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%.

Lipid extraction from microbial and microalgae biomass requires the separation of oil-rich cells from the production media. This downstream procedure represents a major bottleneck in biodiesel production, increasing the cost of the final product. Flocculation is a rapid and cheap system for removing solid particles from a suspension. This natural characteristic is displayed by some microorganisms due to the presence of lectin-like proteins (called flocculins/adhesins) in the cell wall. In this work, we showed, for the first time, that the heterologous expression of the adhesin Cfl1p endows the oleaginous species Cutaneotrichosporon oleaginosus with the capacity of cell flocculation. We used Helm’s test to demonstrate that the acquisition of this trait allows for reducing the time required for the separation of lipid-rich cells from liquid culture by centrifugation without altering the productivity. This improves the lipid production process remarkably by providing a more efficient downstream.

OPA1, a dynamin-related GTPase mutated in autosomal dominant optic atrophy, is essential for the fusion of the inner mitochondrial membrane. Although OPA1 deficiency leads to impaired mitochondrial morphology, the role of OPA1 in central carbon metabolism remains unclear. Here, we aim to explore the functional role and metabolic mechanism of OPA1 in cell fitness beyond the control of mitochondrial fusion. We applied [U-¹³C]glucose and [U-¹³C]glutamine isotope tracing techniques to OPA1-knockout (OPA1-KO) mouse embryonic fibroblasts (MEFs) compared to OPA1 wild-type (OPA1-WT) controls. Furthermore, the resulting tracing data were integrated by metabolic flux analysis to understand the underlying metabolic mechanism through which OPA1 deficiency reprograms cellular metabolism. OPA1-deficient MEFs were depleted of intracellular citrate, which was consistent with the decreased oxygen consumption rate in these cells with mitochondrial fission that is not balanced by mitochondrial fusion. Whereas oxidative glucose metabolism was impaired, OPA1-deficient cells activated glutamine-dependent reductive carboxylation and subsequently relied on this reductive metabolism to produce cytosolic citrate as a predominant acetyl-CoA source for de novo fatty acid synthesis. Prevention of cytosolic glutamine reductive carboxylation by GSK321, an inhibitor of isocitrate dehydrogenase 1 (IDH1), largely repressed lipid synthesis and blocked cell proliferation in OPA1-deficient MEFs. Our data support that, when glucose oxidation failed to support lipogenesis and proliferation in cells with unbalanced mitochondrial fission, OPA1 deficiency stimulated metabolic anaplerosis into glutamine-dependent reductive carboxylation in an IDH1-mediated manner.

Several studies have confirmed the reduction of starch digestibility with anthocyanins in food systems via mechanisms of enzyme inhibition. However, starch-polyphenol interactions may also contribute to this reduction, by modifying food microstructures and physicochemical properties of starch. The interactions among anthocyanins, starch digestibility, and food microstructures are significant to clarify the digestion processes of fortified food systems, but its interrelationship lacks clarity. Hence, we aim to evaluate the effects of black rice anthocyanin extract (BRAE) incorporation on the microstructural changes of wheat bread, in relation to overall digestibility. Overall, BRAE incorporation demonstrated a dose-dependent reduction in starch digestibility. Physicochemical analyses reflected that BRAE incorporation decreased starch gelatinisation and increased crystallinity. Microscopic imaging revealed differentiating microstructural characteristics of starch and gluten with BRAE incorporation, supporting the reduction in digestibility. Our results conclusively demonstrate that BRAE incorporation in bread suppresses starch digestibility not only through enzyme inhibition, but also food microstructural modifications.

Auricularia auricula-judae polysaccharide (AP) has unique molecular structures and multiple bioactivities with excellent gel-forming property and thermal tolerance. However, few researches focus on the interactions between AP and legume starches. In this study, the effects of AP on the pasting, gelatinization, rheology, microstructure, and in vitro digestibility of kidney bean starch (KBST) were evaluated. The pasting, gelling and structural properties of AP-KBST mixtures were characterized by rapid visco analyzer, rheometry, texture analyzer, laser particle analyzer, low-field nuclear magnetic resonance, Fourier transform infrared spectroscopy and scanning electron microscopy, respectively. And an in vitro method was employed to measure the digestibility of AP-KBST composites. The pasting viscosity, swelling degree of starch granules, viscoelasticity, gel strength, cold storage stability and water-retention capacity of KBST were enhanced with increasing AP concentration. The combination of AP and KBST exhibited a higher short-range ordered and a firmer and denser structure than that of KBST alone. Moreover, AP increased the contents of resistant starch and slowly digestible starch, which were positively correlated with the storage modulus and the degree of order, thereby suggesting that the formation of strong and ordered gel network structure by synergistic interactions between AP and KBST was responsible for the reduced starch digestibility.

