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.

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.

Microalgae have a growing recognition of generating biomass and capturing carbon in the form of CO₂. The genus Chlorella has especially attracted scientists’ attention due to its versatility in algal mass cultivation systems and its potential in mitigating CO₂. However, some aspects of how these green microorganisms respond to increasing concentrations of CO₂ remain unclear. In this work, we analyzed Chlorella sorokiniana and Chlorella vulgaris cells under low and high CO₂ levels. We monitored different processes related to carbon flux from photosynthetic capacity to carbon sinks. Our data indicate that high concentration of CO₂ favors growth and photosynthetic capacity of the two Chlorella strains. Different metabolites related to the tricarboxylic acid cycle and ATP levels also increased under high CO₂ concentrations in Chlorella sorokiniana, reaching up to two-fold compared to low CO₂ conditions. The signaling molecules, inositol polyphosphates, that regulate photosynthetic capacity in green microalgae were also affected by the CO₂ levels, showing a deep profile modification of the inositol polyphosphates that over-accumulated by up to 50% in high CO₂ versus low CO₂ conditions. InsP₄ and InsP₆ increased 3- and 0.8-fold, respectively, in Chlorella sorokiniana after being subjected to 5% CO₂ condition. These data indicate that the availability of CO₂ could control carbon flux from photosynthesis to carbon storage and impact cell signaling integration and energy levels in these green cells. The presented results support the importance of further investigating the connections between carbon assimilation and cell signaling by polyphosphate inositols in microalgae to optimize their biotechnological applications.

Hybrid products, in which a portion of meat is replaced with plant proteins, could be an effective solution to reduce meat consumption and its environmental impact, and provide well-balanced nutritious food. This study focused on exploring the potential of creating hybrid meat products by high-moisture extrusion from a mixture of minced beef (with 7 or 17% fat) and two different commercial pea protein ingredients. Hybrid extrudates were successfully produced from a 1:1 mixture of beef and either pea protein isolate (PI) or milled texturised pea protein concentrate (TPC). Differences in the appearance, texture and sensory properties of the hybrid extrudates depended both on ingredient properties and extrusion processing parameters (especially temperature). Extrudates with beef and PI were softer and layered, while extrudates with beef and TPC were harder and had smaller fibres. The fat content of the beef did not significantly affect the textural properties of the extrudates. The extrudates with beef and TPC had a meat-like odour and umami taste, while extrudates with beef and PI had a dominant pea-like taste and odour.

Superheated steam (SS), far-infrared radiation (FIR), and microwave treatment (Mw) are emerging stabilization treatments to improve the shelf life of black rice. However, their effects on the digestibility of cooked black rice remain unclear. Therefore, the structural characteristics of cooked black rice influenced by the above treatments and their effects on in vitro digestibility were compared. Results indicated that cooked SS stabilized black rice (CBR-SS) had the highest crystallinity (9.17%), the lowest porosity, and the completest protein network. Besides, the lowest level of rapidly digestible starch (31.02%) and the highest level of resistant starch (32.75%) were observed in CBR-SS. Consequently, CBR-SS showed the lowest equilibrium hydrolysis rate, kinetic constant, and expected glycemic index during in vitro starch digestion among the cooked stabilized black rice samples. Furthermore, the level of total bioaccessible phenolic compounds and antioxidant activity was the highest in CBR-SS. Therefore, SS stabilization treatment is the most valued as it grants cooked black rice the slowest starch digestibility and the highest level of bioaccessible phenolic compounds.

