Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 µA mM-1cm-2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

Effect of sorbitan monopalmitate on the polymorphic transitions and physicochemical properties of mango butter.

The present study reports the effect of sorbitan monopalmitate (SM) as a crystallization modifier on the physicochemical properties of mango butter (MB). The concentration of SM was varied in the range of 1 and 5 wt%. The addition of SM promoted the aggregation of globular MB crystals. The FTIR patterns did not show any significant changes when SM was added. XRD and DSC analyses confirmed the crystallization of MB crystals in stable ß' and ß (V) polymorphic states. However, SM also introduced imperfections in the crystal lattices of MB. Among all formulations, M2 (SM; 1% w/w) possessed a mechanically stable network structure. The crystallization rate of MB was tailored by SM in a concentration-dependent manner. The solid content was highest in M4 (SM; 5% w/w) at 10 °C and 30 °C among all the oleogels. In gist, SM in manageable quantities can be utilized for preparing custom-tailored MB-based products.

High resolution and high throughput analytical methods for d-tagatose and process related impurities using capillary electrophoresis.

d-tagatose is a low calorie multifunctional rare ketohexose sugar with sweetness similar to that of sucrose and it has high potential benefits for food and pharmaceutical industries. It is found in traces in some fruits as a natural component. In view of its high demand as a substitute for sugar, mass production of d-tagatose through enzymatic conversion of Lactose to d-tagatose is adopted. The existing HPLC method has limitations with respect sensitivity and resolution in quantification and monitoring of d-tagatose in the presence of its process related impurities. In the present investigation a new robust, fast and green analytical technique has been developed based on capillary electrophoresis (CE) for the separation and quantification of d-tagatose in presence of other sugars: Lactose, d-glucose, d-galactose and d-talose. Optimum conditions are found to be: Back Ground Electrolyte (BGE): 36 mM of Na2HPO4 and 130 mM of NaOH; pH: 12.6; voltage: +18 kV for high resolution and -18 kV for high throughput methods with direct UV-Detector at 265 nm. At these optimum conditions, good separation between the sugars is achieved in less than 20 min for high resolution and less than 4 min for high throughput methods. The developed methodology is validated as per ICHQ2R1 guide lines and successfully applied for monitoring d-tagatose during the enzymatic conversion of Lactose/d-galactose to d-tagatose and also to determine the unknown amounts of d-tagatose in crystallized samples and further, it is used in identifying the d-tagatose in fruits.

Protective effects of curcumin on bleomycin-induced changes in lung glycoproteins.

The present study investigated the therapeutic effect of curcumin on bleomycin (BLM)-induced alterations in glycoprotein components in the fibrotic lungs. Analysis of the bronchoalveolar lavage fluid (BALF) demonstrated increased fibronectin content at 3, 5, 7, and 14 days after BLM administration. Similarly, lung tissue fibronectin content revealed a progressive increase at various times (days 3, 5, 7, 14, and 28) during the development of lung fibrosis. In addition, alveolar macrophage release of fibronectin was also elevated in BLM-treated rats. Analysis of carbohydrate moieties of glycoproteins revealed an increase in total hexose, fucose, sialic acid and hexosamine levels at 7, 14, and 28 days after BLM treatment. Furthermore, the activities of lung glycosidases such as N-acetyl-ß-D-glucosaminidase, ß-glucosidase, ß-galactosidase, and ß-fucosidase in the fibrotic rats were elevated. Importantly, curcumin significantly inhibited the BLM-induced increases in BALF and lung fibronectin levels. Treatment of BLM rats with curcumin dramatically suppressed alveolar macrophage release of fibronectin. Curcumin also inhibited the increases in complex carbohydrates and glycosidases in the fibrotic lungs. These findings suggest that BLM-induced lung fibrosis is associated with accumulation of glycoproteins, and curcumin has the ability to suppress the enhanced deposition of glycoproteins in the fibrotic lung.

Signature Ions Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) for Specific and Confident Glycation Site Mapping in Therapeutic Proteins.

