Oxidation Of Vanillin Research Articles

Vanillin production from lignin oxidation in a batch reactor

Vanillin production from lignin oxidation is a biomass-based process that employs a by-product of the pulp and paper industry and air to obtain a high-added value compound. However, lignin is an organic polymer with a structure that depends strongly on the source and the conditions to obtain vanillin should be adjusted for different samples. The objective of this work is to establish a fast and standard protocol to characterize lignin conversion to vanillin by batch oxidation. The experimental technique is coupled with a mathematical model that allows us to fit the data and determine kinetic rate constants under non-isothermal conditions. Two examples of vanillin oxidation with very different lignin sources are presented. The results revealed vanillin yields with respect to the lignin mass ranging from 3.5% to 7.6% for the high and low-molecular weight lignins, respectively.

Oxidation of vanillin by peroxomonosulphate-thermodynamic and kinetic investigation

The oxidation of vanillin by peroxomonosulphate (PMS) in acetic acid–sodium acetate buffered medium was carried out at 308 K. The rate was first order with respect to [vanillin] and [PMS]. The rate increased with increase in pH and the rate was too fast to be measured at pH 5.2. The rate increased with increase in [acetate] and the plot of kobs versus [acetate] was a straight line with positive intercept. Variation of ionic strength had no effect on the rate of the reaction. Effects of polarity were studied with five different solvents and in all the cases, log kobs versus 1/ɛ were linear with negative slope. The reaction had been carried out at four different temperatures and the activation and thermodynamic parameters were calculated. The product of oxidation was confirmed as vanillic acid by IR, 1H NMR and GC-MS spectral analysis. Based on the results obtained a reaction scheme had been proposed and the rate law was derived.

Inhibitory effects of flavonoids on aldehyde oxidase activity

Flavonoids are an important group of natural compounds that can interfere with the activity of some enzymes. In this study, effects of various flavonoids on aldehyde oxidase (AO) activity were evaluated in vitro. AO was partially purified from guinea pig liver. The effects of 12 flavonoids from three subclasses of flavon-3-ol, flavan-3-ol and flavanone on the oxidation of vanillin and phenanthridine as substrates of AO and xanthine as a substrate of xanthine oxidase (XO) were investigated spectrophotometrically. Among the 12 flavonoids, myricetin and quercetin were the most potent inhibitors of both AO and XO. In general, the oxidation of vanillin was more inhibited by flavonoids than that of phenanthridine. Almost all of the flavonoids inhibited AO activity more potently than XO, which was more evident with non-planner flavanols. A planner structure seems to be essential for a potent inhibitory effect and any substitution by sugar moieties reduces the inhibitory effects. This study could provide a new insight into AO natural inhibitors with potential to lead to some food-drug interactions.

Thermal oxidation of vanillin affects its antioxidant and antimicrobial properties

Accelerated oxidation of vanillin was studied by isothermal and non-isothermal differential scanning calorimetry (DSC) in model solutions. Exothermic peaks of DSC thermograms, due to the oxidation of vanillin, were observed. Vanillin oxidation to vanillic acid was confirmed by the detection of vanillic acid in heated vanillin samples using GC–MS. The effect of temperature on vanillin oxidation was studied by conducting DSC experiments with pure vanillin at several different final temperatures and by subsequent determination of vanillin and vanillic acid by GC–MS. Furthermore, the DPPH free radical assay was done on DSC-treated samples as well as on mixtures of vanillin–vanillic acid. The radical-scavenging activity of the samples was increased along with the vanillic acid content. Additionally, the antimicrobial activities and the minimum inhibitory concentrations (MIC) of solutions containing vanillin and vanillic acid against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Enterobacter aerogenes, Escherichia coli and Yersinia enterocolitica were determined by the agar well-diffusion method. All tested samples exhibited inhibitory activity against all of the bacteria. Yet, the higher the vanillic acid concentration, the lower was the MIC of the samples. It is concluded that the thermal treatment of vanillin-containing food may lead to products with improved antioxidant and antimicrobial properties.

Environmentally benign liquid phase oxidation of vanillin over copper containing ternary hydrotalcites

Liquid phase oxidation of vanillin to vanillic acid is carried out over copper containing ternary hydrotalcites using water as a solvent and H2O2 as oxidant at 313 K. The catalytic activity and yield of these samples showed very high conversion and selectivity under environmental friendly conditions.

