the in vivo study, 40 male Wistar rats were induced to anemia during 21 days (depletion phase), then, in repletion phase (14 days), the animals received as iron source (12 ppm): ferrous sulfate; Pajeú; Xiquexique, Aracê; or Tumucumaque. Pajeú had higher levels of complexing compounds than the biofortified cultivars. In Caco-2 cells, the cellular iron uptake index was higher for biofortified cowpeas, particularly Tumucumaque. Concerning the in vivo study, Tumucumaque group had the best hemoglobin regeneration efficiency and relative biological value, and improved colon crypt size. Then, the iron bioavailability was higher in the biofortified cowpeas, especially Tumucumaque, possibly due to the lower content of mineral-complexing compounds and improvement of absorptive area. This work provides important information to support strategies to combat iron deficiency.

This study aimed to clarify the starch digestion characteristics and related physicochemical properties of the newly developed low-GI rice variety, Ditangliangyou 335 (D335), in comparison with two widely grown rice varieties, Xiangzaoxian 45 (X45) and Zhongzao 39 (Z39). The results showed that D335 had an active digestion duration (286 min) that was 101-190 % shorter, a glucose production rate (1.06 mg g⁻¹ min⁻¹) that was 57-73 % slower, and a total glucose production (303 mg g⁻¹) that was 11-19 % less than X45 and Z39. These differences were attributable to the distinct starch physicochemical properties, including amylose content, amylose-to-amylopectin ratio, starch granule size, amylopectin chain length, and starch molar mass, as well as the different pasting properties of rice flour, such as pasting temperature and breakdown viscosity. These findings reveal the starch digestion characteristics and the key physicochemical properties that determine these characteristics in the low-GI rice variety D335.

Obesity is a complex disease that increases the risk of other pathologies. Its prevention and long-term weight loss maintenance are problematic. Gut microbiome is considered a potential obesity modulator. The objective of the present study was to summarize recent findings regarding the relationships between obesity, gut microbiota, and diet (vegetable/animal proteins, high-fat diets, restriction of carbohydrates), with an emphasis on dietary fiber and resistant starch. The composition of the human gut microbiome and the methods of its quantification are described. Products of the gut microbiome metabolism, such as short-chain fatty acids and secondary bile acids, and their effects on the gut microbiota, intestinal barrier function and immune homeostasis are discussed in the context of obesity. The importance of dietary fiber and resistant starch is emphasized as far as effects of the host diet on the composition and function of the gut microbiome are concerned. The complex relationships between human gut microbiome and obesity are finally summarized.

This study aims to improve the quality of vermicelli products by replacing a portion of rice flour with orange-fleshed sweet potato powder at four ratios ranging from 10 % (F1) to 25 % (F4) (5 % interval), while the control sample used 100 % rice flour (F0). The products were analyzed for quality and sensory characteristics. The analysis of five vermicelli production formulas revealed that the product made from formula F3, which contains OFSP powder and rice flour at a ratio of 20:80 %, had a relatively high amount of β-carotene (9.29 μg/g), a bright color (L∗ = 67.15, b∗ = 29.45), and a suitable hardness (46.79 g-force). The F3 sample had better cooking quality than the F0 sample, which was proved by the percentages of weight increase, volume increase, and dry matter loss of 59.88 %, 56.89 %, and 7.08 %, respectively. The estimated PRAL score of the product prepared from formula F3 was −1.45 (PRAL<0), indicating that the vermicelli product is alkaline, which can support and improve human health. A quantity of 100 g of vermicelli contains 25.15 g of carbohydrates, 3.17 g of protein, 1.48 g of lipids, 4.26 g of resistant starch, 2.43 g of fiber, and 1.66 g of ash. The reduction in estimated glycemic index (eGI) was also found, reaching a value of 51.24 for the F3 sample, whereas the eGI of F0 was 58.24 (medium-high range). Furthermore, microstructural image analysis (from a scanning electron microscope) of sample F3 indicated that OFSP powder addition could result in a better gel network structure than occurs in conventional vermicelli. The findings from this study will be a steppingstone to processing various popular food products with enhanced nutrients. This activity can also be developed on a larger production scale, improving the quality of life for communities, especially in areas with chronic vitamin A deficiency.

Processed green banana starch with high resistant starch content is commonly used as a prebiotic in the food industry. Physical treatment of starch including moist heat treatment and annealing brings significant efficiency and safety to the product. This study was carried out to understand better the effects of moist heat treatment (HMT) and annealing on the changes in green banana starch. Moist heat treatment gradually increased the resistant starch content to a peak of 55 wt% at 25% moisture. Meanwhile, annealing rapidly increased resistant starch to the peak of 45 wt% with negligible water usage (3 mL). The starch granules were disrupted by the annealing process which involved vigorous hydrolysis of the starch chains by the annealing process. Both treatments also changed the crystallinity (type B to type C). The swelling and water solubility of starch treated with HMT gradually decreased from 72 to 65 kJ/mol.K with increasing moisture from 0% to 30%. These properties had an insignificant difference with starches treated by annealing with ~65 kJ/mol.K. The trends of oil/water absorption and freeze-thaw stability of the samples were similar to the changes in resistant starch content. Overall, moist heat treatment-maintained granule structure during the treatment of starch. Annealing allowed rapid changes in resistant starch content and significant effects on starch's physical properties.

The study aimed to modify the structure of two major components (i.e., starch and protein) in bean flour to enhance bean flour's techno-functionality. The bean flour was processed by two very different temperature treatments – dry heat (DH) and extrusion. The effect of processing on starch and protein was analyzed by characterizing their physicochemical properties. While processing did not alter, as expected, the total starch (35-47 % (db)) and protein (24-25 % (db)) content of the bean flours, it did significantly change the microstructure and molecular architecture of starch and proteins. DH processing mainly affected protein conformation while minimally affecting starch structure. Conversely, extrusion caused extensive structural modifications of both starch and protein in bean flour. Both DH and extrusion processing decreased intramolecular β-sheet secondary structure of bean protein. Additionally, both unprocessed and thermally processed bean flours were characterized for their techno-functional properties. The heat treatments (DH and extrusion) did not only result in colour variations, but they also modified the particle size distribution, pasting profile, rheological properties, and water absorption and solubility indices of the bean flour. The structure-function relationship of bean flour components (starch and protein) and bean flour's functionality implied that thermal processing treatments led to changes in the protein/starch matrix which were reflected in the techno-functionality of processed bean flours. However, the changes induced by each of the different types of processing were very different, thus, unlocking different opportunities for functionalization of bean flours for food manufacturing and product development applications.

Starch is widely utilized in the food industry, but its native form may have limitations in terms of functionality and nutrition. This study examines the characteristics of native oat starch and explores its potential for chemical modification by cross-linking, with the aim of gaining a deeper understanding of its functionality. Citrate-modified oat starch (COS) is generated by cross-linking native oat starch (NOS) with citric acid. Chemical analysis reveals distinctions between NOS and COS, notably in terms of resistant starch content, which is elevated in COS. X-ray diffraction (XRD) results reveal that NOS exhibits crystalline peaks characteristic of A-type starch, distinguishing it from COS. In contrast, COS displays absent crystalline peaks, attributed to cross-linking. Swelling factor, solubility, and paste clarity along with most of the pasting properties are found to be significantly lower for COS compared to NOS. The scanning electron microscopic images show how cross-linking can alter the morphology of the NOS. The findings from this investigation highlight significant distinctions between NOS and COS. The cross-linking process successfully enhances the resistant starch content and imparted distinctive properties absent in native oat starch. Consequently, there is potential for incorporating modified oat starch as a food ingredient.