Sucrose/D-Glucose Assay Kit

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.

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.

Background: Artificial microbial consortia composed of heterotrophic and photoautotrophic organisms represent a unique strategy for converting light energy and carbon dioxide into high-value bioproducts. Currently, the types of desired bioproducts are still limited, and microbial fitness benefit rendered by paired partner generally needs to be intensified. Exploring novel artificial microbial consortia at a laboratory scale is an essential step towards addressing this unmet need. This study aimed to conduct and analyze an artificial consortium composed of cyanobacterium Synechococcus elongatus FL130 with the filamentous fungus Aspergillus nidulans TWY1.1 for producing fungi-derived secondary metabolite of polyketide neosartoricin B. Results: Polyketide-producing A. nidulans TWY1.1 substantially ameliorated the growth and the survival of sucrose-secreting cyanobacterium S. elongatus FL130 in salt-stressed environments. Besides sucrose, comparable amounts of other carbohydrates were released from axenically cultured FL130 cells, which could be efficiently consumed by TWY1.1. Relative to axenically cultured FL130, less glycogen was accumulated in FL130 cells co-cultured with TWY1.1, and the glycogen phosphorylase gene catalyzing the first step for glycogen degradation had two-fold expression. Different from axenically cultured filamentous fungi, abundant vacuoles were observed in fungal hyphae of TWY1.1 co-cultured with cyanobacterium FL130. Meanwhile, FL130 cells displayed a characteristic pattern of interacting with its heterotrophic partner, densely dispersing along certain hyphae of TWY1.1. Finally, polyketide neosartoricin B was produced from TWY1.1 in FL130-TWY1.1 co-cultures, which was tightly adjusted by nitrogen level. Conclusion: Overall, the results thoroughly proved the concept of pairing cyanobacteria with filamentous fungi to build artificial consortia for producing fungi-derived biomolecules.

In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5’/3’ gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.

Importance of the work: Phitsanulok 2 (PSL2) is a high-yielding rice commonly cultivated in lower northern Thailand. However, it is susceptible to bacterial blight (BB) disease caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo), resulting in major grain yield losses. Objectives: To investigate the BB resistance levels and the effect of nitrogen levels on the sucrose contents in BC₄F₅ lines carrying the Xa21 gene. Materials & Methods: The Xa21 gene was introduced into PSL2 using marker-assisted backcrossing. The five BC₄F₅ lines carrying Xa21 and parental lines were grown with different nitrogen levels, followed by inoculation with XooE. Results: All five BC4F5 lines had the highest resistance to the BB disease at 75 kg N/ha. The lesion lengths of plants treated with 75 kg N/ha, 150 kg N/ha and 225 kg N/ha under greenhouse conditions were in the ranges 2.8–3.9 cm, 5.65–6.22 cm and 7.67–8.25 cm, respectively. In the field experiment, diseased leaf areas were in the range 7.63–8.34% in the BC4F5 lines, which were not significantly different to IRBB21. The sucrose content in the rice leaves increased when a higher rate of nitrogen fertilizer was applied. All BC₄F₅ lines had similar plant heights to PSL2. Three BC₄F₅ lines had greater grain yields per plant than PSL2. Main finding: All BC₄F₅ lines had greater yields and showed similar resistance to BB as IRBB21 under field trails at 75 kg N/ha. This study revealed that an overdose of nitrogen fertilizer enhanced the sucrose content in rice leaves and induced severe BB disease.

Chicory (Cichorium intybus var. foliosum) roots are an agricultural residue and a low cost feedstock for the production of the platform chemical 5-Hxdroxymethylfurfural (HMF). In a first step, inulin and fructose have to be extracted from the roots. The resulting aqueous extract represents the starting material for the HMF production. In the reaction to HMF, inulin has to be hydrolyzed first to fructose. For this reason, two methods to increase the fructose content in these extracts before the reaction were investigated. This was conducted within the framework of integrating acid hydrolysis into a biorefinery process for HMF production. The first method (one-step process) was acid-assisted extraction to directly hydrolyze inulin in the course of the extraction process. Chicory roots were extracted at 60 and 80°C at pH 2 and 4 using buffer solutions. The second approach (two-step process) was aqueous extraction at neutral pH followed by nitric acid hydrolysis of the extract at 60 and 80°C under reduced pH. It was found that in the first approach, the pH of 2 led to a fivefold increase in the fructose content of the extract, resulting from inulin hydrolysis and corresponding to 56% of theoretical fructose yield. For the second approach, it was possible to achieve complete hydrolysis at pH below 2.5 and at 80°C. Separating extraction and hydrolysis was found to be more suitable in terms of including this process step into a biorefinery concept for HMF production. It was possible to reduce the initial inulin content by 95%.

