Total Starch Assay Kit (AA/AMG)

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.

Most commonly used methods for the measurement of starch in food, feeds and ingredients employ the combined action of α‐amylase and amyloglucosidase to hydrolyse the starch to glucose, followed by glucose determination with a glucose oxidase/peroxidase reagent. Recently, a number of questions have been raised concerning possible complications in starch analytical methods. In this paper, each of these concerns, including starch hydrolysis, isomerisation of maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase, enzyme purity and hydrolysis of sucrose and β‐glucans have been studied in detailed. Results obtained for a range of starch containing samples using AOAC Methods 996.11 and 2014 .10 are compared and a new simpler format for starch measurement is introduced. With this method that employs a thermostable α-amylase (as distinct from a heat stable α-amylase) which is both stable and active at 100°C and pH 5.0, 10 samples can be analysed within 2 h, as compared to the 6 h required with AOAC Method 2014.10.

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.

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%.

A procedure for the quantitative analysis of total starch in plant materials has been developed and subjected to a comprehensive interlaboratory study involving 32 laboratories, in accordance with the protocol for collaborative studies recommended by American Association of Cereal Chemists and AOAC International. The method involved treatment of a sample at approximately 95°C with thermostable α-amylase to obtain starch depolymerization and solubilisation. The slurry is then treated with purified amyloglucosidase to give quantitative hydrolysis of the starch fragments to glucose, which is measured with glucose oxidase/peroxidase reagent. Test samples used in the interlaboratory study included modified and native starches, cereal flours and brans, processed cereal products, animal feeds, and plant material. Results were statistically analysed according to AOAC International guidelines (1). The procedure was shown to be highly repeatable (relative standard deviation 2.1-3.9%) and reproducible (relative standard deviation 2.9-5.0%), and on the basis of these results has gained first approval status with AACC (AACC Method 76-13) and approval as AOAC Method 986.11. The method is more robust than a method previously reported (AACC Method 76-12), and 20 samples can be analysed within 2 hr.

The precision of an enzymatic procedure for analysis of total starch in cereal flours and products was determined in a comprehensive inter-laboratory study involving 29 laboratories. Test samples represented a range of sample types, including modified and native starches, cereal flours and brans, processed cereal products, animal feeds, and plant material. Results were statistically analyzed according to AOAC guidelines. The procedure was shown to be highly repeatable (relative standard deviation 1.5-7.3%) and reproducible (relative standard deviation 4.1-11.3%). It is now available, in a slightly modified form, as an assay kit. The assay, therefore, provides a convenient alternative to existing procedures for quantitative measurement of starch in cereal products.

A rapid and quantitative method has been developed for the determination of total starch in a wide range of materials, including high-amylose maize starches and food materials containing resistant starch. The method allows the analysis of 20 samples in 3 h. A single assay can be performed in 2 h. For a range of samples, the total starch values obtained with this method were significantly higher than those obtained with current standard methods. Two assay formats have been developed. In assay format 1, the sample is incubated solubilised with the chaotropic agent dimethyl sulphoxide (DMSO) to gelatinise the starch, which is then solubilised and partially depolymerised by controlled incubation at ∼ 100°C with a defined level of thermostable alpha-amylase. This allowed near-complete solubilisation of most starches. The remaining starch is then solubilised and the starch fragments are converted to maltose and maltotriose by the combined action of highly purified pullulanase and beta-amylase. After volume adjustment and filtration (if necessary), the maltooligo-saccharides are hydrolysed by high-purity amyloglucosidase to glucose, which is measured with a glucose oxidase/peroxidase reagent. This assay format gave quantitative starch determination in all native starch samples, including high-amylose maize starches. In assay format 2, which is applicable to most starches and cereal flours, the DMSO pre-treatment step is omitted. Samples containing glucose and/or maltosaccharide are pre-washed with aqueous ethanol before analysis.

Background and Objectives: Effects of the physicochemical properties of buckwheat flour (BKF), quinoa flour (QF), proso millet flour (PMF), and whole wheat flour (WWF) were evaluated in a pancake product made with unbleached fine wheat pastry flour (FWF). Pancakes were formulated with 25%, 50%, 75%, and 100% (w/w) substitution levels. FWF was used as the control. Findings: The flours varied in composition, affecting their functional properties. BKF was characterized by high total dietary fiber (TDF), FWF had the highest starch content, WWF had the highest protein content, and PMF had the highest peak and onset temperature. The influential functional properties on pancake quality were the content of TDF and insoluble dietary fiber, as well as the water and oil holding capacity. Due to the rapid cooking time in pancakes, the PMF starch did not gelatinize adequately, likely due to the amylose content or shape and size of the starch, resulting in a low-hardness pancake without a continuous matrix. When a pancake was prepared with partial pregelatinized PMF, the pancake had a continuous matrix. Conclusion: BK, QF, and WWF can be incorporated into pancake formulations without flour modification. PMF must be modified before usage in a pancake application to ensure that a continuous matrix is formed.

