α-Amylase Assay Kit (Ceralpha Method)

The primary quality assessor of wheat grain is the Hagberg Falling Number (FN) method. This is a viscometric test surrogate for α-amylase activity. Despite being used for over sixty years, FN has been increasingly scrutinised due to its low throughput, poor reproducibility and inability to differentiate between the causes of low FN including Pre-harvest Sprouting (PHS) and Late Maturity α-Amylase (LMA). Our study describes initial efforts to analyse a specific wheat flour set tailored for the identification of enzymatic candidates that would allow discrimination between PHS and LMA affected grains. Using the sensitive enzyme-coupled assay substrate R-AMGR3, results suggest that α-glucosidase (exo-α-glucosidase) is a potential enzyme marker candidate to specifically detect sprouted but not LMA-affected grain.

A simple method for measurement of the amylolytic activity of malt has been developed and fully evaluated. The method, termed the Maltose Value (MV) is an extension of previously reported work. Here, the MV method has been studied in detail and all aspects of the assay (sample grinding and extraction, starch hydrolysis, maltose hydrolysis and determination as glucose) have been optimised. The method is highly correlated with other dextrinising power methods. The MV method involves extraction of malt in 0.5% sodium chloride at 30°C for 20 minutes followed by filtration; incubation of an aliquot of the undiluted filtrate with starch solution (pH 4.6) at 30°C for 15 min; termination of reaction with sodium hydroxide solution; dilution of sample in an appropriate buffer; hydrolysis of maltose with a specific α-glucosidase; glucose determination and activity calculation. Unlike all subsequent reducing sugar methods, the maltose value method measures a defined reaction product, maltose, with no requirement to use equations to relate analytical values back to Lintner units. The maltose value method is the first viable method in 130 years that could effectively replace the 1886 Lintner method.

Background: The level of available carbohydrates in our diet is directly linked to two major diseases; obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective: The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose and galactose. Method: Non-resistant (digestible) starch is hydrolysed to glucose and maltose by pancreatic α-amylase and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results: A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions: A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (%RSDr) was <3.58% (w/w) across a range of samples containing 44.1 to 88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust and simple to use. Highlights: A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose and galactose; information of value in determining the glycemic index of foods.

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.

Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α-amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65^oC (MIM 65^oC). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times.

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.

In General, the development of methods for measuring α-amylase is pioneered in the clinical chemistry field and then translated to other industries, such as the cereals and fermentation industries. In many instances, this transfer of technology has been difficult or impossible to achieve due to the presence of interfering enzymes or sugars and to differences in the properties of the enzymes being analysed. This article describes many of the commonly used methods for measuring α-amylase in the cereals, food, and fermentation industries and discusses some of the advantages and limitations of each.

This study was conducted to evaluate the method performance of a rapid procedure for the measurement of α-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing α-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable α-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, α-glucosidase, and β-amylase. When the substrate is cleaved by endo-acting α-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of α-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 µmol p-nitrophenyl (in the presence of excess quantities of α-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal α-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.

Enzymes are added to animal feed to increase its digestibility, to remove anti-nutritional factors, to improve the availability of components, and for environment reasons (Campbell and Bedford, 1992; Walsh et al., 1993). A wide-variety of carbohydrase, protease, phytase and lipase enzymes find use in animal feeds. In monogastric diets, enzyme activity must be sufficiently high to allow for the relatively short transit time. Also, the enzyme employed must be able to resist unfavourable conditions that may be experienced in feed preparation (e.g. extrusion and pelleting) and that exist in the gastrointestinal tract. Measurement of trace levels of enzymes in animal feed mixtures is difficult. Independent of the enzyme studied, many of the problems experienced are similar; namely, low levels of activity, extraction problems inactivation during feed preparation, non-specific binding to other feed components and inhibition by specific feed-derived inhibitors, e.g. specific xylanase inhibitors in wheat flour (Debyser et al., 1999).

A procedure for the measurement of fungal and bacterial α-amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end) p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and α-glucosidase, and is absolutely specific for α-amylase. The assay procedure is simple, reliable and accurate.

A new procedure for the assay of cereal α-amylase has been developed. The substrate is a defined maltosaccharide with an α-linked nitrophenyl group at the reducing end of the chain, and a chemical blocking group at the non-reducing end. The substrate is completely resistant to attack by β-amylase, glucoamylase and α-glucosidase and thus forms the basis of a highly specific assay for α-amylase. The reaction mixture is composed of the substrate plus excess quantities of α-glucosidase and glucoamylase. Nitrophenyl-maltosaccharides released on action of α-amylase are instantaneously cleaved to glucose plus free p-nitrophenol by the glucoamylase and α-glucosidase, such that the rate of release of p-nitrophenol directly correlates with α-amylase activity. The assay procedure shows an excellent correlation with the Farrand, the Falling Number and the Phadebas α-amylase assay procedures.