Higher-energy collisional dissociation (HCD) is a well-established fragmentation technique in liquid chromatography tandem mass spectrometry (LC-MS/MS) and is used to study protein post translational modifications (PTMs) during peptide mapping. However, labile PTMs like glycosylation, glycation, sulfonylation, or phosphorylation tend to fragment earlier than peptide backbones under HCD. This leads to complicated MS/MS spectra, compromising data quality and downstream data interpretation. Electron-transfer/higher-energy collision dissociation (EThcD) has been used to analyze PTMs, but important components might be missed because of the increased duty cycle. To address this issue, modification-specific fragment ions formed in HCD experiments could be utilized to trigger EThcD analysis only for modified peptides. The trigger for EThcD was established by applying HCD with a high normalized collision energy, generating multiple informative Amadori derived lysine signature ions from a glycated peptide. These signature ions were then applied to trigger targeted EThcD for lysine glycation identification. This improved approach can further expand the characterization efforts of highly labile PTMs in therapeutic proteins.

Maternal nutrition and stage of early pregnancy in beef heifers: impacts on hexose and AA concentrations in maternal and fetal fluids1.

We examined the hypothesis that maternal nutrition and day of gestation would affect the concentrations of AAs and hexoses in bovine utero-placental fluids and maternal serum from days 16 to 50 of gestation. Forty-nine cross-bred Angus heifers were bred via artificial insemination and fed a control diet (CON = 100% of requirements for growth) or a restricted diet (RES = 60% of CON) and ovariohysterectomized on days 16, 34, or 50 of gestation; nonpregnant controls were not bred and ovariohysterectomized on day 16 of the synchronized estrous cycle. The resulting design was a completely randomized design with a 2 × 3 factorial + 1 arrangement of treatments. Maternal serum, histotroph, allantoic fluid, and amniotic fluid were collected at time of ovariohysterectomy. Samples were then analyzed for concentrations of AAs and intermediary metabolites: alanine (Ala), arginine, asparagine (Asn), aspartate (Asp), citrulline, cysteine, glutamine, glutamate (Glu), glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine (Met), ornithine, phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan, tyrosine (Tyr), and valine (Val). The concentrations of Gly, Ser, and Thr in maternal serum were greater (P ≤ 0.05) in CON compared with RES. Furthermore, day of gestation affected (P ≤ 0.05) concentrations of Asn, Glu, Phe, Thr, and Tyr in maternal serum. Status of maternal nutrition affected the Asp concentration of histotroph where RES was greater (P = 0.02) than CON. In histotroph, Ala, Leu, Met, and Val concentrations were greater (P ≤ 0.05) on day 50 compared with day 16. Additionally, Glu and Pro concentrations in histotroph were greater (P < 0.01) on days 34 and 50 compared with day 16. A day × treatment interaction was observed for the concentration of Val in allantoic fluid where day 34 CON was greater (P = 0.05) than all other days and nutritional treatments. In addition, the concentration of Gln in amniotic fluid experienced a day × treatment interaction where day 34 RES was greater (P ≤ 0.05) than day 34 CON, which was greater (P ≤ 0.05) than day 50 CON and RES. These data support our hypothesis that day of gestation and maternal nutrition affect the concentrations of various neutral and acidic AA in beef heifer utero-placental fluids and maternal serum from days 16 to 50 of gestation.

Quantification of Soluble Sugars and Sugar Alcohols by LC-MS/MS.

Sugars are simple carbohydrates composed primarily of carbon, hydrogen, and oxygen. They play a central role in metabolism as sources of energy and as building blocks for synthesis of structural and nonstructural polymers. Many different techniques have been used to measure sugars, including refractometry, colorimetric and enzymatic assays, gas chromatography, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. In this chapter we describe a method that combines an initial separation of sugars by high-performance anion-exchange chromatography (HPAEC) with detection and quantification by tandem mass spectrometry (MS/MS). This combination of techniques provides exquisite specificity, allowing measurement of a diverse range of high- and low-abundance sugars in biological samples. This method can also be used for isotopomer analysis in stable-isotope labeling experiments to measure metabolic fluxes.

Delaying fat bloom formation in dark chocolate by adding sorbitan monostearate or cocoa butter stearin.

