Sugar Equivalents Research Articles

Mechanistic Investigation of Enzyme Triggered Release from a Xyloglucan Matrix Tablet for Controlled Colonic Drug Delivery

The objective of this study was to investigate the mechanisms underlying drug release from a controlled colonic release (CCR) tablet formulation based on a xyloglucan polysaccharide matrix and identify the factors that control the rate of release for the purpose of fundamentally substantiating the concept and demonstrating its robustness for colonic drug delivery. Previous work demonstrated in vitro limited release of 5-aminosalicylic acid (5-ASA) and caffeine from these tablets in small intestinal environment and significant acceleration of release by xyloglucanase, an enzyme of the colonic microbiome. Targeted colonic drug delivery was verified in an animal study in vivo. In the present work, interaction of the xyloglucan matrix tablets with aqueous dissolution media containing xyloglucanase was found to lead to the spontaneous formation of a hydrated highly viscous gummy layer at the surface of the matrix which had a reduced drug content compared to the underlying regions and persisted with a nearly constant thickness that was inversely correlated to the enzyme concentration throughout the duration of the release process. Enzymatic hydrolysis of xyloglucan was determined to take place at the surface of the matrix leading to matrix erosion and a relation for the rate of enzymatic reaction as a function of bulk enzyme concentration and the concentration of dissolved xyloglucan in the gummy layer was derived. A mathematical model was developed encompassing aqueous medium ingress, matrix metamorphosis due to xyloglucan dissolution and matrix swelling, enzymatic hydrolysis of the polysaccharide and concomitant drug release due to matrix erosion and simultaneous drug diffusion. The model was fitted to data of reducing sugar equivalents in the medium reflecting matrix erosion and released drug amount. Enzymatic reaction parameters and reasonable values of medium ingress velocity, xyloglucan dissolution rate constant and drug diffusion coefficient were deduced that provided an adequate approximation of the data. Erosion was shown to be the overwhelmingly dominant drug release mechanism while the role of diffusion marginally increased at low enzyme concentration and high drug solubility. Changing enzyme concentration had a rather weak effect on matrix erosion and drug release rate as demonstrated by model simulations supported by experimental data, while xyloglucan dissolution was slow and had a stronger effect on the rate of the process. Therefore, reproducible colonic drug delivery not critically influenced by inter- and intra-individual variation of microbial enzyme activity may be projected.

Tablet formulation with dual control concept for efficient colonic drug delivery

Aim of this study was to develop a tablet formulation for targeted colonic drug release by implementing two control mechanisms: A pH-sensitive coating layer based on Eudragit® FS 30 D to prevent drug release in the upper gastrointestinal tract, combined with a matrix based on plant-derived polysaccharide xyloglucan to inhibit drug release after coating removal in the small intestine and to allow microbiome triggered drug release in the colon. In vitro dissolution tests simulated the passage through the entire gastrointestinal tract with a four-stage protocol, including microbial xyloglucanase addition in physiologically relevant concentrations as microbiome surrogate to the colonic dissolution medium. Matrix erosion was monitored in parallel to drug release by measurement of reducing sugar equivalents resulting from xyloglucan hydrolysis. Limited drug release in gastric and small intestinal test stages and predominant release in the colonic stage was achieved. The xyloglucan matrix controlled drug release after dissolution of the enteric coating through the formation of a gummy polysaccharide layer at the tablet surface. Matrix degradation was dependent on enzyme concentration in the colonic medium and significantly accelerated drug release resulting in erosion-controlled release process. Drug release at physiologically relevant enzyme concentration was completed within the bounds of colonic transit time. The dual control concept was applicable to two drug substances with different solubility, providing similar release rates in colonic environment containing xyloglucanase. Drug solubility mechanistically affected release, with diffusion of caffeine, but not of 5-ASA, contributing to the overall release rate out of the matrix tablet.

Dietary Fibre and Organic Acids in Kiwifruit Suppress Glycaemic Response Equally by Delaying Absorption-A Randomised Crossover Human Trial with Parallel Analysis of 13C-Acetate Uptake.