Intact cells, as the smallest unit of whole foods, were isolated from three legume crops and fermented with human faecal inoculum to elucidate the effect of food macro-nutrients compositional difference (starch, proteins and lipids) on in vitro colonic fermentation profiles. After 48 h of fermentation, the highest production of short-chain fatty acids (SCFAs) were observed for the pea cells, abundance in starch (64.9 %, db). In contrast, branch chain fatty acids (BCFAs) were the major metabolites for protein-enriched soybean cells (protein content 56.9 %, db). The peanut cells rich in lipids (49.2 %, db) has the lowest fermentation rate among the three varieties. Correspondingly, pea cells favoured the growth of Bifidobacterium, whereas soybean and peanut cells promoted an abundance of Bacteroides and Shigella, respectively. Furthermore, except the intact pea cells promoting the abundance of butyrate producer Roseburia, a similar fermentation pattern was found between intact and broken cells suggesting that macro-nutrient types, rather than structure, dominate the production of metabolites in colonic fermentation. The findings elucidate how the food compositional difference can modulate the gut microbiome and thus provide the knowledge to design whole food legumes-based functional foods.

The effects of dairy and plant protein addition on microstructural characteristics and in vitro gastro-small intestinal starch digestion characteristics of Chinese steamed breads (CSBs) were studied. Breads containing rennet casein (RC) and a mixture of soy protein isolate and milk protein concentrate (SM) at two different levels (RC I, RC II; SM I, SM II) were prepared. Microstructural characteristics of the undigested and digested control (100% wheat flour) bread and high protein steam bread (HPCSB) versions were compared through scanning electron microscopy. The compact microstructure of HPCSBs displayed a network of proteins wrapped around starch granules and had fewer air cells compared to the control. The addition of both proteins influenced the microstructure of HPCSBs, which in turn affected their textural and starch digestion properties. The in vitro starch digestion of control CSB and HPCSBs confirmed that the addition of proteins is capable of lowering the starch hydrolysis (%). The highest starch hydrolysis was observed for the control wheat bread, followed by SM1 > RC I > SM II and RC II at the end of the small-intestinal digestion. The estimated glycaemic indices (eGI) for all HPCSBs were statistically lower than the control CSB. In comparison to control CSB, the microstructure of HPCSBs appeared more irregular, less porous, and compact during gastric and small intestinal digestion.

Orange-fleshed sweet potato (OFSP) has attracted increasing attention due to its high carotenoid levels, which is beneficial to human health. However, the changes in the chemical composition of OFSP after cooking are not fully elucidated yet. In this study, the contents of starch, soluble sugar, carotenoids, volatile compounds, and metabolites of OFSP before and after cooking (steaming, boiling, and baking) were determined to evaluate the changes in its flavor and chemical composition. After cooking, the contents of starch decreased from 18.15% to 7%, and soluble sugar increased from 11.78% to 39.33%. The baked OFSP was the sweetest one due to the production of most sugars during baking. Additionally, the aroma of baked OFSP was the best due to the abundance of furans, aldehydes, ketones, and monoterpenes, including 6 volatiles produced by carotenoids. However, the steamed OFSP retained more carotenoids, while all carotenoids were reduced 7%-23% after cooking. Furthermore, 593 metabolites were identified in OFSP, of which 82.5% were well preserved after cooking. Overall, understanding of chemical profile changes of OFSP after cooking will provide a theoretical basis for broadening sweet potato processing and application.

In this study, the physicochemical properties of potato starch from different varieties were investigated. Furthermore, the relationships among gelatinization, retrogradation behavior, and impedance characteristics of potato starch gels were evaluated by texture analysis, low-field nuclear magnetic resonance spectroscopy, and electrical impedance spectroscopy. The results indicated amylose content was positively correlated with setback viscosity, and negatively correlated with T_o and ΔH. In addition, impedance values of potato starch gels differed in a frequency-dependent manner. Notably, higher frequencies resulted in low diffusion of ions in prepared gels, which combined with the concentration of mobile ions in free water, led to a gradual decrease in impedance module. Compared with phase values, impedance module showed high correlation with gelatinization parameters (T_o, T_p, and T_c) and viscosity parameters (peak temperature and setback viscosity), more notably at frequencies below 100 Hz. In this context, the electric current flowed through mobile ions that interacted with bound water attached to the starch molecules at lower voltage frequencies, and were repressed by the formation of an ordered and compact gel network during retrogradation. Collectively, these results indicate that impedance spectroscopy can be potentially used as an efficient and reliable method to predict gelatinization and retrogradation behavior of potato starch.