The Online Morphology Control and Dynamic Studies on Improving Vitamin B<Subscript>12</Subscript> Production by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis> with Online Capacitance and Specific Oxygen Consumption Rate

The relationship between the morphological character of Pseudomonas denitrificans and vitamin B₁₂ synthesis based on real-time capacitance measurement and online specific oxygen consumption rate (Q _O2) control was established for enhancing vitamin B₁₂ production. Results demonstrated that the threshold Q _O2 value lower than 2.0 mmol/gDCW/l would greatly stimulate the state transfer from the cell number growth phase to the cell elongation phase and promote rapid vitamin B₁₂ biosynthesis, while the vitamin B₁₂ biosynthesis rate could also be inhibited when the rate of cell’s length-to-width ratio (ratio-LW) was higher than 10:1. Furthermore, the optimal morphology controlling strategy was achieved based on online Q _O2 control, which increases the appropriate active cell numbers at the former phase, and then control the elongation of ratio-LW no more than 10:1 at the vitamin B₁₂ biosynthesis phase. The maximal vitamin B₁₂ production reached 239.7 mg/l at 168 h, which was improved by 14.7 % compared with the control (208 mg/l). This online controlling strategy would be effectively applied for improving industrial vitamin B₁₂ fermentation.     

Determination of B-group vitamins in Turkish honey using ultra-performance liquid chromatography with electrospray ionization coupled to tandem mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry method with electrospray ionization (UPLC–ESI–MS/MS) for analysis of B-group vitamins in honey has been presented. Aim of this study is the characterization of different types of Turkish honeys according to B-group vitamins. Vitamins were determined in 17 different types of Turkish honey samples by UPLC–ESI–MS/MS. Heather honey samples were distinguished among the studied honeys with the richest vitamin content with 286.10?mg/kg, and it is followed by sunflower honey and thyme honey with the total vitamin contents of 206.01 and 163.27?mg/kg, respectively. The presence of vitamin B₁ (thiamine), vitamin B₂ (riboflavin), vitamin B₃ (nicotinamide, B₃N and nicotinic acid, B₃H), vitamin B₆ (pyridoxine), and vitamin B₉ (folic acid) was detected in all the honey samples analyzed. Moreover, vitamin B₅ (pantothenic acid) was observed in most of them. Vitamin B₁₂ (cyanocobalamin) and vitamin B₇ (biotine) were not detected in the studied honey samples. Turkish honey samples showed efficacious vitamin content for the consumers.     

Sequential determination of fat- and water-soluble vitamins in Rhodiola imbricata root from trans-Himalaya with rapid resolution liquid chromatography/tandem mass spectrometry

A rapid method was developed to determine both types of vitamins in Rhodiola imbricata root for the accurate quantification of free vitamin forms. Rapid resolution liquid chromatography/tandem mass spectrometry (RRLC–MS/MS) with electrospray ionization (ESI) source operating in multiple reactions monitoring (MRM) mode was optimized for the sequential analysis of nine water-soluble vitamins (B₁, B₂, two B₃ vitamins, B₅, B₆, B₇, B₉, and B₁₂) and six fat-soluble vitamins (A, E, D₂, D₃, K₁, and K₂). Both types of vitamins were separated by ion-suppression reversed-phase liquid chromatography with gradient elution within 30 min and detected in positive ion mode. Deviations in the intra- and inter-day precision were always below 0.6% and 0.3% for recoveries and retention time. Intra- and inter-day relative standard deviation (RSD) values of retention time for water- and fat-soluble vitamin were ranged between 0.02–0.20% and 0.01–0.15%, respectively. The mean recoveries were ranged between 88.95 and 107.07%. Sensitivity and specificity of this method allowed the limits of detection (LOD) and limits of quantitation (LOQ) of the analytes at ppb levels. The linear range was achieved for fat- and water-soluble vitamins at 100–1000 ppb and 10–100 ppb. Vitamin B-complex and vitamin E were detected as the principle vitamins in the root of this adaptogen which would be of great interest to develop novel foods from the Indian trans-Himalaya.     