Non-sugar components of kiwifruit reduce the amplitude of the glycaemic response to co-consumed cereal starch. We determined the relative contribution of different non-sugar kiwifruit components to this anti-glycaemic effect. Healthy participants (n = 9) ingested equal carbohydrate meals containing 20 g starch as wheat biscuit (WB, 30 g), and the sugar equivalent of two kiwifruit (KFsug, 20.4 g), either intrinsic or added as glucose, fructose and sucrose (2:2:1). The meals were WB+KFsug (control, no non-sugar kiwifruit components), WB + whole kiwifruit pulp (WB+KF), WB + neutralised kiwifruit pulp (WB+KFneut), WB + low-fibre kiwifruit juice (WB+KFjuice) and WB+KFsug + kiwifruit organic acids (WB+KFsug+OA). All meals were spiked with 100 mg sodium [1-13C] acetate to measure intestinal absorption. Each participant ingested all meals in random order. Blood glucose and breath 13CO2 were measured at ingestion and at 15 min intervals up to 180 min. Compared with WB+KFsug, whole kiwifruit pulp (WB+KF) almost halved glycaemic response amplitude (p < 0.001), reduced incremental area under the blood glucose response curve (iAUC) at 30 min (peak) by 50% (p < 0.001), and averted late postprandial hypoglycaemia. All other treatments suppressed response amplitude half as much as whole kiwifruit and averted acute hypoglycaemia, with little effect on iAUC. Effects on 13CO2 exhalation paralleled effects on blood glucose (R2 = 0.97). Dietary fibre and organic acids contributed equally to the anti-glycaemic effect of kiwifruit by reducing intestinal absorption rate. Kiwifruit flesh effectively attenuates glycaemic response in carbohydrate exchange, as it contains fructose, dietary fibre and organic acids.

Estimated Sweetness in US Diet Among Children and Adults Declined From 2001 to 2018: A Serial Cross-Sectional Surveillance Study Using NHANES 2001-2018.

An agreed-upon measure of total dietary sweetness is lacking hindering assessments of population-level patterns and trends in dietary sweetness. This cross-sectional study used 24-h dietary recall data for 74,461 participants aged ≥ 2 y from nine cycles (2001–2018) of the National Health and Nutrition Examination Survey (NHANES) to evaluate trends in the sweetness of the diet in the United States (US). LCS-containing items were matched to a sugar-sweetened counterpart (e.g., diet cola–regular cola or sucralose sugar). The matched pair was used to estimate the sugar equivalents from LCS-sweetened foods or beverages to estimate dietary level sweetness, which was described as grams of approximate sugar equivalent (ASE) per day. Trends in ASE were estimated overall and by subgroup, and trends were further disaggregated by food or beverage category. Overall, LCS sources contributed about 10.5% of ASE. Total ASE declined from 152 g/d to 117 g/d from 2001–2002 to 2017–2018 (p-trend < 0.001), with comparable declines in children and adults. Declines in total ASE were predominantly driven by beverages (−36.7% from 2001–2002 to 2017–2018) and tabletop sweeteners (−23.8%), but not food (−1.5%). Observed trends were robust to sensitivity analyses incorporating random, systematic, and sensory trial informed estimates of sweetness and also an analysis excluding possible under-reporters of dietary energy. This practical approach and underlying data may help researchers to apply the technique to other dietary studies to further these questions.

Characteristics of some beverages adjusted with stevia extract, and persistence of steviol glycosides in the mouth after consumption

With the increase of obesity and diabetes, low-calorie, and natural sweeteners that can be used instead of sugar have become urgent. Stevia rebaudiana is used as an alternative natural sweetener in many countries because it is 200–300 times sweeter than sucrose, has no negative impact on human health, is low-calorie and natural, and is suitable for the use of obesity and diabetes patients. Recently, some studies have been conducted on the favorable properties of steviol glycosides on oral health, as well.In this study, 100 kDa and 5 kDa molecular weight cut-off membranes were used to concentrate stevia extracts, remove herbaceous odor, and remove bitter aftertaste. The enriched extracts obtained were used in the formulation of some beverages and were sensually tested. The sugar equivalent of the obtained retentate flux of 5 kDa nanofiltration was calculated and used in the production of nectar and fruit beverages with different energy levels, and some quality properties were determined. During the sensory evaluation of nectar samples, saliva samples were taken from the panelists at specific intervals. Change in steviol glycoside concentration was detected in saliva samples depending on time. Thus, the persistence of steviol glycosides in the mouth was determined. Consequently, it was proved that whole stevia extracts could be successfully used in products, and rebaudioside A was more persistent in the mouth than stevioside after consumption.