Oxidation of Vanillin by a New Oxidant Diperiodatoargentate(III) in Aqueous Alkaline Medium

The kinetics of oxidation of vanillin (VAN) by diperiodatoargentate (III) (DPA) in alkaline medium at a constant ionic strength of 1.0 mol dm-3 was studied spectrophotometrically. The reaction between DPA and vanillin in alkaline medium exhibits 1:1 stoichiometry (vanillin:DPA). The reaction is first order in [DPA] and has less than unit order both in [VAN] and [alkali]. A decrease in the dielectric constant of the medium increases the reaction rate. An increase in periodate concentration has no effect on the rate. The oxidation reaction in alkaline medium has been shown to proceed via a DPA−vanillin complex, which decomposes slowly in a rate-determining step to give the products. The main products were identified by spot test, IR, and MS studies. The reaction constants involved in the different steps of the mechanism are calculated. The activation parameters with respect to the slow step of the mechanism are computed and discussed. Thermodynamic quantities are also determined.

Oxidation of vanillin by diperiodatocuprate(III) in aqueous alkaline medium: A kinetic and mechanistic study by stopped flow technique

AbstractThe kinetics of oxidation of vanillin (VAN) by diperiodatocuprate(III) (DPC) in alkaline medium at a constant ionic strength of 0.50 mol dm−3 was studied spectrophotometrically. The reaction between DPC and vanillin in alkaline medium exhibits 1:2 stoichiometry (vanillin: DPC). The reaction is of first order in [DPC] and has less than unit order in both [VAN] and [alkali]. Intervention of free radicals was observed in the reaction. Increase in periodate concentration decreases the rate. The oxidation reaction in alkaline medium has been shown to proceed via a monoperiodatocuprate(III)–vanillin complex, which decomposes slowly in a rate‐determining step followed by other fast steps to give the products. The main products were identified by spot test, IR, and MS studies. The reaction constants involved in the different steps of the mechanism are calculated. The activation parameters with respect to slow step of the mechanism are computed and discussed, and thermodynamic quantities are also determined. © 2007 Wiley Periodicals, Inc. 39: 236–244, 2007

Kinetics and Mechanism of the Oxidation of Vanillin by Hexacyanoferrate(III) in Aqueous Alkaline Medium

The title reaction was investigated in aqueous alkaline medium. A first-order dependence in hexacyanoferrate(III) concentration and a fractional order in both vanillin and alkali were obtained at the concentrations studied. The added product, hexacyanoferrate(II), had a retarding effect on the rate of reaction. Ionic strength and dielectric constant of the reaction medium have little effect on the reaction rate. The effect of temperature on the rate of reaction has also been studied and activation parameters have been evaluated. A mechanism based on the experimental results is proposed and the rate law is derived. The reaction constants are calculated and used to regenerate the kobs values, which are compared with the experimental values.

Oxidation of Vanillin and Related Compounds by Sodium N-Chloro-p-toluenesulfonamide in Acid Medium: A Kinetic and Mechanistic Approach

The kinetics of oxidation of vanillin, vanillyl alcohol, vanillylamine hydrochloride and vanillyl mandelic acid (hereafter abbreviated as substrate) by N-chloro-p-toluenesulfonamide or chloramine–T (CAT) in HClO4 medium has been investigated at 30±0.1°C. The oxidation reaction follows identical kinetics with first-order in [CAT]o, fractional order in [substrate]o, and inverse fractional-order in [H+]. Variation of ionic strength and addition of the reaction product, p-toluenesulfonamide, or halide ions had no significant effect on reaction rate. Decrease in dielectric constant of the medium decreases the rate. The solvent isotope effect was studied in D2O medium. The reaction does not induce polymerization of acrylonitrile. Michaelis-Menten type of kinetics has been proposed and activation parameters for the rate-limiting step as well as overall reaction have been computed. The decomposition constants of substrate-oxidant complexes have been evaluated. Under comparable experimental conditions, the rate of oxidation increases in the following order vanillin>vanillylamine hydrochloride>vanillyl alcohol>vanillylmandelic acid. An isokinetic relationship is observed with β=339 K, indicating enthalpy as a controlling factor. Vanillic acid was identified as the oxidation product of each substrate and was confirmed by IR and GC-MS data. A general mechanism involving the formation of a complex between substrate and the conjugate acid (CH3C6H4SO2NHCl) of the oxidant has been proposed. The derived rate law is in agreement with the experimental results.