Sugar content is one of the most important properties of potato tubers as it directly affects their processing and the final product quality, especially for fried products. In this study, data obtained from spectroscopic (interactance and reflectance) and hyperspectral imaging systems were used individually or fused to develop non-cultivar nor growing season-specific regression and classification models for potato tubers based on glucose and sucrose concentration. Data was acquired over three growing seasons for two potato cultivars. The most influential wavelengths were selected from the imaging systems using interval partial least squares for regression and sequential forward selection for classification. Hyperspectral imaging showed the highest regression performance for glucose with a correlation coefficient (ratio of performance to deviation) or r(RPD) of 91.8(2.41) which increased to 94%(2.91) when the data was fused with the interactance data. The sucrose regression results had the highest accuracy using data obtained from the interactance system with r(RPD) values of 74.5%(1.40) that increased to 84.4%(1.82) when the data was fused with the reflectance data. Classification was performed to identify tubers with either high or low sugar content. Classification performance showed accuracy values as high as 95% for glucose and 80.1% for sucrose using hyperspectral imaging, with no noticeable improvement when data was fused from the other spectroscopic systems. When testing the robustness of the developed models over different seasons, it was found that the regression models had r(RPD) values of 55(1.19)-90.3%(2.34) for glucose and 35.8(1.07)-82.2%(1.29) for sucrose. Results obtained in this study demonstrate the feasibility of developing a rapid monitoring system using multispectral imaging and data fusion methods for online evaluation of potato sugar content.

Background: Oxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO₂-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3-an enzyme that is natively associated with transfer of electrons from PSI to O₂, as part of an acclimation strategy towards varying environmental conditions. Results: The effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O₂. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO₂, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the Δflv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background. Conclusions: The observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.

We previously reported that Synechococcus elongatus PCC 7942, engineered with the sucrose transporter CscB, can export up to 85% of its photosynthetically-fixed carbon as sucrose and shows considerable promise as an alternative carbohydrate source. One approach to effectively utilize this cyanobacterium is to generate synthetic, light-driven consortia in which sucrose-metabolizing heterotrophs catalyze the conversion of the low-value carbohydrate into higher-value compounds in co-culture. Here, we report an improved synthetic photoautotroph/chemoheterotroph consortial design in which sucrose secreted by S. elongatus CscB directly supports the bacterium Halomonasboliviensis, a natural producer of the bioplastic precursor, PHB. We show that alginate encapsulation of S. elongatus CscB enhances sucrose-export rates ~2-fold within 66 h, to ~290 mg sucrose L^-1 d^-1 OD₇₅₀^-1 and enhances the co-culture stability. Consortial H. boliviensis accumulate up to 31% of their dry-weight as PHB, reaching productivities up to 28.3 mg PHB L^-1 d^-1. This light-driven, alginate-partitioned co-culture platform achieves PHB productivities that match or exceed those of traditionally engineered cyanobacterial monocultures. Importantly, S. elongatus CscB/H. boliviensis co-cultures were continuously productive for over 5 months and resisted invasive microbial species without the application of antibiotics or other chemical selection agents.

Two sorghum genotypes (red, tannin; white, non-tannin), were evaluated for their potential use in breakfast cereals. Two levels of whole grain sorghum flour (550 g/kg dry mix or 700 g/kg dry mix) were processed per genotype using a pilot-scale, twin screw extruder. A whole grain oat-based cereal was used as a reference. White sorghum cereals (WSC) had significantly (p < 0.05) higher starch, brightness (L*), and yellowness (b*) than red sorghum cereals (RSC). RSC had higher protein and bulk density than the WSC. Cereals made with 700 g sorghum flour/kg were smaller and denser with lower water solubility and absorption indices than those made with 550 g/kg. In vitro protein digestibility of the RSC (43–58%) was significantly reduced compared with the WSC (69–73%) and the reference sample (72%). WSC with 700 g sorghum flour/kg contained significantly more resistant starch than the RSC cereals and the oat reference (208 g/kg starch versus 81–147 g/kg starch, respectively). Overall acceptability and texture of sorghum cereals did not differ significantly from the oat reference, although appearance and aroma liking were significantly reduced. Therefore, non-tannin sorghum has potential to be used in the breakfast cereal industry with minimal impact on nutritional profile and sensory properties.

