α-Amylase (Bacillus licheniformis)

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.

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%.

Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.

An improved enzymic method for the determination of starch damage in wheat flour has been developed and characterized. The proposed method is simple and reliable, and enables up to 20 samples to be measured in duplicate in 2 h. A single assay takes approximately 40 min. The assay protocol is in two phases. In the first, the flour sample is incubated with purified fungal alpha-amylase to liberate damaged starch granules as soluble oligosaccharides. After centrifugation, the oligosaccharides in the supernatant are hydrolysed by amyloglucosidase to glucose in phase 2. The glucose is then quantified with a glucose oxidase/peroxidase reagent. The proposed method therefore avoids potential errors associated with existing standard assays, which employ unpurified amylase preparations and non-specific reducing group methods to quantify the hydrolytic products. Despite the use of purified assay components, the proposed starch damage method did not exhibit an absolute end-point to the action of alpha-amylase in phase 1. This was due to a low rate of hydrolysis of undamaged granules, and is a feature of enzymic methods for starch damage determination. Other amylolytic enzymes, including beta-amylase, isoamylase and pullulanase, and combinations of these enzymes, were evaluated as alternatives to alpha-amylase in the proposed method. These enzymes, when used alone, gave no benefits over the use of alpha-amylase. When used in combination with alpha-amylase, there was a synergistic action on undamaged granules. A test kit based on the assay format described in this paper is the subject of an international interlaboratory evaluation.

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.

High pressure processing (HPP) is a starch modification method generally conducted in water, and little is known about the pressure-induced changes in different media. This study investigated the effects of water versus sodium sulfate on corn, potato, and pea starches subjected to HPP at 690 MPa. HPP in both media reduced gelatinization enthalpy and crystallinity for all starches. HPP in sodium sulfate promoted the transition of common corn and potato starches to C-type crystallites. HPP starches in sodium sulfate generally displayed lower pasting temperatures, higher peak viscosities, and greater breakdowns than in water. Alpha-amylase susceptibility increased for all HPP starches and was generally lower in sodium sulfate than in water. HPP common corn and potato starchs in sodium sulfate displayed a porous structure after α-amylase digestion. The competition of sodium sulfate for water molecules between starch helices induced variations in the properties of HPP starches with different crystalline structures.

The orderly deposition of secondary cell wall (SCW) in plants is implicated in various biological programs and is precisely controlled. Although many positive and negative regulators of SCW have been documented, the molecular mechanisms underlying SCW formation coordinated with distinct cellular physiological processes during plant adaptive growth remain largely unclear. Here, we report the identification of Cellulose Synthase co-expressed Kinase1 (CSK1), which encodes a receptor-like cytoplasmic kinase, as a negative regulator of SCW formation and its signaling cascade in rice. Transcriptome deep sequencing of developing internodes and genome-wide co-expression assays revealed that CSK1 is co-expressed with cellulose synthase genes and is responsive to various stress stimuli. The increased SCW thickness and vigorous vessel transport in csk1 indicate that CSK1 functions as a negative regulator of SCW biosynthesis. Through observation of green fluorescent protein-tagged CSK1 in rice protoplasts and stable transgenic plants, we found that CSK1 is localized in the nucleus and cytoplasm adjacent to the plasma membrane. Biochemical and molecular assays demonstrated that CSK1 phosphorylates VASCULAR-RELATED NAC-DOMAIN 6 (VND6), a master SCW-associated transcription factor, in the nucleus, which reduces the transcription of a suite of SCW-related genes, thereby attenuating SCW accumulation. Consistently, genetic analyses show that CSK1 functions upstream of VND6 in regulating SCW formation. Interestingly, our physiological analyses revealed that CSK1 and VND6 are involved in abscisic acid-mediated regulation of cell growth and SCW deposition. Taken together, these results indicate that the CSK1–VND6 module is an important component of the SCW biosynthesis machinery, which coordinates SCW accumulation and adaptive growth in rice. Our study not only identifies a new regulator of SCW biosynthesis but also reveals a fine-tuned mechanism for precise control of SCW deposition, offering tools for rationally tailoring agronomic traits.