An immediate luminescence enhancement method for determination of vitamin B1 using long-wavelength emitting water-soluble CdTe nanorods

A method for determination of vitamin B₁ has been developed that is based on the enhancement effect of vitamin B₁ on the luminescence of water-soluble CdTe nanorods modified with thioglycolic acid and cysteine. The effect of variables including the size of the nanorods on the enhancement of luminescence have been investigated. A preliminary mechanistic study showed that the passivating action of vitamin B₁ on the surface of the CdTe nanorods is likely to be responsible for the enhancement. Interferences by shortwave fluorescence are effectively eliminated because measurements are performed in the near-infrared. Due to the near-infrared measurement character, the fluorescence interference of vitamin B₂ can be effectively eliminated. Under the optimum conditions, the extent of luminescence enhancement is proportional to the concentration of vitamin B₁ in the range from 0.1 to 3.0 μmol L^?1 and the detection limit is 0.03 μmol L^?1. The relative standard deviation for 2.0 μmol L^?1 vitamin B₁ is 1.3% (n?=?6). The method is highly sensitive and selective, avoids the sample treatment needed in other procedures, and can be applied to the determination of vitamin B₁ in real samples with satisfactory results.     

Quantum chemical study of the effects of interaction of polyhydrated Hg(II) with methylcobalamin — A derivative of vitamin B<Subscript>12</Subscript>

Quantum chemical methods were used to investigate the mechanism of interaction of polyhydrated Hg(II) with methylcobalamin — a derivative of vitamin B₁₂. Migration of the methyl group in the B₁₂·Hg(II) nH₂O cluster, initiating the formation of methylmercury, is considered. The reaction of substitution of methylmercury for the cobalt atom in the corrin fragment of B₁₂ has been studied. The thermodynamic and activation characteristics of these processes have been obtained. The mercury analog of vitamin B₁₂ is shown to exist in two radically different conformations.     

Low-frequency electromagnetic pulses enhance Ca2+ uptake by chick small intestine in vitro

The effect of external EMF on Ca²⁺ uptake by the chick small intestine was investigated in vitro by use of the accumulating preparations of epithelial tissue. The intestinal tissue preparations were exposed to EMF in specific zones of Helmholtz coils. The steady magnetic field (SMF) having an induction value B₀ = 1.14 mT, as well as pulsed EMF—constant pulse rate (f = 80 Hz) and randomized pulse rate (in range of 40–120 Hz)-with amplitude BM = B₀ was used. The fall time of the pulse was changed stepwise in the range 0.15–0.55 ms.It was found that SMF, as well as pulsed EMF significantly enhanced the uptake of Ca²⁺ by the small intestine. A SMF was found to enhance uptake by up to 40–50% for both normal (D) and rachitic chicks. The effects of the induced E_rot depended essentially of vitamin D status of chicks due to the complex action of vitamin D-dependent factors. The effect of randomized pulse EMF was qualitatively similar to that of constant pulse EMF, but quantitatively it was less. Data indicate the contribution of the B-, as well as E-component to the modification of the Ca²⁺ uptake.     

Improved Vitamin B12 Fermentation Process by Adding Rotenone to Regulate the Metabolism of Pseudomonas denitrificans

Our previous research had revealed that the dissolved oxygen limitation was more favorable for vitamin B₁₂ fermentation, due to its inducement to the increased glycolytic flux in Pseudomonas denitrificans. In this paper, a novel strategy was implemented to further investigate the metabolic characteristics of P. denitrificans under different oxygen supply levels, by exogenously adding rotenone (a respiratory chain inhibitor interfering with the oxygen consumption) to the fermentation broths. Compared to the fermentation process without rotenone treatment, it was observed that 5 mg/L rotenone treatment could significantly strengthen the glycolytic flux of P. denitrificans via activating the key glycolytic enzymes (phosphofructokinase and pyruvate kinase), resulting in the accelerated generations of anterior precursors (glutamate and 5-aminolevulinic acid) for vitamin B₁₂ biosynthesis. Although 5 mg/L rotenone treatment had a negative effect on cell growth of P. denitrificans, the vitamin B₁₂ yield was increased from 48.28?±?0.62 mg/L to 54.70?±?0.45 mg/L, which further proved that an increased glycolytic flux in P. denitrificans was a consequence of higher vitamin B₁₂ production.     