The aim of this study was to examine how silages from different grassland species and harvesting frequencies affect feed intake, milk production, and methane (CH₄) emission in dairy cows. We hypothesized that cows consuming silages of more frequent harvest, grass species with greater organic matter digestibility and legumes with lower NDFom concentration would have greater silage dry matter intake and milk yield and thereby lower CH₄ yield and intensity. Forty Norwegian Red cows were allocated to 5 treatments in a cyclic changeover design with 4 21-d periods (14 d of adaptation, 7 d of data collection). The 5 treatments evaluated were silages produced from timothy (Phleum pratense L.) in a 3-cut system (T3), timothy in a 2-cut system (T2), perennial ryegrass (Lolium perenne L.) in a 3-cut system (PR3), red clover (Trifolium pratense L.) in a 3-cut system (RC3) and a mix of T3 and RC3 (50:50 on DM basis) (T3/RC3). The treatments were prepared by mixing silages from each crop over the growing season, proportional to the harvested DM yield of each cut. Cows were offered the mixed silages ad libitum supplemented with a fixed level of concentrate. Gas emissions were measured using 2 Greenfeed units. Milk yield was recorded in the milking robot at each visit, and milk samples were collected at 3 consecutive milkings during the last 7 d of each period. Cows were weighed after each milking, and total-tract digestibility of each diet was estimated using acid insoluble ash as internal marker in fecal grab samples. The data were analyzed using the MIXED procedure of SAS with block, period and treatment as fixed effects and animal within block as random effect. Silage and total DMI did not differ between T3 and T2, but total DMI was lower for PR3 than for T3. There was a quadratic effect of increased proportion of red clover, with highest intakes of T3/RC3 and lower intakes of RC3 than of T3. Energy corrected milk (ECM) yield was lower for T2 than T3, and for PR3 than T3. There was a quadratic effect of increased proportion of red clover, with highest ECM yield in T3/RC3 and lower in RC3 than in T3. Organic matter digestibility was lower for T2 than T3, but it did not differ between T3 and PR3. Including red clover in the diet linearly decreased organic matter digestibility. Methane production (g/d) did not differ between T3 and T2, but CH₄ intensity (g/kg ECM) was greater for T2 than for T3. There was no difference between T3 and PR3 for CH₄ production but yield and intensity were greater for PR3 than T3. Including red clover in the diet linearly increased CH₄ production, yield and intensity with greatest intensity in the 100% red clover diet. In conclusion, changing harvesting frequency for timothy from 2 to 3 harvests per year did not affect CH₄ production or yield, but CH₄ intensity was reduced. Replacing timothy with perennial ryegrass and increased inclusion rate of red clover both increased CH₄ yield and intensity.

Individuals affected by celiac disease or non-celiac gluten sensitivity disease need to adhere to a lifelong gluten-free (GF) diet. However, most of the GF products available in the market are low in nutritional qualities, as they are mainly starch-based. The use of wholegrain cereals in GF bread is therefore a promising strategy to improve its nutritional quality, in particular when using pigmented varieties. Pigmented rice is high in anthocyanins and phenolic acids that can help preventing chronic diseases. This study investigated the applicability of wholegrain pigmented rice (brown, red, black) in developing GF bread using ohmic and conventional heating processes. The addition of pigmented rice to GF breads resulted in higher specific bread volume, lower crumb firmness and relative elasticity, while porosity was increased, although with less unform pore size, compared to control bread from white (polished) rice. Wholegrain pigmented rice addition resulted in significantly higher amounts of total dietary fiber, total phenolic content, and antioxidant activities of GF breads. Overall, this study demonstrated that wholegrain pigmented rice flours have the potential to improve the physical and nutritional values of GF breads.

This study investigated the effect of grape seed (GSd) and grape skin (GSk) tannins on the physicochemical, rheological properties and in-vitro digestibility of starches (corn, pea and wheat) derived from three different botanical sources. Quantification of bound and unbound tannins using MCP and HPLC analysis demonstrated that majority of the tannins were bound to starch molecules. The results of particle size distribution, starch-iodine binding and FTIR studies indicated the development of inclusion complexes through hydrophobic interactions with tannins in pea starch, while other two starches prominently formed non-inclusion complexes via hydrogen bonding. Back extrusion analysis of textural properties indicated that wheat starch-tannin complexes resulted in firmer starch-tannin gels compared to other two starches. Rheological studies revealed an increase in the viscoelastic modulus (G’ and G”) with improved elastic behavior for all starch-tannin gels. Starches complexed with tannins demonstrated strong antioxidant properties and in-vitro starch digestion studies revealed significant reductions in rapidly digestible starch (RDS) and slowly digestible starch (SDS), along with an increase in resistant starch (RS), particularly in pea starch complexed with GSd tannins. This study enhanced our understanding of how GSd and GSk tannins influence the properties of starches from various botanical origins, helping in understanding starch-tannin interactions and enabling the creation of foods with improved texture and digestibility.