Two formulations of dark chocolate were developed by adding cocoa butter stearin (CBSt) or sorbitan monostearate (SMS) and compared to a standard formulation in order to investigate fat bloom formation over time. Fat bloom was monitored by Whiteness Index (WI), melting behavior and polymorphism determinations, in bars stored during 90 days at 20 °C and under oscillating temperature between 20 and 32 °C. All samples stored at 20 °C did not develop fat bloom and the required ß(V) form was maintained. Under oscillating storage condition, samples with CBSt (6.0%, w/w) and SMS (0.15%, w/w) delayed the surface fat bloom formation by at least 45 and 15 days, respectively, compared to standard chocolate, observed visually and through WI increments. The ß(V) to ß(VI) polymorphic transition correlated well with the WI, and also with changes in DSC thermograms, confirming the higher effectiveness of specific triacylglycerol (mainly StOSt) in delaying bloom formation.

Production of D-tagatose and bioethanol from onion waste by an intergrating bioprocess.

The rapid increase of agricultural waste is becoming a burgeoning problem and considerable efforts are being made by numerous researchers to convert it into a high-value resource material. Onion waste is one of the biggest issues in a world of dwindling resource. In this study, the potential of onion juice residue (OJR) for producing valuable rare sugar or bioethanol was evaluated. Purified Paenibacillus polymyxaL-arabinose isomerase (PPAI) has a molecular weight of approximately 53kDa, and exhibits maximal activity at 30°C and pH 7.5 in the presence of 0.8mM Mn2+. PPAI can produce 0.99g D-tagatose from 10g OJR. In order to present another application for OJR, we produced 1.56g bioethanol from 10g OJR through a bioconversion and fermentation process. These results indicate that PPAI can be used for producing rare sugars in an industrial setting, and OJR can be converted to D-tagatose and bioethanol.

Apoplastic Sugar Extraction and Quantification from Wheat Leaves Infected with Biotrophic Fungi.

Biotrophic fungi such as rusts modify the nutrient status of their hosts by extracting sugars. Hemibiotrophic and biotrophic fungi obtain nutrients from the cytoplasm of host cells and/or the apoplastic spaces. Uptake of nutrients from the cytoplasm is via intracellular hyphae or more complex structures such as haustoria. Apoplastic nutrients are taken up by intercellular hyphae. Overall the infection creates a sink causing remobilization of nutrients from local and distal tissues. The main mobile sugar in plants is sucrose which is absorbed via plant or fungal transporters once unloaded into the cytoplasm or the apoplast. Infection by fungal pathogens alters the apoplastic sugar contents and stimulates the influx of nutrients towards the site of infection as the host tissue transitions to sink. Quantification of solutes in the apoplast can help to understand the allocation of nutrients during infection. However, separation of apoplastic fluids from whole tissue is not straightforward and leakage from damaged cells can alter the results of the extraction. Here, we describe how variation in cytoplasmic contamination and infiltrated leaf volumes must be controlled when extracting apoplastic fluids from healthy and rust-infected wheat leaves. We show the importance of correcting the data for these parameters to measure sugar concentrations accurately.

Hydrophilic interaction anion exchange for separation of multiply modified neutral and anionic Dictyostelium N-glycans.

The unusual nature of the N-glycans of the cellular slime mould Dictyostelium discoideum has been revealed by a number of studies, primarily based on examination of radiolabeled glycopeptides but more recently also by MS. The complexity of the N-glycomes of even glycosylation mutants is compounded by the occurrence of anionic modifications, which also present an analytical challenge. In this study, we have employed hydrophilic interaction anion exchange (HIAX) HPLC in combination with MALDI-TOF MS/MS to explore the anionic N-glycome of the M31 (modA) strain, which lacks endoplasmic reticulum α-glucosidase II, an enzyme conserved in most eukaryotes including Homo sapiens. Prefractionation with HIAX chromatography enabled the identification of N-glycans with unusual oligo-α1,2-mannose extensions as well as others with up to four anionic modifications. Due to the use of hydrofluoric acid treatment, we were able to discriminate isobaric glycans differing in the presence of sulphate or phosphate on intersected structures as opposed to those carrying GlcNAc-phosphodiesters. The latter represent biosynthetic intermediates during the pathway leading to formation of the methylphosphorylated mannose epitope, which may have a similar function in intracellular targeting of hydrolases as the mannose-6-phosphate modification of lysosomal enzymes in mammals. In conclusion, HIAX in combination with MS is a highly sensitive approach for both fine separation and definition of neutral and anionic N-glycan structures.

