Erythorbate Research Articles

An Ascorbic Acid Oxidase-based Sensing Platform for Stereoselective Interaction with Ascorbic Acid and Isoascorbic Acid.

A simple enzyme-based nanohybrid material was fabricated via immobilizing ascorbic acid oxidase (AO) on the surface of flower-like electrodeposited gold nanoparticles (dpAu) and reduced graphene oxide (rGO) modified glassy carbon electrodes (GCEs). The composite material was used for stereoselective interaction with ascorbic acid (AA) and isoascorbic acid (IAA). Herein, AO was applied as a stereoselective selector, and the dpAu/rGO nanohybrid not only acted as a supporter for high loading of AO, but also served as the nanomaterial for signal amplification. The results showed obvious peak current differences between AA and IAA, indicating that this strategy could be employed to recognize AA and IAA. Under the optimum conditions, the sensor exhibited a good linear response to AA and IAA in a linear range of 1.0 × 10-4 - 5.0 × 10-3 M. This approach with the merits of simplicity and rapid response provided a promising perspective for identification of AA and IAA.

Quantitative Analysis of L-Ascorbic Acid and Erythorbic Acid in Foods by HPLC, and Confirmation Method by LC-MS/MS

Analysis of L-ascorbic acid (AsA) and erythorbic acid (ErA) in foods is generally performed by HPLC measurement after extraction with metaphosphoric acid solution. But this method can not always measure the concentrations of AsA and ErA precisely due to the presence of interfering compounds, and the reproducibility of retention time is poor. We considered that quantitative analysis by HPLC and confirmation by LC-MS/MS using an identical extraction solvent might be an effective approach for AsA and ErA analysis. Chelate fiber was added to the sample, followed by extraction with acetic acid solution containing ethylenediaminetetraacetic acid disodium salt, purification with Oasis MCX, and 2-fold dilution with methanol. The resulting solution was used for quantification by HPLC using a ZIC-HILIC column and identification by LC-MS/MS. In recovery tests with 8 kinds of foods, the recovery of AsA was over 91%, and that of ErA was over 88%. The RSD was 5.1% or less for both analytes. Analysis of 8 kinds of foods by both methods showed that this method gave better RSD values than the conventional method. AsA and ErA in all samples were confirmed by product ion scanning and selected reaction monitoring of LC-MS/MS.

A Novel 2-Keto-D-Gluconic Acid High-Producing Strain Arthrobacter globiformis JUIM02.

2-Keto-D-gluconic acid (2KGA) is mainly used for industrial production of erythorbic acid, a food antioxidant. In this study, a 2KGA producing strain JUIM02 was firstly identified as Arthrobacter globiformis by morphological observation and 16S rDNA sequencing. The 2KGA synthetic capacity of A. globiformis JUIM02 was evaluated by both fermentation and bioconversion, with 180g/L dextrose monohydrate as substrates, in shake flasks and 5L fermenters. For fermentation, 2KGA titer, yield, molar yield, and productivity of JUIM02 reached 159.05g/L, 0.97g/g, 90.18%, and 6.63g/L/h in 24h. For non-sterile and buffer-free bioconversion by free resting cells (~ 3.2g/L dry cell weight) of JUIM02, these data were 172.96g/L, 1.06g/g, 98.07%, and 5.41g/L/h in 32h. Moreover, JUIM02 resting cells could be repeatedly used. Resting cells stored at 4°C within 30days showed stable bioconversion capacity, with 2KGA titers ≥ 171.50g/L, yields ≥ 1.04g/g, and molar yields ≥ 97.24%. The 2KGA synthetic pathway in A. globiformis, which was rarely reported, was also speculated similar to Pseudomonas and verified preliminarily. In conclusion, A. globiformis JUIM02 is a promising 2KGA industrial-producing strain suitable for various production methods and a suitable object for 2KGA metabolism research of A. globiformis.

A Pt and Pd bimetal nanowire based stereoselective sensor for the discrimination of ascorbic acid and isoascorbic acid

A simple Pt and Pd bimetal nanowire based stereoselective sensor for the recognition of ascorbic acid and isoascorbic acid.