Despite the increased interest in macroalgae protein and fibers, little information is available on their bioaccessibility. The application of an in vitro gastrointestinal digestion model to study the degree of disintegration and release of proteins with expressed bioactivities from wild and cultivated Palmaria palmata and Saccharina latissima was proposed in this study. Macroalgae from the Gulf of St Lawrence, Canada, were submitted to digestive transit times of 2 (oral), 60 (gastric) and 120 (duodenal) minutes. Among wild samples, P. palmata had a higher percentage of disintegration, protein release and degree of hydrolysis than S. latissima. While the least digested sample, wild S. latissima, was the sample with the highest antioxidant activity (210 μmol TE g⁻¹), the most digested sample, cultivated P. palmata, presented the highest ability to inhibit the angiotensin-converting enzyme (ACE), reaching 32.6 ± 1.2% at 3 mg mL⁻¹. ACE inhibitory activity increased from 1 to 3 mg mL⁻¹, but not at 5 mg mL⁻¹. Wild samples from both species showed an ACE inhibition around 27.5%. Data suggested that the disintegration of the samples was influenced by their soluble and insoluble fiber contents. Further information on the bioaccessibility and bioactivity of these macroalgae should consider the characterization of digestion products other than protein, as well as the effects of previous product processing.

A high-throughput method for quantification of water extractable arabinoxylan (WE-AX) and water unextractable arabinoxylan (WU-AX) was adapted for and evaluated on 197 commercial Swedish wheat flours, collected continuously during harvest years 2018 and 2019. In the method, starch was hydrolysed by alpha-amylase and WE-AX was precipitated with 80% ethanol. AX residues were quantified by gas chromatography after acid hydrolysis. The method had a good repeatability (2.1% RSD_r for total AX). Spring wheat flour had a higher WE-AX content (0.68%) and lower WU-AX content (1.19%) than winter wheat flour (0.56% and 1.31%). The variation of total AX content was high for winter wheat flour (1.5-2.2%), with no correlation to ash or protein content. Total AX content differed significantly both between harvest years and locations, indicating an impact from environment on AX composition. Overall, the method enabled high-throughput analysis of wheat flour and can be further used to study how endogenous AX impacts baking quality.

Background and Objective: Nitrogen fertilizer applied as top-dressing to rice plants before flowering affects the yield and quality of rice grains. However, little is known about the effects of varying dosages of nitrogen at this stage on the brewing properties of sake using the rice grains. To explore this topic, we cultivated the sake rice cultivar “Koshitanrei” with low and high levels of nitrogen (equivalent to 10 kg and 30 kg N per hectare, respectively) applied 1 week before flowering and then conducted a small-scale sake brewing test using the harvested rice. Findings: Sake made from rice from the low-N treatment was fermented more efficiently. This yielded a higher volume of sake with a higher alcohol concentration and decreased amino acid concentrations. Conclusions: The use of rice from the low-N treatment enhanced alcohol fermentation, resulting in increased alcohol yield and decreased concentrations of amino acids and related compounds.

Savannahs dominated by grasses with scattered C₃ trees expanded between 24 and 9 million years ago in low latitudes at the expense of forests. Fire, herbivory, drought and the susceptibility of trees to declining atmospheric CO₂ concentrations ([CO₂]_a) are proposed as key drivers of this transition. The role of disturbance is well studied, but physiological arguments are mostly derived from models and palaeorecords, without direct experimental evidence. In replicated comparative experimental trials, we examined the physiological effects of [CO₂]_a and prolonged drought in a broadleaf forest tree, a savannah tree and a savannah C₄ grass. We show that the forest tree was more disadvantaged than either the savannah tree or the C₄ grass by the low [CO₂]_a and increasing aridity. Our experiments provide insights into the role of the intrinsic physiological susceptibility of trees in priming the disturbance-driven transition from forest to savannah in the conditions of the early Miocene.