Static magnetic field (SMF), an innovative and eco-friendly technology, has attracted widespread attention in the field of modified starch physicochemical properties. This study aimed to investigate the effects of SMF treatment on the structural and digestive properties of germinated corn (GC) starch. In vitro digestibility examination of GC starch revealed that SMF treatment (30 mT, 2 h) led to a 12.76% reduction in the rapidly digestible starch (RDS), while slowly digestible starch (SDS) and resistant starch (RS) increased by 45.57% and 15.78%, respectively. Additionally, SMF treatment endowed GC starch with higher crystallinity and improved short-range order. Furthermore, the physicochemical property analysis indicated that SMF treatment decreased the swelling power, solubility, and oil absorption of GC starch by 31.62%, 51.19%, and 25.92%, respectively. These findings support the development of low-glycemic index (GI) foods and demonstrate a potential to modify GC starch structure and reduce starch digestibility through a green pathway.

Conventional bioethanol production from starch-based crops involves high-temperature cooking, which is energy-intensive and degrades the protein quality of distiller's grains (DG), a valuable co-product. This study addresses the critical gap of reducing the energy demand and protein degradation by comparing conventional high-temperature processing with granular starch hydrolyzing enzyme (GSHE) fermentation at low temperatures. Specifically, the novelty lies in optimizing partial starch swelling treatments (50°C, 60°C, 70°C) to enhance ethanol yields while preserving DG protein quality. Using sorghum varieties (normal and waxy) as a model system, we conducted experiments combining low-temperature starch swelling and GSHE fermentation to evaluate their impacts on ethanol yield and protein properties. Waxy sorghum exhibited higher ethanol fermentation efficiency than normal sorghum. Partial starch swelling significantly improved ethanol yield without compromising DG protein quality. GSHE fermentation with starch swelling at 70°C for 30 min achieved the highest ethanol concentration (12.02% v/v) and yield (92.74%) for waxy sorghum. Protein digestibility remained high for both waxy (85.39%) and normal sorghum (85.21%) even at higher swelling temperatures. Surface hydrophobicity of DG proteins increased with temperature, particularly at 95°C during conventional processing. Notably, partial starch swelling improved the lightness (L* values) of sorghum proteins, indicating better quality. Molecular characterization further revealed the specific effects of processing on protein properties. This research highlights the potential of low-temperature starch swelling combined with GSHE fermentation to enhance ethanol production efficiency and protein quality in DG, offering a sustainable alternative to conventional bioethanol processes.

Large quantities of brewer’s spent grains are not fully utilized even as cattle feed. These feedstocks can be used to produce highly functional biomaterials, carbonaceous materials, and additives. In this investigation, four major fractions were isolated and characterized: Hemicellulose A, Hemicellulose B, cellulosic-rich fraction (CRF), and oligosaccharides. Overall, 21.4% Hemicellulose A, 18.5% Hemicellulose B, 17.4% cellulosic rich fraction, and 5.5% pure oligosaccharides were obtained from the hexane-extracted brewery’s spent grains. Detailed physio-chemical analyses of each fraction showed that these fractions can be used to produce useful products such as emulsifiers, carbonaceous materials, modified cellulosic fibers, additives, as well as N-doped chars. Component analyses revealed that, Hemi. A contains high fixed carbon (20 wt.%), followed by hexane extracted material (17.1 wt.%), CRF (14.6 wt.%), and Hemi. B (14.5%). Standard proximate analyses showed that Hemi. A has the highest protein (66 wt.%), which can be utilized as a renewable solid-state N-precursor as dopants during the thermochemical conversion process. The sugar composition revealed that BSG has a typical arabinoxylan structure with a high percentage of arabinose and xylose. It also contains a high percentage of glucose, which may come from the residual β-glucan present in the BSG. FTIR analyses revealed changes in the structure of each fraction. Hence, BSG and extracted fractions exhibit significant potential for waste valorization, contributing significantly to the full utilization of products from the brewing industry.

Enhancing crops productivity to ensure food security is one of the major challenges encountering agriculture today. A promising solution is the use of biostimulants, which encompass molecules that enhance plant fitness, growth, and productivity. The regulatory metabolite zaxinone and its mimics (MiZax3 and MiZax5) showed promising results in improving the growth and yield of several crops. Here, the impact of their exogenous application on soil and rice root microbiota was investigated. Plants grown in native paddy soil were treated with zaxinone, MiZax3, and MiZax5 and the composition of bacterial and fungal communities in soil, rhizosphere, and endosphere at the tillering and the milky stage was assessed. Furthermore, shoot metabolome profile and nutrient content of the seeds were evaluated. Results show that treatment with zaxinone and its mimics predominantly influenced the root endosphere prokaryotic community, causing a partial depletion of plant-beneficial microbes at the tillering stage, followed by a recovery of the prokaryotic community structure during the milky stage. Our study provides new insights into the role of zaxinone and MiZax in the interplay between rice and its root-associated microbiota and paves the way for their practical application in the field as ecologically friendly biostimulants to enhance crop productivity.