1H-Tetrazole-5-amine Immobilized on Substituted Polymer Gel/Silica as a New Stationary Phase for Hydrophilic Interaction Chromatography

1H-Tetrazole-5-amine immobilized on substituted polymer gel/silica as a new stationary phase has been newly suggested for application in hydrophilic liquid chromatography (HILIC). The newly investigated column demonstrated a satisfactory retention of very polar compounds. The column showed mixed separation modes: reversed-phase (RP) in water-rich mobile phases and HILIC at high concentrations of acetonitrile content (> 60–80%) in aqueous–organic mobile phases. A continuous change in retention was observed at decreasing concentration of acetonitrile in water, the rise resulting in characteristic U-shape plots of retention factors versus the concentration of acetonitrile in the mobile phase, with minima corresponding to the transition between the mechanisms controlling the retention. The retention mechanism of the new column was investigated by the models (partitioning and surface adsorption) through adjustment of the water/acetonitrile ratio in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of tested analytes. The retention behavior of tested compounds on three different stationary phases (Atlantis HILIC—silica phase; TSKgel Amide-80—amide phase; Kromasil 60-5 DIOL—diol phase) was investigated to compare the selectivity with the new DCpak PTZ HILIC prototype column. The applicability of the new HILIC column for the chromatography of polar compounds was proven by the separation of mixtures of nucleobases (uracil, thymine, guanine, cytosine, and adenine) and organic acids (ascorbic, isoascorbic, pantothenic, and nicotinic acid). The new DCpak PTZ HILIC column could be a good alternative for the separation of the polar compounds.

Development of a non-sulphite oxygen scavenger for monoethylene glycol (MEG) used as gas hydrate inhibitor

The maximum limit for dissolved oxygen in monoethylene glycol (MEG) used as gas hydrate inhibitor is usually set to 10–20 ppb. Exposure to low quality blanket gas or leaks may lead to oxygen levels up to 1 000 ppb. Oxygen scavenging is an attractive means to reduce the concentration, but oxygen scavengers (OSs) developed for water treatment have limitations in MEG solutions. They react slowly in MEG at low temperature when the pH is less than 9 and some of them are incompatible with other production chemicals. Screening of commonly used reducing agents in lean (90%) MEG showed that erythorbic acid was more effective than the alternatives at pH approximately 8. Adding manganese (II) as a catalyst and diethylaminoethanol (DEAE) improved the performance considerably. The final OS formulation contained 17% erythorbic acid, 25% DEAE and 0.5% MnCl2. It worked well in MEG solutions at 20 and 50 °C when erythorbic acid was converted to erythorbate. When dosed to give 200 ppm erythorbate in solution, it successfully decreased the oxygen concentration from 1 000 to 10 ppb in less than 30 min. The performance was also satisfactory in lean MEG with moderate concentrations of an imidazoline based corrosion inhibitor.

Structure and catalytic mechanism of monodehydroascorbate reductase, MDHAR, from Oryza sativa L. japonica.

Ascorbic acid (AsA) maintains redox homeostasis by scavenging reactive oxygen species from prokaryotes to eukaryotes, especially plants. The enzyme monodehydroascorbate reductase (MDHAR) regenerates AsA by catalysing the reduction of monodehydroascorbate, using NADH or NADPH as an electron donor. The detailed recycling mechanism of MDHAR remains unclear due to lack of structural information. Here, we present the crystal structures of MDHAR in the presence of cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), and complexed with AsA as well as its analogue, isoascorbic acid (ISD). The overall structure of MDHAR is similar to other iron-sulphur protein reductases, except for a unique long loop of 63–80 residues, which seems to be essential in forming the active site pocket. From the structural analysis and structure-guided point mutations, we found that the Arg320 residue plays a major substrate binding role, and the Tyr349 residue mediates electron transfer from NAD(P)H to bound substrate via FAD. The enzymatic activity of MDHAR favours NADH as an electron donor over NADPH. Our results show, for the first time, structural insights into this preference. The MDHAR-ISD complex structure revealed an alternative binding conformation of ISD, compared with the MDHAR-AsA complex. This implies a broad substrate (antioxidant) specificity and resulting greater protective ability of MDHAR.

A Novel Solid‐state Electrochemiluminescent Enantioselective Sensor for Ascorbic Acid and Isoascorbic Acid

AbstractA novel, stable, solid‐state and stereoselective electrochemiluminescence (ECL) sensor has been designed to enantioselectively discriminate ascorbic acid (AA) and isoascorbic acid (IAA) by immobilizing Ru(bpy)32+ (Ru), thiolated β‐cyclodextrin (β‐CD‐SH) and gold/platinum hybrid nanoparticles supported on multiwalled carbon nanotube/silica coaxial nanocables (GP‐CSCN) on glassy carbon electrode. All chemical compounds could be immobilized on the surface of electrode stably through nafion film, and high stereoselectivity could be introduced to the sensor via the synergistic effects of the β‐CD‐SH and GP‐CSCN nanomaterials. When the developed sensor interacted with AA and IAA, obvious difference of ECL intensities was observed, and a larger intensity was obtained from AA, which indicated that this strategy could be employed to enantioselectively recognize AA and IAA. As a result, ECL technique might act as a promising method for recognition of chiral compounds.