Exopolysaccharides in the rhizosphere: A comparative study of extraction methods. Application to their quantification in Mediterranean soils

Quantifying and characterizing Extracellular Polymeric Substances (EPS) and especially exopolysaccharides (EPSac) is an issue for understanding the hydro-physical and biological functioning of the rhizosphere. However, few comparative studies of extraction techniques have been carried out on soils and none on calcareous Mediterranean soils. Three soil-bound EPS extraction techniques, i.e. Cation Exchange Resin (CER), EDTA and NaOH + Formaldhyde (NaOH + F) were compared on three contrasted Mediterranean soils. CER presented the lowest extraction efficiency of EPSac, but also the lowest contamination of EPS by extractants and extracellular compounds. Contamination with intracellular compounds was low and similar with the three methods. Mid-Infrared (MIR) spectra enabled the best discrimination of the EPS extracts when they were prepared with CER. CER is then identified to be the suitable extraction technique of EPS (including EPSac) from soils, including calcareous soils. This technique was applied on rhizospheric and bulk soils harvested in an experimental field of tomato cultivation. A rhizospheric effect was highlighted during the growth of plants of two cultivars with both the soil-bound EPSac amounts (total sugar equivalent of extracted EPS) and the MIR spectra of extracted EPS. Extraction of soil-bound EPS and their further analysis by spectral and chemometric approaches is a promising way for relating EPS chemical characteristics to their biological and hydric impacts within the plant rhizosphere in a context of agro-ecological transition and climatic change.

Obtaining glucosed syrups from husks of Banana (Musa paradisiaca L.) by enzymatic hydrolysis of cellulases

The objective was to determine the effect of the application of the fungal cellulase enzyme on the silk banana peels (Musa paradisiaca) to obtain glucosed syrup. The enzymatic hydrolysis of banana shells at 30% (w / v) was studied at a constant temperature of 50 ° C. The independent variables to be controlled were the concentration of the fungal cellulase enzyme (0.5%, 1% and 1.5% w / v) and the hydrolysis time (18, 24 and 30 hours). The dependent variables were the percentage of Reducer Sugar and the percentage of Dextrose Equivalent (D.E.) of the enzymatically hydrolysed samples. The experimental design was completely randomized with factorial model 32. Glucosed syrups were obtained from silk plantain hulls by the enzymatic hydrolysis of the fungal cellulase. The highest percentages of Reducer Sugar and Dextrose Equivalent were presented in the Cellulase Concentration at 1.5% (w / v) during a hydrolysis time of 30 hours with average values ​​of 15.5% of reducing sugar percentage and 30.9% of percentage of Dextrose Equivalent. In the enzymatic hydrolysis with fungal cellulase at 1.5% (w / v) and 30 hours a higher value-added product was obtained, the operating conditions and the process steps were defined that should be adapted for the production of glucosed syrup from of banana peels (Musa paradisiaca).

Subcritical water hydrolysis of rice straw in a semi-continuous mode

Rice straw is an agricultural residue rich in cellulose and hemicelluloses. Therefore, the objective of this study was to obtain fermentable sugars and bioproducts from this biomass by subcritical water hydrolysis (SWH) in a scaleable semi-continuous mode. Temperatures of 180 °C, 220 °C and 260 °C, and liquid/solid mass ratios of 7.5 g water/g straw and 15 g water/g straw were evaluated on the reducing sugar yield (YRS), efficiency of conversion (E), kinetic profiles, composition of inhibitors and bioproducts, and physicochemical characteristics of remaining solid material. The pressure was set at 25 MPa and the reaction time was evaluated up to 15 min. The thermogravimetric analysis and Fourier Transform Infrared Spectroscopy provided additional information on the bulk and surface modification of the residual biomass. The highest YRS (33.4 ± 4.3 g/100 g dry straw) and E (72.1 ± 4.6 g sugar equivalents in glucose per 100 g of carbohydrates) were obtained at 220 °C and liquid/solid mass ratio of 7.5 g water/g straw. The analysis of the hydrolyzed solution indicated xylose (0.29–7.81 wt.%), arabinose (0.35–4.85 wt.%), cellobiose (0.04–3.41 wt.%), glucose (0.05–1.54 wt.%), and levulinic acid (0.04–0.77 wt.%). The fermentable sugars and bioproducts can be applied in several industrial fields, especially for the production of bioethanol and other higher value-added chemical compounds. Furthermore, SWH performed in semi-continuous mode is a green technology within the biorefinery concept that can be operated at industrial scale for processing lignocellulosic biomass.

