Complex Mixture Of Sugars Research Articles

Do Soluble Phosphates Direct the Formose Reaction towards Pentose Sugars?

The formose reaction has been a leading hypothesis for the prebiotic synthesis of sugars such as ribose for many decades but tends to produce complex mixtures of sugars and often tars. Channeling the formose reaction towards the synthesis of biologically useful sugars such as ribose has been a holy grail of origins-of-life research. Here, we tested the hypothesis that a simple, prebiotically plausible phosphorylating agent, acetyl phosphate, could direct the formose reaction towards ribose through phosphorylation of intermediates in a manner resembling gluconeogenesis and the pentose phosphate pathway. We did indeed find that addition of acetyl phosphate to a developing formose reaction stabilized pentoses, including ribose, such that after 5 h of reaction about 10-fold more ribose remained compared with control runs. But mechanistic analyses using liquid chromatography-mass spectrometry showed that, far from being directed towards ribose by phosphorylation, the formose reaction was halted by the precipitation of Ca2+ ions as phosphate minerals such as apatite and hydroxyapatite. Adding orthophosphate had the same effect. Phosphorylated sugars were only detected below the limit of quantification when adding acetyl phosphate. Nonetheless, our findings are not strictly negative. The sensitivity of the formose reaction to geochemically reasonable conditions, combined with the apparent stability of ribose under these conditions, serves as a valuable constraint on possible pathways of sugar synthesis at the origin of life.

Development and Validation of HPLC-DAD Method with Pre-Column PMP Derivatization for Monomeric Profile Analysis of Polysaccharides from Agro-Industrial Wastes.

The instrumental analysis of complex mixtures of sugars often requires derivatization to enhance the method’s selectivity and sensitivity. 1-Phenyl-3-methyl-5-pyrazolone (PMP) is a common sugar derivatization agent used in high-performance liquid chromatography (HPLC). Although many C18 column applications for PMP–sugar derivative analysis have been developed, their transferability is not straightforward due to variations in column chemistry and preparation technology. The aim of this study was to develop and validate an application for Zorbax Extend C18 columns for the analysis of 8 neutral and 2 acidic sugars commonly found in plant polysaccharides. The method was further compared to well-established alditol acetates and m-hydroxydiphenyl methods and employed for sugar profiling of selected agro-industrial wastes. The most influential separation factors were the mobile-phase pH and acetonitrile content, optimized at 8.0 and a 12–17% gradient, respectively. The method showed excellent linearity, repeatability and intermediate precision. High sensitivity was achieved, especially for neutral sugars, with an accuracy error range of 5–10% relative standard deviation. The sugar profiling results were highly correlated to the reference method for neutral sugars. The HPLC method was highly applicable for the evaluation of polysaccharides in selected wastes and showed advantages in terms of simplicity, accuracy in acidic sugar determination and suitability for their simultaneous analysis with neutral sugars.

Use of Raman and Raman optical activity to extract atomistic details of saccharides in aqueous solution.

Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

A Novel Technique to Characterize and Quantify Crystalline and Amorphous Matter in Complex Sugar Mixtures

Sugar-rich foods are the complex mixtures of different carbohydrates in amorphous and crystalline forms. They are mostly not in equilibrium, and transition from amorphous form to crystalline form is very likely during their storage. It is essential to know the crystalline/amorphous fraction of sugars to study these undesirable changes. The existing techniques are not adequate owing to the nonideal interactions among these sugars. In this work, we propose a novel technique, which first segregates crystals from the amorphous matter by dispersing the food material in an appropriate solvent. This is followed by separation of crystals by filtration, and further quantification and characterization by high-performance liquid chromatography, X-ray diffraction, differential scanning calorimeter and microscopy. Non-centrifugal sugar and fondant, which are mixtures of sucrose, glucose and fructose, are chosen as representative examples to demonstrate the efficacy of the proposed method. On an average, 70–90% of the sucrose in NCS samples was found to be in crystalline state.