The high cost of protein feeds and growing concern for the environment have motivated dairy producers and nutritionists to focus their attention on reducing nitrogen (N) losses on dairy farms. It is well recognized that reducing the N content of cattle diets is the single most important factor to increase the efficiency of N use. However, effectively lowering the N content of diets requires the nutritionist to know the availability of N in feeds so as to not negatively affect milk production or overfeed N. To provide reliable data for nutritionists, a new assay to estimate unavailable N in the intestine (uN) was developed. To determine whether uN could be used as a replacement for acid detergent insoluble nitrogen (ADIN) in diet formulation, we conducted a replicated pen study to evaluate the effect of total-tract uN on the performance of high-producing dairy cattle. One hundred twenty-eight cattle that were 97 to 147 d in milk at the beginning of the experiment were allocated into 8 pens of 16 cows, and pens were randomly allocated to 2 dietary treatments. Cattle were fed 1 of 2 isonitrogenous and isocaloric diets that were also equal in neutral detergent fiber, deviating only in the inclusion of 2 different blood meals (BM) used in each diet. The uN contents of the 2 BM were 9% (low uN) and 34% (high uN) total N content as predicted by the assay, whereas when measured as ADIN, no difference in indigestibility was observed. The inclusion of BM was on an isonitrogenous basis, and the predicted difference in uN was 39 g/d or 5.8% of N intake, representing the formulated difference in available N between the 2 treatments. There was no effect of uN on dry matter or N intake, which averaged 27.3 kg/d and 668 g/d for both treatments, respectively. Milk yield and energy-corrected milk were 1.6 and 1.9 kg/d greater for cows fed the low uN diet compared with those fed the high uN diet. The lower uN diet was also associated with greater milk protein yield, greater milk fat yield, and greater milk urea N. The Cornell Net Carbohydrate and Protein System (version 6.5) was used to evaluate the application of the uN measurement by replacing ADIN in BM with the uN value in the inputs for the BM. All other cow and feed chemistry data were inputted as measured in the experiment. The predictions of metabolizable protein-allowable milk demonstrated that using the uN values in place of ADIN increased the accuracy of the prediction and enabled the model to predict the first-limiting nutrient provided all other feed, cattle, and management characteristics were also defined.

The purpose of this study was to investigate the effects of different polyphenol-rich herbal teas on reducing the in vitro starch digestibility of white bread and evaluation of predicted glycemic indexes. Generally, except for the goji berry treatment, all herbal teas reduced the starch digestibility and predicted glycemic index of white bread. Compared to untreated white bread, the rapidly digestible starch levels were decreased by 10% and 12% in the turmeric tea treatment. In addition, hydrolysis indexes were decreased by 12% and 10% in the black tea treatment compared to untreated white bread. The turmeric treatment on white bread reduced the predicted glycemic index more than other teas. It is thought that the curcumin in turmeric has more inhibitory effects on α-amylase activity than other teas. We also demonstrated that dietary polyphenols such as anthocyanins and catechins found in herbal teas might reduce starch digestion by inhibiting α-amylase and α-glucosidase thereby lowering the glycemic index of foods.

Duckweeds are the smallest flowering plants on Earth. They grow fast on water’s surface and produce large amounts of biomass. Further, duckweeds display high adaptability, and species are found around the globe growing under different environmental conditions. In this work, we report the composition of 21 ecotypes of fourteen species of duckweeds belonging to the two subfamilies of the group (Lemnoideae and Wolffioideae). It is reported the presence of starch and the composition of soluble sugars, cell walls, amino acids, phenolics, and tannins. These data were combined with literature data recovered from 85 publications to produce a compiled analysis that affords the examination of duckweeds as possible food sources for human consumption. We compare duckweeds compositions with some of the most common food sources and conclude that duckweed, which is already in use as food in Asia, can be an interesting food source anywhere in the world.

The production of marine foods is on the rise, and shrimp is one of the most widely consumed. As a result, a considerable amount of shrimp waste is generated, becoming a hazardous problem. Shrimp waste is a rich source of added-value components such as proteins, lipids, chitin, minerals, and carotenoids; however, new bioprocesses are needed to obtain these components. This work aimed to characterize the chemical and nutraceutical constituents from the liquor of shrimp waste recovered during a lactic acid fermentation process using the novel substrate sources whey and molasses. Our results showed that the lyophilized liquor is a rich source of proteins (25.40 ± 0.67%), carbohydrates (38.92 ± 0.19%), minerals (calcium and potassium), saturated fatty acids (palmitic, stearic, myristic and lauric acids), unsaturated fatty acids (oleic acid, linoleic, and palmitoleic acids), and astaxanthin (0.50 ± 0.02 µg astaxanthin/g). Moreover, fermentation is a bioprocess that allowed us to obtain antioxidants such as carotenoids with an antioxidant capacity of 154.43 ± 4.73 µM Trolox equivalent/g evaluated by the ABTS method. Our study showed that liquor from shrimp waste fermentation could be a source of nutraceutical constituents with pharmaceutical applications. However, further studies are needed to separate these added-value components from the liquor matrix.