This study aims to determine the effect of dietary inclusion of fenugreek seed gum (FSG), rich in galactomannans, on nutrient apparent digestibility and caecal environment, as well as on in vitro caecal fermentation of Tunisian growing rabbits. Three experimental diets were formulated, including 0, 0.25 and 0.5% of FSG (FSG0, FSG0.25 and FSG0.5, respectively) for the in vivo trial and 0, 0.125, 0.25, 0.5 and 100% of FSG (FSG0, FSG0125, FSG0.25, FSG0.5 and FSG100, respectively) for the in vitro trial. In the in vivo trial, 45 weaned rabbits 31 d old (15 per treatment) were housed in individual cages until 94 d of age. Apparent digestibility coefficients were determined at two ages, from 38 to 41 and from 56 to 59 d old, and caecal traits were recorded after slaughtering. In the in vitro trial, the five experimental diets were incubated with a rabbit caecal inoculum. Gas production was measured and modelled until 72 h and the fermentation traits were measured. Apparent faecal digestibility coefficients of main nutrients and main caecal environment traits were not significantly affected by the dietary inclusion of FSG (P>0.05). However, animals fed with FSG showed lower caecal pH (-0.15; P<0.05) values. Regarding the in vitro fermentation, FSG100 increased asymptotic gas production (+11.25, P<0.001), sharpness of the switching characteristic of the profile (+1.98, P<0.001) and the maximum substrate degradation rate (RM) (+0.188, P<0.001), but decreasing the time after incubation at which half of the asymptotic amount of gas has been formed (-5.86, P<0.001) and at which RM occurs (-4.53, P<0.01). Likewise, FSG100 significantly decreased caecal pH (-1.035, P<0.001), lactic acid (-9.51, P<0.069) and N-NH₃ concentrations (-12.81, P<0.001). Meanwhile, it increased the total volatile fatty acids (VFA) production (+43.15, P<0.001). Gradual dietary inclusion of FSG from 0 to 0.5% only significantly increased total VFA production in the caecum (+100 mmol/L per percentage point of FSG inclusion; P<0.05). In conclusion, FSG is highly and rapidly in vitro fermented by rabbit caecal bacteria. However, dietary inclusion of FSG up to 0.5%, might be insufficient to affect the apparent digestibility and fermentation profile of growing rabbits to a great extent.

O. Warburg conducted one of the first studies on tumor energy metabolism. His early discoveries pointed out that cancer cells display a decreased respiration and an increased glycolysis proportional to the increase in their growth rate, suggesting that they mainly depend on fermentative metabolism for ATP generation. Warburg's results and hypothesis generated controversies that are persistent to this day. It is thus of great importance to understand the mechanisms by which cancer cells can reversibly regulate the two pathways of their energy metabolism as well as the functioning of this metabolism in cell proliferation. Here, we made use of yeast as a model to study the Warburg effect and its eventual function in allowing an increased ATP synthesis to support cell proliferation. The role of oxidative phosphorylation repression in this effect was investigated. We show that yeast is a good model to study the Warburg effect, where all parameters and their modulation in the presence of glucose can be reconstituted. Moreover, we show that in this model, mitochondria are not dysfunctional, but that there are fewer mitochondria respiratory chain units per cell. Identification of the molecular mechanisms involved in this process allowed us to dissociate the parameters involved in the Warburg effect and show that oxidative phosphorylation repression is not mandatory to promote cell growth. Last but not least, we were able to show that neither cellular ATP synthesis flux nor glucose consumption flux controls cellular growth rate.

Levan, a β-2,6-glycosidic linked fructan, is a promising alternative for the inulin dominated fructan market. Although levan is already used in some cosmetic products, the commercial availability of the fructan is still limited. Here we show that Gluconobacter japonicus LMG 1417 is a potent levan-forming organism and a promising platform for the industrial production of levan. The levansucrase LevS1417, which is produced by G. japonicus LMG 1417 and secreted by a signal-peptide-independent pathway, exhibited extraordinary high activity (4726 ± 821 U mg⁻¹ at 50 °C). A cell-free levan production based on the supernatant of the investigated strain led to a final levan yield of 157.9 ± 7.6 g L⁻¹. The amount of secreted levansucrase was more than doubled by plasmid-mediated homologous overproduction of LevS1417 in G. japonicus LMG 1417. Accordingly, the space-time yield of cell-free levan production was doubled using the plasmid-bearing mutant.