Hydrolysis of Cellulose by Soluble Clostridium Thermocellum and Acidothermus Cellulolyticus Cellulases

The goal of this work was to clone, express, characterize and assemble a set of soluble thermostablecellulases capable of significantly degrading cellulose. We successfully cloned, expressed, and purified eleven Clostridium thermocellum (Cthe) cellulases and eight Acidothermuscellulolyticus(Acel) cellulases. The performance of the nineteen enzymes was evaluated on crystalline (filter paper) and amorphous (PASC) cellulose. Hydrolysis products generated from these two substrates were converted to glucose using beta-glucosidase and the glucose formed was determined enzymatically. Ten of the eleven Cthe enzymes were highly active on amorphous cellulose. The individual enzymes all produced &lt;10% reducing sugar equivalents from filter paper. Combinations of Cthe cellulases gave higher conversions, with the combination of CelE, CelI, CelG, and CelK converting 34% of the crystalline cellulose. All eight Acel cellulases showed endo-cellulase activity and were highly active on PASC. Only Acel_0615 produced more than 10% reducing sugar equivalents from filter paper, and a combination of six Acel cellulases produced 32% conversion. Acel_0617, a GH48 exo-cellulase, and Acel_0619, a GH12 endo-cellulase, synergistically stimulated cellulose degradation by the combination of Cthe cellulases to almost 80%. Addition of both Acel enzymes to the Cthe enzyme mix did not further stimulate hydrolysis. Cthe CelG and CelI stimulated cellulose degradation by the combination of Acel cellulases to 66%.

Evaluation of date seed oil and date molasses as novel carbon sources for the production of poly(3Hydroxybutyrate-co-3Hydroxyhexanoate) by Cupriavidus necator H16 Re 2058/pCB113

Cupriavidus necator H16 Re2058/pCB113 could effectively utilize oil sources to synthesize P(3HB-co-3HHx). In this study, P(3HB-co-3HHx) biosynthesis procedure with optimally controlled (between 2–28) 3HHx mol% was established for C. necator H16 Re2058/pCB113 by regulating the amounts of dual carbon sources supplied; date molasses and date seed oil. Alone, date molasses supplied at 20 g/L total sugar equivalents to recombinant C. necator resulted in pure P(3HB) biosynthesis, with accumulation up to 28 wt% of cell dry weight (CDW), and effective total biomass of 4 g/L. Whereas, date seed oil supply alone or in combination with date molasses resulted in P(3HB-co-3HHx) biosynthesis. P(3HB-co-3HHx) with as high as 28 mol% 3HHx, up to 80 wt% of CDW and total biomass of 7.6 g/L were produced using 10 g/L date seed oil as the sole carbon source. The results of PHA production in the presence of date seed oil as a sole carbon source was obtained with a modest yield at a range of 0.38–0.62 g-PHA per g-date oil used. Gel permeation chromatography (GPC) analyses of these P(3HB-co-3HHx) copolymers exhibited weight average molecular weights (Mw) between 500 − 690 kDa. Differential scanning calorimetry (DSC) analyses showed Tm values of 157 °C and 85 °C for P(3HB) and P(3HB-co-28 mol% 3HHx), respectively. NMR analyses (1H and 13C) of P(3HB-co-3HHx) copolymers confirmed the incorporation of 3HHx monomers between 2–28 mol% depending on the supplied dual carbon source composition.

Standardization of recipe for the preparation of low calorie RTS beverage from pineapple (Ananas comosus) using sugar substitutes

The investigation on standardization of recipe for preparation of low clorie RTS beverage from pineapple ( Ananas comosus ) using sugar substitutes was conducted. The experiment was laid out in Factorial Completely Randomized Design with aspartame, sucralose and their combination with cane sugar (sucrose) with 15 per cent juice and 0.3 per cent acidity. The RTS beverage with sucrose was the control. Sugar substitutes were used in place of sugar based on sugar equivalents. The prepared RTS beverages were analysed for chemical composition and sensory quality attributes. Among different combination sugar substitutes, sample D prepared by using 50 % sucrose + 50% sucralose was reached with highest sensory scores for overall acceptability.

The fructanolytic abilities of the rumen bacterium Butyrivibrio fibrisolvens strain 3071.