Hydrothermally induced changes in the properties of MFC and characterization of the low molar mass degradation products

The aim of this study was to determine hydrothermally induced changes in the physico-chemical properties of a fine microfibrillated cellulose (MFC). The MFC was prepared from a monocomponent endoglucanase-treated never-dried birch kraft pulp which had been run several times through a microfluidizer in order to obtain a sample substantially free from fibers or fiber fragments. The fine MFC was subjected to hydrothermal treatment at 150,^{circ }hbox {C} for 0.5, 2.5, 4.5, 8.5 and 20.5 h, after which the viscosity, water-retention capacity, surface charge, discoloration and formation of hydrolysis products were determined. The viscosity, surface charge and water-retention capacity of MFC decreased as a result of the treatment but in oscillatory measurements the storage and loss moduli increased. Hydrothermal treatment also caused discoloration of the sample which could not be fully washed away with water. Surface analysis with XPS revealed no significant changes in the surface structure of the dried MFC cakes but the filtrates after the hydrothermal treatment were complex mixtures of sugars, organic acids and furans. The main degradation products detected were xylose and xylobiose, but isomerization products of sugars (xylulose, fructose) were also found in the filtrates. In addition numerous organic acids, including formic, glycolic, lactic and xylonic acid, were identified in the filtrates, showing that both acid and alkaline catalyzed reactions occur due to dissociation of water into hbox {H}^{+} and hbox {OH}^{-}.

Development of an advanced derivatization protocol for the unambiguous identification of monosaccharides in complex mixtures by gas and liquid chromatography

The separation and analysis of complex monosaccharide mixtures is highly challenging and requires typically carefully selected derivatization procedures to avoid changes in the sample composition. Here we present in a comparative study several single- and two-step derivatization approaches for LC and GC separations using a set of reference compounds ranging from C1 building block such as formaldehyde to C6 monosaccharides. Separation conditions have been optimized resulting in the simultaneous separation of 15 unbranched aldoses. By parallel derivatization using hydroxylamine hydrochloride (HACl)/ N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and O-ethylhydroxylamine hydrochloride (EtOx)/ N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and comparative GC measurements we developed a protocol for the unambiguous identification and separation of aldoses, ketoses, alditols and aldonic acids, which commonly occur in complex sugar mixtures as reaction by-products or decomposition products. In particular this procedure helps to deconvolute overlapping analytes and facilitates quantification. Additionally, the method presented here has been investigated in regard to storage life, detection limits, quantification and MS analysis. The broad applicability of this method to different sample matrices is shown for the analysis of food samples and complex aldol reaction mixtures in the formose reaction, which is of great relevance in the context of the origin of life.

Experimental Investigation of Nanofiltration Process for the Separation of Complex Sugar Mixtures Containing Mono- and Multivalent Salts

The energy consumption of sugar producing industry is enormous mainly due to the thermal energy demand of multi-stage evaporation. However, alternative processes have been studied to reduce the high operating cost. Membrane technology is a well-established process for water treatment. Reverse osmosis (RO) and nanofiltration (NF) have also been examined for the pre-concentration of clarified thin sugar juice to reduce the role of evaporation in the sugar production process. RO/NF can be used to remove water from the aqueous solution at a temperature below its boiling point and without phase change. It has a positive effect on the quality of the clarified thin sugar juice because sugar juice is degraded at high temperatures. The clarified thin sugar juice contains 15 °Brix (15 w%) of sugar and about 0.25 - 0.40 w% of the ionic compounds. In the present work the effect of the presence of monovalent and multivalent salts on the separation performance of a nanofiltration membrane during sugar concentration has been studied experimentally. The sugar rejection was not affected much by the salt concentration but the rejection of monovalent or multivalent ions was found to be highly dependent on the sugar concentration.

New insights into sugar characterization in complex plant matrices by high-performance thin-layer chromatography

ABSTRACTHigh-performance thin-layer chromatography (HPTLC) is an innovative, green, reliable, and rapid method for the characterization of complex sugar mixtures. For research into sustainable nontimber forest products, Adansonia digitata dry fruit pulp in this proof-of-concept study was used to demonstrate this method due to its reported high nutritional value and prebiotic activity. Chromatographic separations were performed on (20 × 10 cm2) HPTLC glass plates with diol stationary phase, using a 15-step gradient by mixing solutions acetone/acetonitrile 1:1, and ultrapure water for enzymatic degradation products. Instead, monosaccharide moieties were separated on HPTLC silica gel 60 F254 over isocratic mode with 80 mm of eluent front composed of acetonitrile/acetic acid/water 63/33/5 v/v/v. Identification and quantification were performed by densitometry acquisitions at 400 nm after opportune derivatization. Our research highlighted that this technique can be used as a standard method to gain new insights into inulin determinations, with several advantages over existing conventional liquid chromatography. The development of innovative methods for the characterization of biopolymers is crucial, for food and nutraceutical industry and for quality control of phytochemicals. Characterization of such materials, up until now, has been done by mass difference and so accurate analytical methods were lacking.

Continuous Ethanol Fermentation of Pretreated Lignocellulosic Biomasses, Waste Biomasses, Molasses and Syrup Using the Anaerobic, Thermophilic Bacterium Thermoanaerobacter italicus Pentocrobe 411

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47–0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2–2.7 g/L/h and a total sugar conversion of 90–99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.