A fenugreek seed gum, extracted from Trigonella foenum-graecum seeds and rich in galactomannan, was chemically and physically characterised and its prebiotic potential for young rabbits was evaluated in vitro, both as pure fenugreek seed gum and when included up to 20 g/kg in rabbit diets rich in soluble and insoluble fibre. Fenugreek seed gum was resistant to pepsin and pancreatin digestion but was totally fermented by rabbit caecal bacteria. Fenugreek seed gum linear inclusion up to 20 g/kg in diets rich in soluble fibre has led to a reduction in the solubility of some nutrients during in vitro enzymatic phase and an increase in the fermented fraction. Fenugreek seed gum satisfies two essential conditions of a prebiotic: resistance to enzymatic digestion and being totally fermented by caecal bacteria.

The LacLM-type β-galactosidase from Lactobacillus helveticus DSM 20075 expressed in both Escherichia coli (EcoliBL21Lhβ-gal) and Lactobacillus plantarum (Lp609Lhβ-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by β-galactosidase from L. helveticus, together with three GOS mixtures produced using β-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, L. reuteri (Lr-GOS), L. bulgaricus (Lb-GOS), and Streptococcus thermophilus (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using β-galactosidases (β-gal I and β-gal II) from Bifidobacterium breve, was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional ¹H NMR spectroscopy. β-Galactosidases from lactic acid bacteria and B. breve displayed a preference to form β-(1→6)- and β-(1→3)-linked GOS. The GOS mixtures produced by these enzymes consisted of mainly DP2 and DP3 oligosaccharides, accounting for ~90% of all GOS components. GOS mixtures obtained with β-galactosidases from lactic acid bacteria and B. breve were quite similar to the commercial GOS III mixture in terms of product spectrum and showed a broader product spectrum than the commercial GOS V mixture. These GOS mixtures also contained a number of GOS components that were absent in the commercial Vivinal GOS (V-GOS).

Octenyl succinic anhydride modified waxy maize starch after heat-moisture treatment (HT-OSAS) is a heat-stable slowly digestible starch (SDS), but the mechanism is poorly understood. Tea polyphenols (TP), as a probe with hydrophobic moiety, were employed to study the structural basis for its slow digestion. The content of RS (8.91%) in the HT-OSAS was significantly increased by TP in a dose-dependent manner (19.06% at TP of 15%) while there is a slight decrease of SDS content (34.85-30.63%). However, after retrogradation for one week, TP significantly reduced the SDS content (29.79%-15.67%). Additionally, TP increased the hydrodynamic radius of HT-OSAS (60-102 nm), but reduced its emulsifying activity (-35%), gelatinization enthalpy (-45%), and viscosity (-66%), demonstrating an inhibitory effect of TP on the association of HT-OSAS molecules, and a compact physical structure of HT-OSAS formed through OSA-mediated hydrophobic interaction is likely the basis to its slow digestion property. Novel strategies to densify the physical structure of starch could improve the nutritional quality of starch with a slow digestion property.

An aerobic denitrifier was newly isolated and identified by VITEK^® 2 Compact System and MALDI-TOF MS as P. stutzeri strain D1. Sequence amplification indicates that the denitrification genes napA, nirS, norB and nosZ are present in a novel strain D1, as well as in reference strain ATCC 17588. Strain D1 had capability to fully remove 3 g/L of nitrate (as KNO3) in 48 h, while the reference strain completed this task in 60 h. Single factor experiments indicate that the optimal conditions for biomass production were: temperature of 30°C, pH value of 7 and inoculum volume of 5 vol.%. Scaling up of biomass production of both denitrifiers was successfully performed in 3 and 7 L laboratory bioreactors by reaching 9 log CFU/mL of the viable cells. The results demonstrate the feasibility of using investigated P. stutzeri strains in denitrification processes and the simplicity of the up-scaling of biomass production for the treatment of large areas contaminated with nitrate.

The digestibility of starch in cooked ordinary rice grains and cooked texturized rice grains was compared using an in vitro gastrointestinal tract (GIT), which involved sequentially exposing samples to simulated oral, gastric, and small intestinal regions. The total extent of starch hydrolysis (%) of texturized rice was slightly lower than that of ordinary rice by the end of the GIT, probably because of its higher resistant starch content. Furthermore, the rate of starch hydrolysis in the small intestine was much slower for texturized rice than ordinary rice. Gel electrophoresis and microstructure analysis suggested that the proteins in the texturized rice formed a cross-linked network around the starch molecules. This network may have retarded the ability of digestive enzymes to access the starch, thereby reducing the hydrolysis rate. These results have important implications for the design of starch-based foods with a lower glycemic index, and therefore improved nutritional profiles.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

β-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ~2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant β-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The K_m and v_max values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol O-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of ONPG with K_i,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and ONPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55°C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of β-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.