Near-infrared (NIR) diffuse reflectance has been extensively and successfully applied on quality assurance for fruits, vegetables, and food products. This study is principally aimed to extract the primary wavelengths related to the prediction of glucose and sucrose for potato tubers (of Frito Lay 1879 (FL), a chipping cultivar, and Russet Norkotah (RN), a table use cultivar, and investigating the potential of classification of potatoes based on sugar levels important to the frying industry. Whole tubers, as well as 12.7 mm slices, were scanned using a NIR reflectance spectroscopic system (900–1685 nm). To extract the most influential wavelength in the studied range, interval partial least squares (IPLS), and genetic algorithm (GA) were utilized. Partial least squares regression (PLSR) was applied for building prediction models. Prediction models for RN showed stronger correlation than FL with r(RPD) (correlation coefficient (ratio of reference standard deviation to root mean square error of the model)) values for whole tubers for glucose being as high as 0.81(1.70), and 0.97(3.91) for FL and RN; in the case of sliced samples the values were 0.74(1.49) and 0.94(2.73) for FL and RN. Lower correlation was obtained for sucrose with r(RPD) for whole tubers as high as 0.75(1.52), 0.92(2.57) for FL and RN; and the values for sliced samples were 0.67(1.31) and 0.75(1.41) for FL and RN respectively. Classification of potatoes based on sugar levels was conducted and training models were built using different classifiers (linear discriminant analysis (LDA), K-nearest neighbor (Knn), partial least squares discriminant analysis (PLSDA), and artificial neural network (ANN)), in addition to classifier fusion. To obtain more robust classification models for the training data, 4-fold cross validation was used and results were tested using separate sets of data. Classification rates of the testing set for whole tubers, based on glucose, were as high as 81% and 100% for FL and RN. For sliced samples, the rates were 83% and 81% for FL and RN. Generally, lower classification rates were obtained based on sucrose with values of whole tubers of 71%, and 79% for FL and RN, and for sliced samples the rates were 75%, and 82% which follows a similar trend as PLSR results. This study presents a potential of using selected wavelengths and NIR reflectance spectroscopy to effectively evaluate the sugar content of potatoes and classify potatoes based on thresholds that are crucial for the frying industry.

The bioindustrial production of fuels, chemicals, and therapeutics typically relies upon carbohydrate inputs derived from agricultural plants, resulting in the entanglement of food and chemical commodity markets. We demonstrate the efficient production of sucrose from a cyanobacterial species, Synechococcus elongatus, heterologously expressing a symporter of protons and sucrose (cscB). cscB-expressing cyanobacteria export sucrose irreversibly to concentrations of >10 mM without culture toxicity. Moreover, sucrose-exporting cyanobacteria exhibit increased biomass production rates relative to wild-type strains, accompanied by enhanced photosystem II activity, carbon fixation, and chlorophyll content. The genetic modification of sucrose biosynthesis pathways to minimize competing glucose- or sucrose-consuming reactions can further improve sucrose production, allowing the export of sucrose at rates of up to 36.1 mg liter^-1 h illumination^-1. This rate of production exceeds that of previous reports of targeted, photobiological production from microbes. Engineered S. elongatus produces sucrose in sufficient quantities (up to ~80% of total biomass) such that it may be a viable alternative to sugar synthesis from terrestrial plants, including sugarcane.

A direct plate bioassay procedure was applied for rapid and quantifiable assessment of the influence of various nutritional parameters on pediocin production by Pediococcus acidilactici NRRL B5627. Solid-state cultivation of the microorganism was done on MRS-based media over 3-and 6-hours incubation periods. Nutritional parameters assessed included the carbon source (glucose, sucrose, fructose, galactose, glycerol), and various salts (NH₄PO₄, CaCl₂, KH₂PO₄, MnSO₄•H₂O). Glucose was found to be the optimal carbon source while glycerol exhibited the most suppressive effect. Using glucose as the carbon source, addition of various salts, in amounts used in liquid media commonly applied in the cultivation of the pediococci, was assessed with respect to bacteriocin production on a per cell basis. Experimental data obtained showed that several nutritional parameters repress pediocin production by P. acidilactici, while the direct plate assay proved to be a good pilot assay prior to conducting more intensive kinetic analysis in liquid cultivation.

• Leaf senescence can be described as the dismantling of cellular components during a specific time interval before cell death. This has the effect of remobilizing N in the form of amino acids that can be relocalized to developing seeds. High levels of carbohydrates have previously been shown to promote the onset of the senescence process. • Carbohydrate accumulation in barley (Hordeum vulgare) plants was induced experimentally by steam-girdling at the leaf base, occluding the phloem, and gene regulation under these conditions was investigated using the Affymetrix Barley GeneChip array and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). • Transcript levels of plastidial (aminopeptidases, cnd41) and vacuolar (thiol and serine) proteases clearly increase in girdled leaves. Of special interest are cnd41, a plastidial aspartyl peptidase that has been implicated in Rubisco degradation in tobacco; and cp-mIII, a highly upregulated carboxypeptidase. SAG12, hexokinases and other senescence-specific genes are also upregulated under these conditions. • Applying a genomic approach to the innovative experimental system described here significantly enhances our knowledge of leaf proteolysis and whole-plant N recycling.