D-Glucose HK Assay Kit

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.

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.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

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.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

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 inclusion complexes are gaining interest due to their enhanced functional properties and potential health benefits. Both freeze-thawing and ultrasound are recognized as effective physical methods to induce the formation of V-type starch inclusion complexes. However, the combined effects of these treatments on starch complex formation have not been fully elucidated. This study aims to investigate the synergistic impact of ultrasound and freeze-thaw treatments on the production of corn starch-glycerin monostearin complexes (GMS), examining their structural and digestive properties. The findings, supported by X-ray diffraction (XRD) and complexing index analyses, indicate a synergistic effect from the combined treatment in creating V-type starch–lipid inclusion complexes. Scanning electron microscopy (SEM) shows that the application of freeze-thaw cycles and ultrasound leads to the release of amylose molecules, enhancing their interaction with lipid molecules. The starch-lipid complexes formed through this process exhibit a highly ordered structure and increased resistance to enzymatic digestion in an in vitro model. These results offer valuable insights into the development of starch-based foods with slower digestibility, which could have implications for dietary management and the functional food industry.

Dissolved organic matter (DOM) plays a central role in soil carbon (C) dynamics, serving as both a substrate for microbial decomposers and a source of material stabilised via physical protection in molecular aggregates and associations with mineral particles. It is well established that soil microorganisms play a key role in mineral-associated C aggregates; however, their impacts on molecular aggregates is not clearly understood. Here, we examined the ability of an ectomycorrhizal fungus (Paxillus involutus) and a saprotrophic fungus (a strain of Gloeophyllum), two major functional groups of fungal decomposers in forest ecosystems, to decompose and process the molecular and colloidal size fractions of DOM. DOM was extracted by water from boreal forest soil, and the chemical composition and colloidal properties were followed over 11 days using nuclear magnetic resonance (NMR) spectroscopy and small-angle light and X-ray scattering techniques. Both fungi decompose various organic compounds into their molecular fractions in the presence of an energy source (i.e. glucose). The decomposition rate was significantly higher for Gloeophyllum than for P. involutus. When glucose was depleted, Gloeophyllum continued to decompose more complex carbohydrates, whereas the decomposition activity of P. involutus almost stopped. A large proportion of the C in the DOM was found in organic colloids. At later stages, Gloeophyllum but not P. involutus, significantly affected the colloids by promoting the formation of larger aggregates. Thus, saprotrophic fungi activity can significantly influence the colloidal properties of DOM. Our results support the view that ectomycorrhizal fungi decompose some of the soil organic C however, their overall capacity for DOM decomposition and transformation is significantly lower than that of saprotrophic fungi.

Ubiquinol or coenzyme Q (CoQ) is a lipid-soluble electron carrier in the respiratory chain and an electron acceptor for various enzymes in metabolic pathways that intersect at this cofactor hub in the mitochondrial inner membrane. The reduced form of CoQ is an antioxidant, which protects against lipid peroxidation. In this study, we have optimized a UV-detected HPLC method for CoQ analysis from biological materials, which involves a rapid single-step extraction into n-propanol followed by direct sample injection onto a column. Using this method, we have measured the oxidized, reduced, and total CoQ pools and monitored shifts in the CoQ redox status in response to cell culture conditions and bioenergetic perturbations. We find that hypoxia or sulfide exposure induces a reductive shift in the intracellular CoQ pool. The effect of hypoxia is, however, rapidly reversed by exposure to ambient air. Interventions at different loci in the electron transport chain can induce sizeable redox shifts in the oxidative or reductive direction, depending on whether they are up- or downstream of complex III. We have also used this method to confirm that CoQ levels are higher and more reduced in murine heart versus brain. In summary, the availability of a convenient HPLC-based method described herein will facilitate studies on CoQ redox dynamics in response to environmental, nutritional, and endogenous alterations.