Erythorbyl laurate as a potential food additive with multi-functionalities: Interfacial characteristics and antioxidant activity

The interfacial characteristics and antioxidant activities of erythorbyl laurate were investigated to provide information on practical applications as a multi-functional food additive. The critical micelle concentration (CMC) of erythorbyl laurate was 0.101mM and its foam stability was three times (half-life 24.33±0.94h) higher than that of Tween 20 (8.00±1.63h). In free radical scavenging assay, the negligible decrease in EC50 of erythorbyl laurate compared to erythorbic acid manifested that C-5 selective esterification of erythorbic acid with an acyl group (lauric acid) did not reduce the inherent antioxidant activity of the donor (erythorbic acid). Erythorbyl laurate formed lipid peroxides slower (i.e. retarded oxidation) in an emulsion system than did erythorbic acid. The localization of erythorbyl laurate as an emulsifier allowed the antioxidant molecules to be concentrated at the oil-water interface where oxidation is prevalent, which led to more effective retardation of lipid oxidation.

Potential Application of the Oryza sativa Monodehydroascorbate Reductase Gene (OsMDHAR) to Improve the Stress Tolerance and Fermentative Capacity of Saccharomyces cerevisiae.

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is an important enzyme for ascorbate recycling. To examine whether heterologous expression of MDHAR from Oryza sativa (OsMDHAR) can prevent the deleterious effects of unfavorable growth conditions, we constructed a transgenic yeast strain harboring a recombinant plasmid carrying OsMDHAR (p426GPD::OsMDHAR). OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen peroxide by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool following the accumulation of antioxidant enzymes and molecules, metabolic enzymes, and molecular chaperones and their cofactors, compared to wild-type (WT) cells carrying vector alone. The addition of exogenous AsA or its analogue isoascorbic acid increased the viability of WT and ara2Δ cells under oxidative stress. Furthermore, the survival of OsMDHAR-expressing cells was greater than that of WT cells when cells at mid-log growth phase were exposed to high concentrations of ethanol. High OsMDHAR expression also improved the fermentative capacity of the yeast during glucose-based batch fermentation at a standard cultivation temperature (30°C). The alcohol yield of OsMDHAR-expressing transgenic yeast during fermentation was approximately 25% (0.18 g·g-1) higher than that of WT yeast. Accordingly, OsMDHAR-expressing transgenic yeast showed prolonged survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsMDHAR expression increases tolerance to reactive oxygen species-induced oxidative stress by improving cellular redox homeostasis and improves survival during fermentation, which enhances fermentative capacity.

The Application of Poly(Glutathione Disulfide)-poly(L-lysine) Multilayer Films for the Enantioselective Interaction with Ascorbic Acid and Isoascorbic Acid

Poly(glutathione disulfide)-poly(L-lysine) (PGSSG-PLY) multilayer films prepared by electropolymerization via cyclic voltammetry were constructed as a new chiral interface for recognition of ascorbic acid (AA) and isoascorbic acid (IAA). The morphology of the films was characterized by scanning electron microscopy (SEM), the electrochemical behaviors of the films were studied via chemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Meanwhile, differential pulse voltammetry (DPV) was used to study the interactions between PGSSG-PLY modified glassy carbon electrodes and AA or IAA. A strong interaction with AA was observed on the chiral interface. The results proved that PGSSG film could amplify eletrochemical signal, while PLY film could provide more recognition sites to discriminate AA and IAA enantiomers with satisfying recognition efficiency. The possible formation of the films on the electrode surface was described. And the influences of electropolymerization cycles and pH to the recognition process were explored. Under the optimum conditions, the method showed an acceptable linear response to AA and IAA in a range from 1.0 × 10−7 to 5.0 × 10−3 M with a low detection limit of 3.3 × 10−8 M (S/N = 3). The modified electrodes also displayed good stability and reproducibility.