Functional analysis of the vanillin pathway in a vdh-negative mutant strain of Pseudomonas fluorescens AN103

The catabolism of ferulic acid in Pseudomonas fluorescens AN103 proceeds via vanillin, vanillic acid, and protocatechuic acid. The inactivation of the vanillin dehydrogenase of P. fluorescens AN103, which catalyzes the oxidation of vanillin to vanillic acid, was pursued as an approach for developing a biotechnological process for the production of biovanillin. In order to inactivate the vdh gene, a plasmid construction was made in which an omega element conferring resistance to kanamycin (ΩKm) was inserted into a truncated vdh gene (Δ vdh). Subsequently, this construct was used to replace the functional vdh gene with the disrupted Δ vdhΩKm in P. fluorescens AN103 by homologous recombination using a triparental mating approach. The successful inactivation of the gene was confirmed both by spectrophotometric and specific activity staining of crude cell extracts after native electrophoresis. However, inactivation of the vdh gene in P. fluorescens AN103 did not result in the expected accumulation of vanillin in media despite the absence of other enzymes exhibiting vanillin dehydrogenase activity. Further characterization showed that the P. fluorescens AN103 vdhΩKm mutant was unable to grow on vanillin as well as on ferulic acid as a sole carbon source. The inability to degrade ferulic acid was attributed to the lack of 4CL activity resulting from the inactivation of the vdh gene.

Vanillin production by recombinant strains of Escherichia coli

Vanillin production from ferulate was studied using different recombinant strains of Escherichia coli. To prevent the occurrence of aerobic conditions and then possible product oxidation, tests were performed in Erlenmeyer flasks under mild mixing (150 rpm). Among other transformants, E. coli JM109(pBB1) appeared to be the best vanillin producer, being able to convert no less than 95% of starting ferulate to the product within 1h. This yield decreased down to 72% after 72h, likely because of a non-specific oxidase activity responsible for vanillin oxidation to vanillate.

Purification and characterization of a microbial dehydrogenase: a vanillin:NAD(P)+ oxidoreductase.

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native Mr value estimated by gel-filtration chromatography gave a value of 210,000. The enzyme made use of NAD+ less effectively than NADP+. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD+ or NADP+. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.

Radical scavenging properties of a flavouring agent–Vanillin

Using pulse radiolysis technique, the one-electron oxidation of vanillin (V-OH) with azide radicals, at pH 6 and 9 resulted in the formation of vanillin phenoxyl radical with k = 6.7 × 107 and 2.5 × 109 dm3 mol-1 s-1, respectively. The transient absorption spectra of the vanillin phenoxyl radical (V-O•) formed either at pH 6 or 9, showed a λmax at 410 nm. At pH 5, the •OH radicals seem to form an adduct with vanillin, λmax at 430 nm and k(•OH + V-OH) = 3.3 × 109 dm3 mol-1 s-1, while at pH 9, the •OH radical reaction resulted in the formation of vanillin phenoxyl radical with λmax at 410 nm and k(•OH + V-O-) = 6.6 × 109 dm3 mol-1 s-1. The reactivity of NO2•radicals with vanillin is lower by orders of magnitude signifying an incomplete reaction. In general, the rate constants for the reaction of •OH, N•, NO• radicals with vanillin were higher at pH 9 than at the lower pH. Its reactivity with other one-electron oxidants like CCl3OO•, CHCl2OO• and CH• radicals and the ability to chemically repair tryptophanyl and guanosyl radicals with k = 1.5 - 4 × 107 dm3 mol-1 s-1 indicate its antioxidative behaviour.