Consumer demand for healthy snack products has led to increased efforts to modify glycemic responses of these products. The formation of starch-ferulic acid (FA) complexes through acidified water steeping leads to an increase in RS content and reduction of glycemic responses in animal models. However, the application of these methodologies to commercially relevant phenolic-rich flours (black rice, BR; purple maize, PM) and their impacts on starch digestibility and phenolic bioaccessbility remain unclear. Using an acidified water steeping method previously reported in pure starch, potato starch (PS), BR and PM flour slurries were prepared with FA at a 20:1 and 8:1 ratio in HCl-acidified water (pH 2). The steeping process reduced phenolic content (~75%) and increased starch digestibility. Ferulic acid loading capacity on PS (22–124 mg/g starch) was greater than for PM and BR flours (8–40 mg/g starch). Ferulic acid bioaccessibility decreased with an increase in its amount in PS and flours. Steeping enhances cyanidin 3-O-glucoside bioaccessibility, while addition of FA decreases its bioaccessibility. The increase in starch digestibility of phenolic-rich flours after steeping with and without FA can be attributed to the limited loading capacity of FA and loss of native phenolics during the steeping process.

Limited attention is given to the efficacy of protocols for the estimation of infant intake of milk components when investigating their impact on infant outcomes. We compared the actual measured intake of human milk components with estimations derived from 15 protocols to determine the most reliable approach for estimating intake of HM leptin, adiponectin, insulin, glucose, and total lipid. Twenty mothers who were 3-5 months postpartum completed a 24 h milk profile study with pre-/post-feed milk samples collection. The true infant intake (control group) based on 24 h milk intake (MI) was compared to estimated infant intakes using concentrations from five sampling protocols that were multiplied by one of true infant MI, considered mean MI (800 mL), or global mean MI (766 mL). The mean measured concentrations of six samples (three sets of pre- and post-feed samples, from morning (06:00-09:00), afternoon (13:00-16:00), and evening (19:00-22:00)) multiplied by the true infant MI, mean considered MI, and global mean MI produced the most accurate estimates of infant intake of these components. Therefore, in the absence of 24 h measurements and sampling, a sampling protocol comprising three sets of pre-/post-feed samples provides the most reliable infant intake estimates of HM leptin, adiponectin, insulin, glucose, and total lipid.

Anthocyanin (ACN) fortification of commonly consumed foods is significant as a dietary strategy against the development of metabolic complications by delivering ACNs at high doses. However, its bioactivity and translated metabolic effects in the presence of varying food matrices and macro-constituents is particularly unclear. This end-to-end study investigates the metabolic effects of black rice ACN extract (BRAE) fortification—from in-vitro enzyme inhibitory activities and digestibility, to downstream in vivo impacts on GI, postprandial glycemia and lipidemia. The in vivo effects were investigated in two separate crossover randomised controlled trials (RCT) of 24 healthy participants each-the first RCT determined the postprandial blood glucose, insulin, and ACN bioavailability to a starch-rich single food over 2 h, while the second RCT determined the postprandial blood glucose, insulin, lipid panel, and lipoprotein particles and subfractions to a starch- and fat-rich composite meal over 4 h. In-vitro findings confirmed the inhibitory activities of major black rice ACNs on carbohydrases (p = 0.0004), lipases (p = 0.0002), and starch digestibility (p < 0.0001). in vivo, a 27-point mean GI reduction of wheat bread was observed with BRAE fortification, despite a non-significant attenuation in postprandial glycemia. Conversely, there were no differences in postprandial glycemia when fortified bread was consumed as a composite meal, but acute lipid profiles were altered: (1) improved plasma HDL-c, ([0.0140 mmol/L, 95% CI: (0.00639, 0.0216)], p = 0.0028), Apo-A1 ([0.0296 mmol/L, 95% CI: (0.00757, 0.0515)], p = 0.0203), and Apo-B ([0.00880 mmol/L, 95% CI: (0.00243, 0.0152)], p = 0.0185), (2) modified LDL and HDL subfractions (p < 0.05), and (3) remodelled lipid distributions in HDL and LDL particles. This end-to-end study indicates the potential of ACN fortification in GI reduction and modulating postprandial lipoprotein profiles to starch- and fat-rich composite meals.