Enantioselective recognition of ascorbic acid and isoascorbic acid on HS-β-cyclodextrin/gold nanoparticles/hollow carbon microspheres hybrid modified electrodes

The HS-β-cyclodextrin/gold nanoparticles/hollow carbon microspheres hybrids were prepared to develop a simple strategy for electrochemical chiral analysis.

HPLC-UV method for the simultaneous determinations of ascorbic acid and dehydroascorbic acid in human plasma

A high performance liquid chromatography (HPLC) paired with UV-vis detection method to determine ascorbic acid and its oxidation product, dehydroascorbic acid, in human plasma was developed. Ascorbic acid in human plasma was extracted and stabilized using 10% metaphosphoric acid, and was analyzed by a Symmetry C18 column with 5 mM Hexadecyltrimethylammonium bromide and 50 mM KH2PO4 solution as the mobile phase (1.0 mL/min flow rate). Isoascorbic acid served as the internal standard and ultraviolet detector wavelength was 254 nm and 265 nm. Dehydroascorbic acid concentration was calculated from the differences in ascorbic acid concentration before and after reduction by dithiothreitol reagent. Quantification for ascorbic acid in human plasma was linear from 1–100 μg/mL. The interand intra-day precisions and accuracy were determined and the results were found to be within ±15%. This method was successfully applied to a human pharmacokinetic study of ascorbic acid as well as dehydroascorbic acid after oral administration of 4,000 mg vitamin C tablets to healthy Korean volunteers.

Scientific Opinion on the re‐evaluation of erythorbic acid (E 315) and sodium erythorbate (E 316) as food additives

Scientific Opinion on the re‐evaluation of erythorbic acid (E 315) and sodium erythorbate (E 316) as food additives

Oxidative stress-mediated antitumor activity of erythorbic acid in high doses

Oxidative stress-mediated antitumor activity of erythorbic acid in high doses

Analysis of Ascorbic Acid and Isoascorbic Acid in Orange and Guava Fruit Juices Distributed in Thailand by LC-IT-MS/MS

This work describes the determination of ascorbic acid (AA) and isoascorbic acid (IAA) in fruit juices by liquid chromatography coupled with ion trap tandem mass spectrometry (LC-IT-MS/MS), with a reversed phase column (C18) and simple isocratic conditions of 0.1 % formic acid. The negative ion mode of electrospray ionization (ESI) and the MS/MS transition of m/z 175 were used for the quantitation of AA- and IAA-generated product ions with m/z 115. The method was validated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy. Good linearity was achieved with 0.6 and 1.8 μg/mL for the LOD and LOQ, respectively. The intraday and intermediate precisions were approximately 4 and 7 %, respectively. Recovery percentages ranged from 88 to 108 %. All validation parameters were found to be within the acceptable range for both AA and IAA. Hence, the proposed method is suitable for simultaneously determining AA and IAA. Fourteen fruit juice samples were analyzed including fruit juices from supermarkets, local markets, and laboratory preparations. Ten fruit juice samples (100 %) from different brands in the supermarkets were investigated for AA and IAA content. AA could be detected in all the samples, ranging from 5.2 ± 2 to 44.4 ± 1.3 μg/mL, and the values of vitamin C in 4 guava samples were less than the values specified by the manufacturer. IAA was observed in 4 of 10 samples ranging from 3.4 ± 0.9 to 78.3 ± 3.9 μg/mL, and the highest value of IAA was approximately two fold higher than the highest value of AA. For freshly prepared fruit juices, AA was detected in both local market- and laboratory-prepared juices. The value ranged from 13.2 ± 0.9 to 105.1 ± 1.3 μg/mL. In addition, the AA stability after opening the package was evaluated. The results showed that after 4 days of storage in the refrigerator, approximately 30 and 15 % losses of AA were observed for the orange and guava juices, respectively. Fresh fruit juices were thus demonstrated to be good sources of AA, with the highest value observed for guava juice prepared in the laboratory.