TiO2‐assisted Photocatalytic Degradation of Vitamin B12 in Aqueous Solution:Influencing Factors,Kinetics, and Reaction Investigations

The photodegradation (λ=365 nm) of the biomolecule vitamin B₁₂, catalyzed by the photocatalyst TiO₂ nanoparticles (NPs), has been investigated in aqueous suspension. The photodegradation process of vitamin B₁₂ has been monitored by means of electronic absorption (Abs), Fourier‐transform infrared (FT‐IR), and resonance Raman (RR) spectroscopies, respectively. The results show that only under UV illumination in the presence of TiO₂ is there effective degradation, and the photocatalytic degradation of vitamin B₁₂ is strongly influenced by the amount of TiO₂ NPs, the pH, and the initial concentration of vitamin B₁₂. The photocatalytic reaction kinetics of vitamin B₁₂ conforms to a Langmuir‐Hinshelwood isotherm model. Changes involving the three structural units of the carbon‐metal bond C–Co, the organic corrin macrocycle combined with the benzimidazole nucleotide, and the inorganic CN in the vitamin B₁₂ molecule during the photocatalytic degradation are also discussed.     

Simultaneous Spectrophotometric Determination of Group B Vitamins Using Parallel Factor Analysis: PARAFAC

In this work, a simple and rapid analytical procedure was applied for simultaneous determination of folic acid (vitamin B₀), thiamin (vitamin B₁), riboflavin (vitamin B₂) and pyridoxal (vitamin B₆) based on the absorbance data in the pH range 2.0‐12.0 at 25 °C using parallel factor analysis (PARAFAC). The effect of the pH as the most important factor on the sensitivity of the determination was studied. The spectral data were recorded in 400‐650 nm intervals and a 2‐12 pH range for all four vitamins. The calibration set was constructed in the concentration ranges of 4‐22, 1‐20, 6‐26, and 4‐20 μg mL^?;1 for B₆,B₂,B₁ and B₀, respectively. The root mean squares errors of prediction for the prediction set, (RMSEP), are 0.65, 0.63, 1.13 and 0.34 for B₀,B₁,B₂ and B₆, respectively. The recovery percent for the validation set are in the range of 90.6 to 107.0%. The effect of the experimental conditions and diverse species were discussed. The optimum values of these factors were searched according to the relative standard deviation of the prediction set of mixtures solutions.     

A new liquid membrane electrode for determination of vitamin B1 in multivitamin preparations

Summary A new liquid membrane electrode was developed which is highly selective and sensitive for vitamin B₁. It is based on the thiamine-picrolonate ion-pair complex as an electroactive material in nitrobenzene solvent. The electrode has a remarkable selectivity for vitamin B₁ in the presence of vitamins B₂, B₆, B₁₂ and nicotinamide. The results obtained for the determination of vitamin B₁ in various multivitamin preparations at levels as low as 1 g/ml show an average recovery of 98% (mean standard deviation 1.8%) and are favourably compared with those obtained by the official methods.

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Neue Flüssig-Membranelektrode zur Bestimmung von Vitamin B₁ in Multivitaminpräparaten

Zusammenfassung Eine neue, für Vitamin B₁ hochselektive Flüssig-Membranelektrode wurde entwickelt. Sie beruht auf dem Thiamin-Picrolonat-Ionenpaarkomplex als elektroaktives Material im Lösungsmittel Nitrobenzol. Die Elektrode besitzt eine beachtliche Selektivität für Vitamin B₁ in Gegenwart der Vitamine B₂, B₆, B₁₂ und Nicotinamid. Bei Bestimmungen in verschiedenen Multivitaminpräparaten mit Konzentrationen bis herab zu 1 g/ml erhielt man eine durchschnittliche Wiederfindung von 98% (mittlere Standardabweichung 1,8%). Die erhaltenen Resultate zeigen gute Übereinstimmung mit den nach amtlichen Methoden erhaltenen Werten.

     

Cathodic adsorption stripping analysis of vitamin B12

Amalgam formation preconcentration is usually performed in electrochemical stripping analysis, but adsorption also can be employed for preconcentration. Vitamin B₁₂ is adsorbed on a mercury electrode in the potential region between ca. ?0.3 and ?1.5 V vs. SCE. Vitamin B₁₂ also yields a polarographic catalytic wave at ca. ?1.6 V. It is considered that the adsorbed protonated reduced form of vitamin B₁₂ plays an important role in producing the catalytic current. Stripping analysis for vitamin B₁₂ employing adsorption preconcentration was found to be possible. Ammonium acetate was used as the supporting electrolyte. It was found that the peak for vitamin B₁₂ was maximal in 0.3 M ammonium acetate. The suitable preconcentration potential was ?1.45 V. The preconcentration time was 5 min while stirring the solution. The scan rate used was 50 mV/s, considering the pen speed of the X-Y recorder. The optimum temperature was 35°C. Under the optimum conditions described above, the calibration curve was linear in the concentration range up to 0.1 μ M. The detection limit was 2 n M.     