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction.

OBJECTIVE: To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. RESULTS: Fructose at 1 M (180 g l-1) was converted to 0.8 M (144 g l-1) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l-1 h-1. No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. CONCLUSION: This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

In vitro antioxidative and immunological activities of polysaccharides from Zizyphus Jujuba cv. Muzao.

Zizyphus jujuba has been used as a traditional Chinese medicinal herb since ancient time. Polysaccharides have been found to be important bioactive compounds in the jujube. This work was designed to investigate the physicochemical characteristics and bioactivities of polysaccharides purified from Zizyphus jujuba cv. Muzao. Water-soluble polysaccharides were extracted using an ultrasonically assisted extraction method. The purified polysaccharides (HJP) were obtained by deproteinization and decoloration. Three main fractions (HJP1, HJP2 and HJP3) were isolated using DEAE-Sepharose fast flow ion-exchange chromatography. The purified polysaccharides were found to consist of mannose, rhamnose, galactose, galacturonic acid, glucose and arabinose at various levels for different fractions. The HJP and its three main fractions displayed DPPH radical scavenging activities as well as relatively strong reducing power and HJP had stronger activities than homogeneous compositions. Moreover, the results from in vitro immunological activities studies indicated that HJP could improve the phagocytosis activity of THP-l cells and had effect on the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6). In conclusion, the polysaccharides from Zizyphus jujuba cv. Muzao were discovered to have antioxidative and immunological activities.

Effects of Organogel Hardness and Formulation on Acceptance of Frankfurters.

Different organogel formulations used as beef fat (BF) replacement (0%, 20%, 40%, 60%, and 80%) were utilized to optimize the mechanical properties of frankfurters. Organogels, made of canola oil (CO), included different concentrations of ethyl cellulose (EC) and sorbitan monostearate (SMS). They consisted of: 8% EC + 1.5% SMS referred to as organogel-I (OG-I), 8% EC + 3.0% SMS (OG-II), and 10% EC + 1.5% SMS (OG-III), which were found promising in a previous study when used at 100% replacement. Replacement of BF with organogels at all levels could bring down the very high hardness values (texture profile analysis and sensory) of frankfurters prepared using CO by itself, relative to the BF control. OG-I and OG-II quantity had no significant effect on hardness and springiness, being similar in many cases to the BF and lower than the CO control. Shear force values of all organogel treatments were not significantly different from one another, and were between the BF and CO controls. Smokehouse yield showed a pattern of decreasing losses with increasing organogel replacement level. Sensory analysis revealed that using CO by itself significantly increased hardness, but structuring the oil (via organogelation), brought it down to the BF control value in all OG-I and OG-II formulations. Juiciness was significantly reduced by using liquid oil but increased with raising the amount of organogels. Oiliness sensation increased with higher organogel substitution and was actually higher than the beef control. The study demonstrates the potential use of vegetable oil structuring in replacing the more saturated BF in emulsion-type meat products.

Distinct Geographical Distribution of the Miscanthus Accessions with Varied Biomass Enzymatic Saccharification.

Miscanthus is a leading bioenergy candidate for biofuels, and it thus becomes essential to characterize the desire natural Miscanthus germplasm accessions with high biomass saccharification. In this study, total 171 natural Miscanthus accessions were geographically mapped using public database. According to the equation [P(H/L| East) = P(H/L∩East)/P(East)], the probability (P) parameters were calculated on relationships between geographical distributions of Miscanthus accessions in the East of China, and related factors with high(H) or low(L) values including biomass saccahrification under 1% NaOH and 1% H2SO4 pretreatments, lignocellulose features and climate conditions. Based on the maximum P value, a golden cutting line was generated from 42°25' N, 108°22' E to 22°58' N, 116°28' E on the original locations of Miscanthus accessions with high P(H|East) values (0.800-0.813), indicating that more than 90% Miscanthus accessions were originally located in the East with high biomass saccharification. Furthermore, the averaged insolation showed high P (H|East) and P(East|H) values at 0.782 and 0.754, whereas other climate factors had low P(East|H) values, suggesting that the averaged insolation is unique factor on Miscanthus distributions for biomass saccharification. In terms of cell wall compositions and wall polymer features, both hemicelluloses level and cellulose crystallinity (CrI) of Miscanthus accessions exhibited relative high P values, suggesting that they should be the major factors accounting for geographic distributions of Miscanthus accessions with high biomass digestibility.