Oxidative stress-mediated antitumor activity of erythorbic acid in high doses

Intravenous (iv) infusion of high-dose ascorbic acid (AA) has been used as a treatment for cancer patients. The tumoricidal action of AA occurs due to its prooxidant effect. Erythorbic acid (EA), one of the AA epimers, has reduced vitamin C activity, while the antioxidant activity of EA is similar to that of AA. Currently, other physiological and pharmacological functions of EA are not well known. We examined the cytotoxicity of EA to murine colon carcinoma (colon-26) cells and the antitumor activity of EA in tumor-bearing mice. Cytotoxic activity of EA to colon-26 cells was evaluated by using the calcein-AM assay. EA showed the same cytotoxic activity to colon-26 cells as that of AA. The cytotoxicity of EA was shown to be caused by oxidative stress. Next, colon-26 tumor-bearing mice were iv administered EA and AA on alternate days for 4 times, and tumor growth rates were measured. Tumor growth was significantly inhibited by administration of high-dose EA in vivo as well as AA. Finally, the in vivo biodistribution and clearance of EA and AA were investigated in tumor-bearing mice. Endogenous AA in the tumor was consumed to resist oxidative stress caused by reactive oxygen species that was generated by administered EA. These results indicated that the oxidative stress-mediated antitumor activity is one of the pharmacological functions of high-dose iv EA.

Engineered Expression Vectors Significantly Enhanced the Production of 2-Keto-D-gluconic Acid by Gluconobacter oxidans.

2-Keto-D-gluconic acid (2KGA), a precursor of the important food antioxidant erythorbic acid, can be produced by Gluconobacter oxidans. To genetically engineer G. oxidans for improved 2KGA production, six new expression vectors with increased copy numbers based on pBBR1MCS-5 were constructed via rational mutagenesis. The utility of the mutant vectors was demonstrated by the increased ga2dh mRNA abundance, enzyme activity, and 2KGA production when the ga2dh gene was overexpressed using these vectors. Among the obtained constructs, G. oxidans/pBBR-3510-ga2dh displayed the highest oxidative activity toward gluconic acid (GA). In a batch biotransformation process, the G. oxidans/pBBR-3510-ga2dh strain exhibited 2KGA productivity (0.63 g/g CWW/h) higher than that obtained using strain G. oxidans/pBBR-ga2dh (0.40 g/g CWW/h). When sufficient oxygen was supplied during the biotransformation, up to 480 g/L GA was exhausted in 45 h by the G. oxidans/pBBR-3510-ga2dh strain and approximately 486 g/L 2KGA was produced, generating the productivity of 0.54 g/g CWW/h.

Purification and enzymatic characterization of membrane-bound d-gluconate dehydrogenase from Arthrobacter globiformis

Arthrobacter globiformis C224 is an industrial 2-keto-gluconic acid (2KGA) producer and currently used for erythorbic acid production in China. In the present study, a membrane-bound gluconate dehydrogenase (GADH) with specific activity of 326.06U/mg and purification fold of 412 was purified from A. globiformis using a five-step procedure including ultrasonication, phase separation with Triton X-114, ammonium sulfate fractionation, DEAE-sepharose fast flow and hydroxyapatite column chromatography. The GADH was identified as to be flavin adenine dinucleotide (FAD)-dependent and consisted of three subunits with molecular mass of 66,000Da, 44,000Da and 23,000Da. The optimal pH and temperature of A. globiformis GADH were 5.0 and 40°C, respectively. It had the stable activity at pH of 5.0–7.0 or below 50°C, and the strict substrate specificity for d-gluconate with the Km of 3.15mmolL−1, 1.04mmolL−1 at pH 5.0 and pH 6.0, respectively. The GADH activity also was significantly influenced by metal ions, organic solvents, and organic acids. Our study will benefit for better understanding of 2KGA production process by A. globiformis C224.

Formation and mitigation of N-nitrosamines in nitrite preserved cooked sausages

Literature on formation and mitigation of N-nitrosamine (NA) and especially non-volatile NA (NVNA) in meat products is scarce and the present study is therefore a relevant contribution to the field. We found positive correlation between the levels of N-nitrosopiperidine (NPIP), N-nitrosohydroxyproline (NHPRO), N-nitrosoproline (NPRO), N-nitrosothiazolidine-4-carboxylic acid (NTCA) and N-nitroso-2-methyl-thiazolidine-4-carboxylic acid (NMTCA) and the amount of nitrite added to cooked pork sausages. The levels studied were 0, 60, 100, 150, 250 and 350mgkg−1. The levels of N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR) remained at or below limit of quantification. Erythorbic acid inhibited the formation of NHPRO, NPRO, NPIP and NTCA. This inhibition was for NTCA and NMTCA counteracted by addition of free iron. Ascorbyl palmitate had less inhibitory effect than erythorbic acid and a combination of the two provided no further protection. Increasing the black pepper content increased the levels of NPIP and NMTCA. Only slight effects of increased fat content and addition of tripolyphosphate were observed.