Study of the voltammetric behavior of vitamin B<Subscript>2</Subscript> and the development of a procedure for its determination in breast milk

The electrochemical behavior of vitamin B₂ at a glassy-carbon electrode is studied. Some physicochemical parameters of the electrode reaction (pK values for different forms of vitamin B₂ and the number of electrons involved in the electrode process) are calculated. A rapid procedure is developed for the quantitative chemical determination of vitamin B₂ in breast milk by differential voltammetry.     

Thermodynamics of Sr(NO3)2 and Cd(NO3)2 in mixed solvents from viscosity data

The viscosities of Sr(NO₃)₂ and Cd(NO₃)₂ have been determined in dioxane, glycol and methyl alcohol+water mixtures at 10, 20 and 30% by weight. The B values have been computed at different temperatures both from the Jones—Dole and Das's equation. From the B values, the effective rigid molar volume, its change with % of organic solvent, temperature and the ion—solvent interaction have been inferred. Activation parameters have also been calculated and the structure breaking effect has been deduced.     

Wittig reaction with ion-supported Ph3P

Ion-supported Ph₃P, 4-(diphenylphosphino)benzyltrimethylammonium bromide A and N-methyl-N-[4-(diphenylphosphino)benzyl]pyrrolidinium bromide B, were used for the Wittig reaction. Ion-supported phosphonium salts A1 and B1, which were prepared from the reactions of ion-supported Ph₃P A and B with ethyl bromoacetate, respectively, reacted with aromatic and aliphatic aldehydes in the presence of K₂CO₃ to give the corresponding α,β-unsaturated ethyl esters in good yields with high purity by simple filtration of the reaction mixture and subsequent removal of the solvent from the filtrate. Similarly, ion-supported phosphonium salts A2 and B2, which were prepared from the reactions of ion-supported Ph₃P A and B with p-methylbenzyl bromide, respectively, reacted with aromatic and aliphatic aldehydes in the presence of NaH to provide the corresponding p-methylstyrene derivatives in good yields with high purity by simple filtration of the reaction mixture and the subsequent removal of the solvent from the filtrate. In both reactions, the co-product, ion-supported Ph₃PO, could be obtained quantitatively by simple filtration, and was converted into the corresponding ion-supported Ph₃P A and B again in high yields using dimethyl sulfate, followed by the reduction with LiAlH₄. Recovered and regenerated ion-supported Ph₃P A and B could be reused for the same Wittig reaction while maintaining good yields of ethyl (E)-3-(4′-chlorophenyl)-2-propenoate and 1-(4′-chlorophenyl)-2-(4″-methylphenyl)ethene with high purity by simple filtration and removal of the solvent from the filtrate.     

Organic cyclizations in microemulsions catalyzed by a cobalt corrin-polyion-scaffold on electrodes

Metallopolyion films of cobalt corrin vitamin B₁₂ hexacarboxylate and poly(l-lysine) [B₁₂(COOH)₆–PLL] covalently attached to carbon electrodes were used to cyclize n-bromoalkyl-2-cyclohexenones in microemulsions with good catalytic efficiency. Trans-1-decalone was obtained in up to 60% yields at reasonable current efficiency. Turnover rates were 30–85 fold greater for films compared to dissolved catalyst. There was no dependence of turnover rate on microemulsion composition, suggesting that cleavage of the alkyl-cobalt bond in the alkyl-Co^III intermediate is rate determining. 5-Endo-trig cyclization giving 4-hyndrindanone is disfavored by Baldwin's rules, but B₁₂(COOH)₆–PLL films in microemulsions still gave 24% yields at relatively high turnover numbers. It is possible that proton donors at film reaction sites protonate a carbanion intermediate to give competitive product 2-propyl-2-cyclohexen-1-one to decrease the yield of 4-hyndrindanone.     

Fragmentation of vitamin B12 in aerosol matrix-assisted laser desorption ionization

Aerosol matrix-assisted laser desorption ionization (MALDI) with a reflection time-of-flight mass spectrometer was used to study fragmentation of vitamin B₁₂. Six MALDI matrices were used: 2,5-di-hydroxy benzoic acid (gentisic acid), 4-nitroaniline, 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapic acid), 3,4-di-hydroxy cinnamic acid (caffeic acid), trans-4-hydroxy-3-methoxy cinnamic acid (ferulic acid), and α-cyano-4-hydroxy cinnamic acid (4-HCCA). Mass spectra were obtained with a 355-nm pulsed Nd:YAG laser at irradiances between 0. 1 and 5 GW/cm² (between 3- and 150-mJ pulse energy). Loss of CN was a major product of prompt ion source fragmentation and the ratio of fragmented to intact analyte was found to be strongly dependent on matrix and weakly dependent on laser irradiance. Additionally, free cobalt ions and cobalt ions bound to small methanol clusters were observed in the mass spectra. The cobalt removal from the corrin ring of vitamin B₁₂ results from direct photon absorption by vitamin B₁₂, but is enhanced by the presence of matrix.     