There is an inadequate understanding of the daily variations in hormones and macronutrients in human milk (HM), and sample collection protocols vary considerably from study to study. To investigate changes in these milk components across 24 h, 22 lactating women collected small milk samples before and after each breastfeed or expression from each breast. Test weighing was used to determine the volume of HM consumed in each feed. The concentrations of leptin, adiponectin, insulin, fat, and glucose were measured, and the intakes were calculated. A linear mixed model was fitted to assess within-feed and circadian variation in HM feed volume and concentration, and intakes of several components. The average infant intake of HM was 879 g/24 h. Significantly higher pre-feed concentrations were found for adiponectin and glucose and lower post-feed concentrations were found for insulin and fat. Significant circadian rhythms were displayed for leptin, adiponectin, insulin, glucose (both concentration and intake), fat concentration, and milk volume. These findings demonstrate the necessity for setting up standardised and rigorous sampling procedures that consider both within-feed and circadian variations in HM components to gain a more precise understanding of the impacts of these components on infant health, growth and development.

The impact of the solubility of phenolic compounds (PCs) from highland barley (HB) on their antioxidant property and protein binding affinity was investigated. HB-PCs from the phenolic extract (HBE) were analyzed through the UPLC-QTOF-MS, and five fractions were further divided based on their water solubility. Representative compounds of proanthocyanidins, (+)-catechins, and (−)-epicatechin from water fraction (HB–W, 197.67 ± 4.8 μmol GAE/100 g) were significantly correlated with the antioxidant activity of the HBE. In contrast, the PCs enriched in the acetone fraction (HB-A, 60.2 ± 3.1 μmol GAE/100 g) with a structure of 2-phenyl chromogen ketone group (such as rutin, kaempferol, hesperidin, and quercetin) were positively correlated with the protein binding affinity. Further analysis showed that the water-insoluble HB-A fraction significantly lowered the starch digestibility (37.5%) and postprandial glycemia (17.8%) compared to HB-W fractions. Thus, the solubility of PCs is intimately associated with their biological functions as antioxidants or protein-binding ligands, indicating PCs with strong antioxidant properties or high protein binding affinity are structurally distinct from each other, and the physical properties of phenolic compounds might be an important factor to further the understanding of their health functions and mechanisms.

We previously isolated a mutant of Saccharomyces cerevisiae strain 85_9 whose glycerol assimilation was improved through adaptive laboratory evolution. To investigate the mechanism for this improved glycerol assimilation, genome resequencing of the 85_9 strain was performed, and the mutations in the open reading frame of HOG1, SIR3, SSB2, and KGD2 genes were found. Among these, a frameshift mutation in the HOG1 open reading frame was responsible for the improved glycerol assimilation ability of the 85_9 strain. Moreover, the HOG1 gene disruption improved glycerol assimilation. As HOG1 encodes a mitogen-activated protein kinase (MAPK), which is responsible for the signal transduction cascade in response to osmotic stress, namely the high osmolarity glycerol (HOG) pathway, we investigated the effect of the disruption of PBS2 gene encoding MAPK kinase for Hog1 MAPK on glycerol assimilation, revealing that PBS2 disruption can increase glycerol assimilation. These results indicate that loss of function of Hog1 improves glycerol assimilation in S. cerevisiae. However, single disruption of the SSK2, SSK22 and STE11 genes encoding protein kinases responsible for Pbs2 phosphorylation in the HOG pathway did not increase glycerol assimilation, while their triple disruption partially improved glycerol assimilation in S. cerevisiae. In addition, the HOG1 frameshift mutation did not improve glycerol assimilation in the STL1-overexpressing RIM15 disruptant strain, which was previously constructed with high glycerol assimilation ability. Furthermore, the effectiveness of the HOG1 disruptant as a bioproduction host was validated, indicating that the HOG1 CYB2 double disruptant can produce L-lactic acid from glycerol.