Chiral separation of D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent and a phenylboronic acid complex.

A novel method was developed for D/L-isomeric separation of aldopentoses and aldohexoses as their (S)-(+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole derivatives using phenylboronate buffer containing sodium dodecyl sulfate as a background electrolyte. The combination of derivatization with a chiral labeling reagent and micellar electrokinetic chromatography with phenylboronate made possible the efficient separation of D/L isomers as well as epimeric isomers of aldopentoses and aldohexoses. Laser-induced fluorescence detection permitted the micromolar-level determination of monosaccharide derivatives. The limit of detection was 105 amol (300 nM), and the repeatabilities of the migration times and peak area responses were 0.8 % and 7.9 % (relative standard deviation; n = 6), respectively. The method was applied to the determination of D/L- galactose in red seaweed.

Lignin extraction distinctively enhances biomass enzymatic saccharification in hemicelluloses-rich Miscanthus species under various alkali and acid pretreatments.

In this study, one- and two-step pretreatments with alkali and acid were performed in the three Miscanthus species that exhibit distinct hemicelluloses levels. As a result, one-step with 4% NaOH or two-step with 2% NaOH and 1% H2SO4 was examined to be optimal for high biomass saccharification, indicating that alkali was the main effecter of pretreatments. Notably, both one- and two-step pretreatments largely enhanced biomass digestibility distinctive in hemicelluloses-rich samples by effectively co-extracting hemicelluloses and lignin. However, correlation analysis further indicated that the effective lignin extraction, other than the hemicelluloses removals, predominately determined biomass saccharification under various alkali and acid pretreatments, leading to a significant alteration of cellulose crystallinity. Hence, this study has suggested the potential approaches in bioenergy crop breeding and biomass process technology.

Steam explosion distinctively enhances biomass enzymatic saccharification of cotton stalks by largely reducing cellulose polymerization degree in G. barbadense and G. hirsutum.

In this study, steam explosion pretreatment was performed in cotton stalks, leading to 5-6 folds enhancements on biomass enzymatic saccharification distinctive in Gossypium barbadense and Gossypium hirsutum species. Sequential 1% H2SO4 pretreatment could further increase biomass digestibility of the steam-exploded stalks, and also cause the highest sugar-ethanol conversion rates probably by releasing less inhibitor to yeast fermentation. By comparison, extremely high concentration alkali (16% NaOH) pretreatment with raw stalks resulted in the highest hexoses yields, but it had the lowest sugar-ethanol conversion rates. Characterization of wall polymer features indicated that biomass saccharification was enhanced with steam explosion by largely reducing cellulose DP and extracting hemicelluloses. It also showed that cellulose crystallinity and arabinose substitution degree of xylans were the major factors on biomass digestibility in cotton stalks. Hence, this study has provided the insights into cell wall modification and biomass process technology in cotton stalks and beyond.

Complete hexose isomer identification with mass spectrometry.

The first analytical method is presented for the identification and absolute configuration determination of all 24 aldohexose and 2-ketohexose isomers, including the D and L enantiomers for allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, and tagatose. Two unique fixed ligand kinetic method combinations were discovered to create significant enough energetic differences to achieve chiral discrimination among all 24 hexoses. Each of these 24 hexoses yields unique ratios of a specific pair of fragment ions that allows for simultaneous determination of identification and absolute configuration. This mass spectrometric-based methodology can be readily employed for accurate identification of any isolated monosaccharide from an unknown biological source. This work provides a key step towards the goal of complete de novo carbohydrate analysis.