To determine if Butyrivibrio fibrisolvens strain 3071 is able to use fructose polymers for growth and to identify the enzymes involved in their digestion. Strain 3071 utilized 97, 89, 85 and 60% of sucrose, timothy grass fructan, inulin oligosaccharides and inulin, respectively, in the growth medium. A cell extract from timothy grass fructan-grown bacteria was used for identification of fructanolytic enzymes by anion exchange chromatography, gel filtration, zymography and thin-layer chromatography. The bacterium synthesizes a specific endolevanase and a nonspecific β-fructofuranosidase. Both enzymes occurred in two forms differing in molecular weight. The β-fructofuranosidase was not able to digest long-chain inulin or timothy grass fructan, but degraded inulin oligosaccharides and sucrose. Addition of 1,4-dithioerythritol to an enzyme solution did not affect the activity of endolevanase(s), but increased the ability of β-fructofuranosidase to digest sucrose. The digestion of timothy grass fructan by endolevanase(s) was described by Michaelis-Menten kinetics in which Km =2·82gl(-1) and Vmax =4·01μmoles reducing sugar equivalents×mg(-1) ×min(-1) . Strain 3071 synthesizes enzymes enabling it to use grass fructans for growth. Butyrivibrio fibrisolvens strain 3071 can be considered a member of the rumen fructanolytic guild.

Isoglucose: what are its prospects post-quotas?

After the end of the EU sugar quota system from 1 October 2017, expansion of isoglucose production will be possible. Currently production is limited to 720,000 t in nine EU countries, which is divided among six producers. Production is concentrated in Central and Southeastern Europe, far away from the main consumption areas, where high sugarbeet yields make sugar production very competitive. The possibilities for isoglucose production expansion are discussed. An 1 mn t production increase, to about 2 mn t of sugar equivalent, seems likely after 2017 despite the high beet sugar production surplus currently seen on the EU market.

The Impact of the U.S. Sugar Program Redux

We analyze the various welfare costs, transfers, trade, and employment consequences of the current U.S. sugar program for U.S. consumers, other sugar users, sugar refiners, cane and beet growing and processing industries, other associated agricultural sectors, and world markets. The removal of the sugar program would increase U.S. consumers' welfare by $2.9 to $3.5 billion each year and generate a modest job creation of 17,000 to 20,000 new jobs in food manufacturing and related industries. Imports of sugar containing products would fall dramatically, especially confectioneries substituting for domestic inputs under the sugar program. Sugar imports would rise substantially to 5–6 million short tons raw sugar equivalent. World sugar price increases would be minor, equivalent to about 1 cent per pound.

Bacterial consortia constructed for the decomposition of Agave biomass

Research has shown that a greater variety of enzymes, as well as variety of microorganisms producing enzymes, can have an overall synergistic effect on the decomposition of lignocellulosic biomass for the production of value-added bio-products. Here, 8 cellulase-degrading bacterial isolates were selected to develop co-, tri-, and tetra-cultures for the decomposition of lignocellulosic biomass. Glucose and xylose equivalents released from imitation biomass media containing 0.5% (w/v) beechwood xylan and 0.5% (w/v) Avicel was measured using di-nitrosalicylic acid for all consortia, along with cell growth and survival. Thereafter, 6 co- and 2 tri-cultures with greatest decomposition were examined for ability to degrade Agave americana fiber. Interestingly, when strains were paired up in co-culture, four pairs: G+5, G+A, C+A1, and G+A1 produced high reducing sugars in 24 h: 6 µM, 8 µM, 8 µM, and finally, 6 µM, respectively. From 4 co-cultures with highest reducing sugar equivalents, tri- and tetra-cultures were produced. The bacterial consortia which had the highest reducing sugars detected were 2 tri-cultures: G + A1 + A4 and G + A1 + 5, displaying levels as high as 9 µM and 5 µM in day 1, respectively. All co- and tri-cultures maintained high cell survival for 14 days with 0.5 g ground Agave. Upon evaluating Agave dry weight after treatment, it was evident that almost half the biomass could be decomposed in 14 days. Scanning electron microscopy of treated Agave supported decomposition when compared with the control. These bacterial consortia have potential for further study of value-added by-product production during metabolism of lignocellulosic biomasses.