Production of lipid from depolymerised lignocellulose using the biocontrol yeast, Rhodotorula minuta: The fatty acid profile remains stable irrespective of environmental conditions

The oleaginous yeast Rhodotorula minuta has been used previously as a biocide agent and for the production of β‐carotene. In addition, R. minuta has been shown to produce up to 40% lipids, while demonstrating a faster growth rate than the similar oleaginous yeasts; Lipomyces starkeyii and R. glutinis. In this study, this promising yeast was evaluated for its potential to produce glyceride lipids under the harsh conditions and complex sugar mixtures produced from depolymerised lignocellulose. The fatty acid profile of R. minuta was not found to change significantly irrespective of the environmental conditions and contained approximately 20% palmitic acid, 5% stearic acid, 60% oleic acid and 15% linolenic acid. R. minuta was found to grow on a range of sugars, and could consume xylose and glucose when both sugars were present; however, R. minuta was found to be highly sensitive to inhibitors, such as furfurals and organic acids, formed under the harsh lignocellulose depolymerisation conditions. Accordingly R. minuta did not grow well on biomass depolymerised with an acid pre‐treatment stage. However, R. minuta was cultured successfully on food waste depolymerised with no additional acids, producing up to 19 g/l cell mass with a lipid content of up to 25% of the dry cell weight.Practical applications: While high oil productivity, fast growth rates and a suitable fatty acid composition are key traits for the economic viability of SCO production, it is essential that the organism can be cultured and produce lipids from low‐cost substrates. This requires the ability to assimilate the nutrients present in the desired hydrolysate, as well as maintain high‐growth rates in the presence of inhibitory compounds produced during the hydrolysis process. In this paper, we demonstrate that the yeast R. minuta has industrial applicability in being able to convert depolymerised waste food to lipids with a profile akin to rapeseed oil.Rhodotorula minuta lipid cultured on depolymerised food waste.

Separation and Quantitative Determination of Carbohydrates in Microbial Submerged Cultures Using Different Planar Chromatography Techniques (HPTLC, AMD, OPLC)

Carbohydrates are the principal sources of nutrient and energy in large-scale submerged fermentation processes. A method for detection and quantification of sugar levels are very advantageous because they can be considered as key indicators in determining the yields and the productivity of the process. For this reason, the objective of this study is to develop and validate a simple and relatively economical analytical method for detecting complex sugar mixtures in fermentation broth based on High-Performance Thin-Layer Chromatography (HPTLC). HPTLC is a widely used, fast and accurate method of separating complex mixtures. The proposed method involved the chromatographic separations of xilo-, galacto-, fructo-oligosaccharides mixtures at different molecular weights, tri- and disaccharides (raffinose, sucrose, lactose), and the corresponding monosaccharides (xylose, fructose, galactose) on HPTLC plates, using different eluent mixtures and elution conditions. The documentation of plates was performed using TLC visualization device and the images of plates were processed using a digital processor. HPTLC methods development using instrumental techniques as OPLC (Over Pressure Liquid Chromatography) and AMD (Automated Multiple Development) has been also described, as to simultaneously monitor several samples in the same elution, with significant time and solvent savings. Four different carbohydrates complex mixtures were analyzed using HPTLC techniques, as to optimize the quality of the separation among components. The methods set up were then applied for quantitative determination of sugars. As a model of submerged fermentations, a strain of Bifidobacterium spp. was used, a saccharolytic bacterium with probiotic activities in the human gut, able to anaerobically ferment complex sugar mixtures. Results could be easily extended to other fermentation processes.

Separation of complex sugar mixtures on a hydrolytically stable bidentate urea-type stationary phase for hydrophilic interaction near ultra high performance liquid chromatography

A stationary phase bearing both bridged bis-ureido and free amino groups (USP-HILIC-NH2 -2.5SP) for high-speed hydrophilic interaction liquid chromatography separations was prepared using a one-pot two-step procedure starting from 2.5 μm totally porous silica particles. Highly polar compounds, such as polyols, hydroxybenzoic acids, and sugars, were successfully analyzed in shorter times and with higher peak efficiency, when compared to results obtained with a bidentate urea-type column packed with 5 μm particles. Increased sugarophilicity and better peak shape were attested for the USP-HILIC-NH2 -2.5SP column (100 × 3.2 mm id) when compared with two commercially available UHPLC columns, namely an acquity BEH amide packed with totally porous 1.7 μm microparticles and a HILIC Kinetex column packed with core-shell 2.6 μm particles. Finally, the new column was employed in the separation of complex mixture of sugars (mono-, di-, and oligosaccharides) and in the analysis of beer samples. The resulting chromatograms showed good selectivity and overall resolution, while the catalyzing effect of the free amino moieties resulted in excellent peak shapes and in the absence of split peaks due to sugar anomerization phenomena.