Short-term germination has been applied to different conventional and unconventional legume seeds to increase nutritional values, beneficial health effects, and techno-functional characteristics for diverse food applications. Nonetheless, the potential impact on black turtle beans (BTBs) is limited in literature. This study examines the compositional, bioactivity, functional, pasting, thermal, and color characteristics of BTBs over a 72 h germination period. On a time-dependent basis, germination increased (p ≤ 0.05) α-amylase activity, protein, insoluble, soluble, and total dietary fiber, resistant starch, minerals, vitamins (B₁, B₂, B₆ and C), as well as the majority of the essential amino acids, protein digestibility, phytochemicals, and antioxidant activity of BTB flour. Germination caused significant reductions in the level of crude fat, total starch, digestible starch, and antinutritional compounds (tannin, phytic acid, and trypsin inhibitors). Germination also modified techno-functional attributes (functional, pasting, thermal, and color characteristics) of BTB flour. Germination also caused interactions with some functional groups, as demonstrated by the FTIR (Fourier transform infrared) spectroscopy. This study revealed that germination could serve as an affordable and natural means to enhance the functionality of BTB flour by improving its nutritional quality, bioactivity, and techno-functional characteristics for the formulation of new food ingredients that meet the current food requirements of the people. PRACTICAL APPLICATION: Short-term germination improved the nutritional value, physicochemical attributes, bioactivity, and techno-functional characteristics of black turtle bean flours. The germinated black turtle bean flour could serve as a novel ingredient for the development of nutritious foods with potential health benefits.

Orange is one of the most consumed fruits worldwide, generating significant byproducts and wastes. By following the principles of green chemistry, the reuse of orange by-products can be made sustainable. In this context, natural deep eutectic solvents (NADES) have emerged as a promising alternative. The present study aims to analyze the physicochemical characteristics and biological effects of orange peel extracts obtained with choline chloride-based NADES. An investigation was conducted on the extracts, including a chromatographic analysis of phenolic and volatile compounds, a spectroscopic FTIR analysis, as well as the evaluation of their antioxidant capacity, their antimicrobial activity on different pathogens, and their capacity to modulate the activity of digestive enzymes. Both chromatographic approaches and FTIR studies indicated that different NADES had various extraction efficiencies towards phenolic compounds, affecting the antioxidant capacity of extracts. The volatile profile of NADES extracts was primarily composed of alcohols, aldehydes, and terpenes, whereas the ethanolic extract (50% (v/v), used as a control) exhibited a higher abundance of terpenes. All extracts were demonstrated to enhance pepsin enzyme activity without affecting that of chymotrypsin. Only choline chloride: glycerol: citric acid gave an extract capable of inhibiting trypsin and amylase activity, as well as the proliferation of pathogenic microorganisms. In conclusion, choline chloride-based NADES may represent a sustainable method for reusing orange byproducts, as they are more effective in extracting valuable bioactive compounds from orange peel when compared to old-fashioned organic solvents.

Wheat is one of the most extensively cultivated cereals, with its pre-harvest quality being critical for subsequent food production. In-field sprouting, triggered by sudden climatic changes, alpha-amylase levels in wheat rise, negatively impacting its quality. This study aims to examine germination progression at the microstructural level. Canadian Western Red Spring (CWRS) wheat was germinated under controlled germination conditions for 6, 12, 18, 24, and 36 h. Alpha-amylase activity, the Falling Number, bulk density, starch crystallinity and microstructural analysis by X-ray micro-computed tomography (micro-CT) and scanning electron microscopy (SEM) were measured. The decline in the Falling Number and the rise in alpha-amylase activity confirmed the progress of germination. Bulk density significantly (p < 0.05) decreased in the germinated kernels, whereas porosity significantly (p < 0.05) increased after 24-h germination. X-ray micro-CT and SEM images showed considerable structural changes at 36 h of germination. Although no significant differences in the cross-sectional area were observed across the kernels as germination progressed, pores were detected in the kernel crease after 36 h. Likewise, the 36-h germinated sample showed a significantly thicker outer layer in the apical region of the kernel, likely due to the detachment of the outer layer. Notable changes in starch and protein structures were observed after 18 h, while starch crystallinity increased throughout germination. Overall, the results indicate that germination-induced alpha-amylase activity significantly impacts the microstructure of wheat kernels, particularly by increasing the outer layer thickness at the apical part of the kernel.

Improving feed efficiency (FE) is essential for the swine industry's economic and environmental sustainability. Genetic selection, particularly through estimating breeding values for feed conversion ratio (EBV_FCR), is a common strategy to enhance FE. However, the biological mechanisms underlying phenotypic variations in FE between pigs with different EBV_FCR values are not fully understood. This study investigates these mechanisms by examining growth performance, nutrient and energy digestibility, and fecal microbiota composition and functionality of pigs at the nursery stage. The study involved 128 pigs, weaned at 21 d (±2 d) and with an initial body weight of 6.87 kg (±0.34 kg). These pigs, selected from dam and sire lines with divergent EBV_FCR values, were randomly assigned to 32 pens with four pigs each. Pigs were fed a corn and soybean meal-based diet, divided into two feeding phases of 2 wk each, under similar rearing conditions. Results indicated no significant differences in average daily feed intake (ADFI), average daily body weight gain (ADG), or feed efficiency (FE, gain:feed) between pigs from different EBV_FCR lines (P > 0.05). Similarly, nutrient digestibility showed no significant variation (P > 0.05). While the overall fecal microbiota taxonomic composition was similar between the groups, there was a trend toward higher beta diversity in the microbiota of pigs from parents with lower EBV_FCR (high efficiency pigs, H pigs) (P < 0.083). Carbohydrate and amino acid metabolism were predominant in all pigs, regardless of genetic background, with similar predicted microbiota functionality across groups. The study concluded that genetic differences based on parents divergent EBV_FCR did not affect growth performance, nutrient utilization, or microbiota characteristics at the nursery stage. This suggests that while EBV_FCR based genetic selection does not impact early-stage performance or microbiome responses, its effects may differ in older pigs, warranting further research.