Voltammetric determination of vitamin B<Subscript>6</Subscript> (pyridoxine) at a graphite screen-printed electrode modified with graphene oxide/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>@SiO<Subscript>2</Subscript> nanocomposite

The preparation and study of electrochemical properties of a graphite screen-printed electrode (SPE) modified with the GO/Fe₃O₄@SiO₂ (GO is graphene oxide) nanocomposites are described. The morphologies of the GO/Fe₃O₄@SiO₂ nanocomposites were examined by scanning electron microscopy. The electrochemical oxidation of vitamin B₆ (pyridoxine) on SPE modified with the GO/Fe₃O₄@SiO₂ nanocomposite was investigated by cyclic voltammetry, differential pulse voltammetry, and chronoamperometry. Under optimum conditions (pH 7.0), the vitamin B₆ oxidation at the surface of the modified SPE occurs at a potential about 190 mV less positive than that at the unmodified SPE. A linear voltammetric response for vitamin B₆ was obtained in the concentration range 1.0?10 ⁶—9.0?10 ⁴ mol L^–1 with a detection limit of 5.2?10 ⁷ mol L^–1 using differential pulse voltammetry. The developed sensor was also successfully applied for determination of trace level of vitamin B₆ in both the standard vitamin B₆ sample and biological samples (urine).     

Dipstick based immunochemiluminescence biosensor for the analysis of vitamin B12 in energy drinks: A novel approach

In this article, we describe a dipstick based immunochemiluminescence (immuno-CL) biosensor for the detection of vitamin B₁₂ in energy drinks. The method is a direct competitive type format involving the immobilization of vitamin B₁₂ antibody on nitrocellulose membrane (NC) followed by treatment with vitamin B₁₂ and vitamin B₁₂–alkaline phosphatase conjugate to facilitate the competitive binding. The dipstick was further treated with substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate (CDP-Star) to generate chemiluminescence (CL). The number of photons generated was inversely proportional to the vitamin B₁₂ concentration. After systematic optimization, the limit of detection was 1 ng mL⁻¹. The coefficient of variation was below 0.2% for both intra- and inter-assay precision. Vitamin B₁₂ was extracted from energy drinks with recovery ranged from 90 to 99.4%. Two different energy drinks samples were analyzed, and a good correlation was observed when the data were compared with a reference enzyme linked immuno sorbent assay (ELISA) method. The developed method is suitable for an accurate, sensitive, and high-throughput screening of vitamin B₁₂ in energy drinks samples. The dipstick technique based on immuno-CL is suitable for the detection of several analyte in food and environmental samples.     

Nicotinamide solvation state in aqueous dimethyl sulfoxide

The solvation state of biologically active compound vitamin B₃, viz., 3-pyridinecarboxamide, in an aqueous-dimethyl sulfoxide solvent of a variable composition was studied by ¹H and ¹³C NMR and IR spectroscopy. Below X _DMSO ?0.65 molar fraction, the solvation of the N heteroatom due to hydrogen bonds with water molecules weakens. At X _DMSO > 0.65 molar fraction, almost no changes are observed in the solvate state of the N heteroatom. The ¹H NMR spectra indicate that the degree of conjugation of the carbamide group with the heterocycle increases with an increase in the DMSO concentration. The structures of the dimethyl sulfoxide and mixed aqueous-dimethyl sulfoxide solvates of nicotinamide were optimized by the B3LYP/6311++(DP) method, and their ¹³C chemical shifts (GIAO) and IR spectra were obtained. According to the IR spectroscopic data, the number of hydrogen bonds involving the carbamide group decreases on going from H₂O to DMSO.     

Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV

Background: In order to study the biosynthesis of vitamin B₁₂, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates.Results: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-l-methionine were included in the incubation, the product was precorrin-4. Both precorrin-3B and precorrin-4 are known precursors of vitamin B₁₂ and their availability has allowed new mechanistic studies of enzymic transformations.Conclusions: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B₁₂. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B₁₂ biosynthesis.