Chemometric Discrimination of Different Tomato Cultivars Based on Their Volatile Fingerprint in Relation to Lycopene and Total Phenolics Content

The characteristic flavour of tomato is given by a complex mixture of sugars, acids, amino acids, minerals and volatile metabolites. Of these, volatile compounds are considered to greatly influence the flavour of tomato fruits. The volatile aroma compounds and phytochemical content of tomatoes are dependent on genotype, environmental conditions and cultural practices, and can thus be used for cultivar discrimination. To assess the possibility of using the volatile profile of tomato to fingerprint and discriminate different tomato cultivars based on an 'in-tube extraction' technique coupled to gas chromatography, combined with mass spectrometry (ITEX/GC-MS) and a chemometric approach. Using the ITEX/GC-MS technique, 61 volatiles were analysed and separated from tomato cultivars, with 58 being identified. The main volatiles identified in all tomato cultivars were: hexanal, trans-2-hexenal, 1-hexanol, 3-pentanone, 3-methylbutanol, 2-methylbutanol, 3-methylbutanal and 6-methyl-5-hepten-2-one. The lycopene content and total phenolic compound content of the tomato cultivars varied between 36.78 and 73.18 mg/kg fresh weight (fw) and from 119.4 to 253.7 mg of gallic acid equivalents (GAE) per kilogram fresh weight, respectively. Volatile fingerprint and phytochemical composition led to a good differentiation between tomato cultivars, with the first two principal components explaining 89% of the variance in the data. The tomato cultivars studied were easily discriminated based on their characteristic volatile profile that was obtained using the reliable ITEX/GC-MS technique. Principal component analysis revealed, in addition to volatile compounds, the important role played by the total phenolic content in tomato cultivar discrimination, which is highly correlated with phenotypic and biochemical differences between tomato cultivars.

Evolutionary engineering of Saccharomyces cerevisiae for enhanced tolerance to hydrolysates of lignocellulosic biomass

Lignocellulosic biomass has become an important feedstock to mitigate current ethical and economical concerns related to the bio-based production of fuels and chemicals. During the pre-treatment and hydrolysis of the lignocellulosic biomass, a complex mixture of sugars and inhibitors are formed. The inhibitors interfere with microbial growth and product yields. This study uses an adaptive laboratory evolution method called visualizing evolution in real-time (VERT) to uncover the molecular mechanisms associated with tolerance to hydrolysates of lignocellulosic biomass in Saccharomyces cerevisiae. VERT enables a more rational scheme for isolating adaptive mutants for characterization and molecular analyses. Subsequent growth kinetic analyses of the mutants in individual and combinations of common inhibitors present in hydrolysates (acetic acid, furfural, and hydroxymethylfurfural) showed differential levels of resistance to different inhibitors, with enhanced growth rates up to 57%, 12%, 22%, and 24% in hydrolysates, acetic acid, HMF and furfural, respectively. Interestingly, some of the adaptive mutants exhibited reduced fitness in the presence of individual inhibitors, but showed enhanced fitness in the presence of combinations of inhibitors compared to the parental strains. Transcriptomic analysis revealed different mechanisms for resistance to hydrolysates and a potential cross adaptation between oxidative stress and hydrolysates tolerance in several of the mutants.

Dynamic flux balance modeling of S. cerevisiae and E. coli co-cultures for efficient consumption of glucose/xylose mixtures

Current researches into the production of biochemicals from lignocellulosic feedstocks are focused on the identification and engineering of individual microbes that utilize complex sugar mixtures. Microbial consortia represent an alternative approach that has the potential to better exploit individual species capabilities for substrate uptake and biochemical production. In this work, we construct and experimentally validate a dynamic flux balance model of a Saccharomyces cerevisiae and Escherichia coli co-culture designed for efficient aerobic consumption of glucose/xylose mixtures. Each microbe is a substrate specialist, with wild-type S. cerevisiae consuming only glucose and engineered E. coli strain ZSC113 consuming only xylose, to avoid diauxic growth commonly observed in individual microbes. Following experimental identification of a common pH and temperature for optimal co-culture batch growth, we demonstrate that pure culture models developed for optimal growth conditions can be adapted to the suboptimal, common growth condition by adjustment of the non-growth associated ATP maintenance of each microbe. By comparing pure culture model predictions to co-culture experimental data, the inhibitory effect of ethanol produced by S. cerevisiae on E. coli growth was found to be the only interaction necessary to include in the co-culture model to generate accurate batch profile predictions. Co-culture model utility was demonstrated by predicting initial cell concentrations that yield simultaneous glucose and xylose exhaustion for different sugar mixtures. Successful experimental validation of the model predictions demonstrated that steady-state metabolic reconstructions developed for individual microbes can be adapted to develop dynamic flux balance models of microbial consortia for the production of renewable chemicals.