Fermentative l-(+)-lactic acid production from non-sterilized rice washing drainage containing rice bran by a newly isolated lactic acid bacteria without any additions of nutrients

A newly isolated lactic acid producing bacterium Lactobacillus rhamnosus M-23, from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. The maximum lactate yield attained was 59 g/l with a productivity of 1.23 g/l/h and a product optical purity of 95% corresponding to a conversion of 0.85 g of lactic acid per gram of sugar equivalent.

Hydrolytic potential of Trichoderma sp. strains evaluated by microplate-based screening followed by switchgrass saccharification

Bioconversion of lignocellulosic biomass to fuel requires a hydrolysis step to obtain fermentable sugars, generally accomplished by fungal enzymes. Large-scale screening of different microbial strains would provide optimal enzyme cocktails for any target feedstock. The aim of this study was to screen a large collection of Trichoderma sp. strains for the hydrolytic potential towards switchgrass (Panicum virgatum L.). Strains were cultivated in a small-scale system and assayed in micro-plates for xylanase and cellulase activities. The population distributions of these traits are reported after growth on switchgrass in comparison with cellulose. The distribution profiles suggest that the growth on switchgrass strongly promotes xylanase production. The IK4 strain displayed the highest xylanase activity after growth on switchgrass (133U/mL). Enzymes (10FPU/g substrate) from IK4 were compared with those from 2 cellulolytic Trichoderma strains and a commercial enzyme in saccharification time-course experiments on untreated and pretreated switchgrass and on an artificial substrate. Samples were analysed by DNS assay and by an oxygraphic method for sugar equivalent or glucose concentration. On the untreated substrate, IK4 enzymes even outperformed a 5-fold load of commercial enzyme, suggesting that xylanase or accessory enzymes are a limiting factor on this type of recalcitrant substrate. On the other substrates, IK4 preparations showed intermediate behaviour if compared with the commercial enzyme at 10FPU/g substrate and at 5-fold load. IK4 also nearly halved the time to release 50% of the hydrolysable sugar equivalents (T50%), with respect to the other preparations at the same enzymatic load. DNS assay and oxygraphic method gave highly correlated results for the 3 saccharified substrates. The study suggests that accessory enzymes like xylanase play a key role in improving the performance of cellulase preparations on herbaceous lignocellulosic feedstocks like switchgrass.

Synthesis of a Novel Benzoyl Adenosine Analog Containing a 1, 4-Dioxane Sugar Analog and the Synthesis of a Corresponding Uracil Adenine Dinucleotide

Adenosine analogs in which the sugar unit was replaced by a 1,4-dioxane sugar equivalent, were prepared by coupling the 1,4-dioxane sugar analog as its anomeric acetates, with N6-benzoyl protected adenine. The 1,4-dioxane system was obtained in an enantioselective synthesis from (R,R)-dimethyl tartrate. Using standard phosphorimidite methodology, the adenine analog was further reacted with a 1,4-dioxane uridine analog to give the corresponding, protected dinucleotide, set-up for further condensation into larger oligonucleotides.

Automated saccharification assay for determination of digestibility in plant materials

BackgroundCell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies.ResultsWe developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared.ConclusionsThe automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.

Internally Protected Amino Sugar Equivalents from Enantiopure 1,2‐Oxazines: Synthesis of Variably Configured Carbohydrates with C‐Branched Amino Sugar Units

A stereodivergent synthesis of differently configured C2-branched 4-amino sugar derivatives was accomplished. The Lewis acid mediated rearrangement of phenylthio-substituted 1,2-oxazines delivered glycosyl donor equivalents that can directly be employed in glycosidation reactions. Treatment with methanol provided internally protected amino sugar equivalents that have been transformed into the stereoisomeric methyl glycosides 28, ent-28, 29, ent-29 and 34 in two simple reductive steps. Reaction with natural carbohydrates or bicyclic amino sugar precursors allowed the synthesis of homo-oligomeric di- and trisaccharides 44, 46 and 47 or a hybrid trisaccharide 51 with natural carbohydrates. Access to a bivalent amino sugar derivative 54 was accomplished by reaction of rearrangement product 10 with 1,5-pentanediol. Alternatively, when a protected L-serine derivative was employed as glycosyl acceptor, the glycosylated amino acid 60 was efficiently prepared in few steps. In this report we describe the synthesis of unusual amino sugar building blocks from enantiopure 1,2-oxazines that can be attached to natural carbohydrates or natural product aglycons to produce new natural product analogues with potential applications in medicinal chemistry.