Diet-related noncommunicable diseases impose a heavy burden on human health worldwide. Rice is a good target for diet-related disease prevention strategies because it is widely consumed. Liu et al. demonstrated that increasing the number of cell layers and thickness of putative aleurone in ta2-1 (thick aleurone 2-1) mutant rice enhances simultaneously the content of multiple micronutrients. However, the increases of aleurone-associated nutrients were not proportional to the increases in the aleurone thickness. In this study, first, cytohistological analyses and transmission electron microscopy demonstrated that the multilayer in ta2-1 exhibited aleurone cell structural features. Second, we detected an increase in insoluble fibre and insoluble bound-phenolic compounds, a shift in aleurone-specific neutral non-starch polysaccharide profile, enhancement of phytate and minerals such as iron, zinc, potassium, magnesium, sulphur, and manganese, enrichment of triacylglycerol and phosphatidylcholine but slight reduction in free fatty acid, and an increase in oleic fatty acid composition. These findings support our hypothesis that the expanded aleurone-like layers in ta2-1 maintained some of the distinctive aleurone features and composition. We provide perspectives to achieve even greater filling of this expanded micronutrient sink to provide a means for multiple micronutrient enhancements in rice.

A collection of tropical medicinal plants from East Malaysia's rainforests are used by indigenous tribes for their curative properties. Despite their purported healing properties, these forest plant species are largely unexplored and hence remain virtually unknown to the outside world. In this study, antidiabetic properties of Psychotria viridiflora, a plant used to treat diabetes by a local community in Sarawak, Malaysia were investigated. Ethyl acetate (EA) extract of P. viridiflora stem was found to exhibit high starch hydrolase inhibition activity with an IC₅₀ value of 15.4 ± 2.1 μg/ml against porcine α-amylase and an IC₅₀ value of 32.4 ± 3.7 μg/ml against rat intestinal α-glucosidase. A complex mixture of A-type oligomeric proanthocyanidins containing (epi)fisetinidol, (epi)afzelechin, (epi)guibourtinidol, and (epi)catechin were found. These compounds may be responsible for the starch hydrolase inhibition activity. Ethyl acetate (EA) extract of P. viridiflora stem was incorporated into wheat and rice flour to reformulate noodles with slow digestibility and was assessed under in vitro simulated gastrointestinal conditions. A dose-dependent effect on digestibility was observed for both noodles upon incorporation of 1-6% (w/w) of EA extract, with noodles containing 6% (w/w) extract exhibiting the greatest reduction in digestibility. As compared to rice noodles containing 6% extract (31.16% inhibition), wheat noodles with the same extract concentration had a smaller decline in digestibility (27.25% inhibition) after 180 min. Overall, our findings highlight the potential of P. viridiflora in the prevention of postprandial hyperglycaemia.

Cellulosic ethanol is an alternative for increasing the amount of bioethanol production in the world. In Brazil, sugarcane leads the bioethanol production, and to improve its yield, besides bagasse, sugarcane straw is a possible feedstock. However, the process that leads to cell wall disassembly under field conditions is unknown, and understanding how this happens can improve sugarcane biorefinery and soil quality. In the present work, we aimed at studying how sugarcane straw is degraded in the field after 3, 6, 9, and 12 months. Non-structural and structural carbohydrates, lignin content, ash, and cellulose crystallinity were analyzed. The cell wall composition was determined by cell wall fractionation and determination of monosaccharide composition. Non-structural carbohydrates degraded quickly during the first 3 months in the field. Pectins and lignin remained in the plant waste for up to 12 months, while the hemicelluloses and cellulose decreased 7.4 and 12.4%, respectively. Changes in monosaccharide compositions indicated solubilization of arabinoxylan (xylose and arabinose) and β-glucans (β-1,3 1,4 glucan; after 3 months) followed by degradation of cellulose (after 6 months). Despite cellulose reduction, the xylose:glucose ratio increased, suggesting that glucose is consumed faster than xylose. The degradation and solubilization of the cell wall polysaccharides concomitantly increased the level of compounds related to recalcitrance, which led to a reduction in saccharification and an increase in minerals and ash contents. Cellulose crystallinity changed little, with evidence of silica at the latter stages, indicating mineralization of the material. Our data suggest that for better soil mineralization, sugarcane straw must stay in the field for over 1 year. Alternatively, for bioenergy purposes, straw should be used in less than 3 months.