Quantification of Sugar Mixtures with Near-Infrared Raman Spectroscopy and Multivariate Data Analysis. A Quantitative Analysis Laboratory Experiment

An experiment for Raman spectroscopy in combination with multivariate data analysis was designed and implemented in the undergraduate analytical chemistry laboratory curriculum. Raman spectroscopy is currently considered among the most important nondestructive routine optical analysis tools. An experiment is presented demonstrating the capability of Raman spectroscopy in combination with principal components analysis and regression (PCA/PCR), which was applied to determine the composition of complex sugar mixtures that cannot be adequately quantified using conventional univariate analysis methods.

An HPTLC-AMD method for understanding the metabolic behavior of microorganisms in the presence of mixed carbon sources. The case ofBifidobacterium adolescentisMB 239

The intestinal microflora Bifidobacteria has been adapted to utilize, as a source of energy, complex carbohydrates that escape hydrolysis by human digestive enzymes, for example fructo-oligosaccharides (FOS). The ability of intestinal microflora to utilize carbohydrates in complex mixtures is of particular interest, even though few data have been reported. This paper presents the results obtained using an innovative analytical approach based on instrumental HPTLC-AMD (automated multiple development) to investigate the metabolic behavior of Bifidobacterium adolescentis MB 239 as a case study. Raffinose, FOS (sucrose, 1-kestose, nystose, fructosyl-nystose), lactose, and their monomeric moieties glucose, galactose, and fructose were simultaneously present as carbon sources in the solution to be fermented by the bacterium. The method proposed has enabled quantitative monitoring of sugar concentrations during the entire time-course of the processes. The complex sugar mixtures were separated by use of acetonitr...

Production of a lactose-free galacto-oligosaccharide mixture by using selective enzymatic oxidation of lactose into lactobionic acid

We report a novel and efficient way of producing lactose-derived galacto-oligosaccharides (GOS) that do not contain remaining lactose and monosaccharides. The initial sugar mixture was obtained by enzymatic transformation at 70°C of a lactose solution of 270 g/liter using recombinant β-glycosidase from the Archaeon Sulfolobus solfataricus. At the optimum reaction time for kinetically controlled transgalactosylation, it contained 46% monosaccharides, 13% lactose and 41% GOS. Lactose was selectively oxidised into lactobionic acid by using fungal cellobiose dehydrogenase which displays a ≈ 100-fold preference for reaction with lactose compared to reaction with GOS. Oxidation of lactose was coupled to reduction of 2,6-dichloro-indophenol which was added in catalytic concentrations. The oxidised redox mediator was regenerated continuously by fungal laccase-catalysed reduction of molecular oxygen into water. Ion exchange chromatographies were employed to remove lactobionic acid, other ions and monosaccharides. The final product contained 97% GOS, 1.2% lactose and 2.1% monosaccharides. The yield accounted for 25% of original lactose. An enzymatic assay for lactose has been developed. It is robust and allows sensitive quantification of the analyte in complex sugar mixtures containing large excesses of monosaccharides and GOS.

A sensitive and selective method for the analysis of complex mixtures of sugars and linear oligosaccharides

2-Aminoacridone has been used to derivatise a variety of sugars and polysaccharides via the formation and reduction of a Schiff base. Separation of these carbohydrate derivatives was achieved with high selectivity and sensitivity using micellar electrokinetic capillary chromatography (MECC) followed by laser induced fluorescence detection.

The prebiotic synthesis of carbohydrates: A reassessment

Questions concerning the significance of previous work on the formose reaction have led us to reexamine the question of the prebiotic synthesis of sugars. The results of new experiments lead to the following conclusions: The formose reaction is a geochemically plausible reaction which depends on neither basic conditions nor on the presence of trace amounts of carbohydrate impurities. However, this process is not a plausible source of ribose nor of any other individual sugar. In contrast to the nonspecific formation of complex mixtures of sugars via the formose reaction, the reduced sugar pentaerythritol is formed with great selectivity by the ultraviolet irradiation of 0.1 M formaldehyde. This compound may have played an important role in chemical evolution.