Biphasic Sonoelectroanalysis: Simultaneous Extraction from, and Determination of Vanillin in Food Flavoring

Biphasic sonoelectroanalysis is employed in the detection of vanillin (4-hydroxy-3-methoxybenzaldehyde). Ethyl acetoacetate is characterized as an electrochemical and sonoelectrochemical solvent and it is shown that quantitative electrochemical measurements can be made in this solvent of relatively low dielectric constant. The electrochemical oxidation of vanillin in this medium is shown to give oxidative linear sweep voltammetric signals sensitive to microadditions. In addition, the near reversible kinetics of vanillin in ethyl aceto-acetate facilitate sensitive square-wave voltammetry. Electrode passivation leads to typically diminishing peak heights for the same concentration of vanillin, but by employing ultrasound between each scan, peak heights are maintained by quantitative depassivation of the electrode surface. Microadditions of 0.05 mM vanillin to ethyl acetoacetate yield a linear calibration plot with r=0.9987 and a detection limit of 0.016 mM. The determination of vanillin in natural vanilla essence is then achieved by the exploitation of biphasic sonoelectrovoltammetry in aqueous ethanolic vanilla pod extract and ethyl acetoacetate. In contrast with silent voltammetry, ultrasound facilitates emulsification and extraction of vanillin in the extract permitting an analytical square-wave voltammetric signal to be obtained. Microadditions to two separate samples of vanilla essence facilitate quantification of vanillin in the extract. Close agreement with a blind analysis of the samples using HPLC-UV is observed with a limit of detection in the biphasic medium of 0.020 mM. Biphasic sonoelectroanalysis removes the need for sample degradation or a separation step, which would lengthen and complicate the analytical protocol. It can therefore be concluded that biphasic sonoelectroanalysis demonstrates an attractive alternative to currently accepted techniques.

Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene.

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD(+)-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (omega Km) or gentamycin (omega Gm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3'-O-phosphotransferase gene and the gentamycin-3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by omega Km or omega Gm, and the functional vdh gene was replaced by vdh omega Km or vdh omega Gm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HR vdh omega Km, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identify of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase.

Convenient Preparation and Quantification of 5,5'-Diferulic Acid.

5,5'-Diferulic acid (5,5'-DFA), which is one of the cross-linking residues in plant cell walls, was prepared by using a facile procedure. The phenol oxidation of vanillin with FeCl3 gave divanillin, which was further devoted to a Perkin reaction to give the desired product. It was found on (13)C-NMR that the chemical shift of C-5 of ferulic acid (FA) clearly shifted downfield, when this carbon is quaternarized by the oxidative dimerizaton to 5,5'-DFA, while those of other carbons of 5,5'-DFA are fundamentally same as those of FA. Also prepared was [9,9'-(13)C2]-5,5'-DFA, which was proved to be a good internal standard on GC-MS quantification of endogenous 5,5'-DFA from plant tissues.

Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly-isolated strain of Pseudomonas fluorescens.

A soil bacterium, designated Pseudomonas fluorescens AN103, was isolated based on its ability to grow on ferulic acid as a sole source of carbon and energy. In addition, this strain was found to metabolize a number of related phenolic substrates which contained a hydroxyl group at the para position of the aromatic ring. During growth on ferulic acid, transient accumulation of vanillic acid and trace amounts of protocatechuic acid were detected in the culture medium. Washed cells grown on ferulic acid readily oxidized vanillin, vanillic acid and protocatechuic acid, the three putative intermediates of the metabolic pathway. The side-chain cleavage of ferulic acid to produce vanillin was demonstrated in vitro for the first time and this enzyme-catalysed reaction was shown to have an essential requirement for CoASH, ATP and MgCl2. This conversion involved a two-step process involving a CoA ligase followed by the side-chain cleavage. The addition of NAD increased the oxidation of vanillin to vanillic acid and had an overall effect of increasing the rate of ferulic acid cleavage. The application of 13C-NMR studies in vitro revealed acetyl-CoA as the C2 side-chain cleavage product. High levels of inducible ferulate-CoA ligase and NAD-linked vanillin dehydrogenase were detected and a novel pathway for ferulic acid metabolism in this organism is proposed.

Oxidation behaviour of vanillin in dairy products

The stability of vanillin in dairy products was investigated by means of highperformance liquid chromatography. While vanillin was oxidised to vanillic acid in fresh and pasteurised milk, it was stable in all other milk products investigated (UHT milk, pasteurised and UHT cream, yoghurt, curd and butter). The oxidation of vanillin to vanillic acid was shown to be pH-dependent. The relatively thermolabile enzyme xanthine oxidase was shown to be partly responsible for the formation of vanillic acid. However, peroxidase, which is also present in dairy products, did not lead to vanillic acid. Oxidation to the dimeric product, divanillin, occurred in the presence of peroxidase and hydrogen peroxide. The oxidation rate of synthetic vanillin to vanillic acid was very similar to that of vanillin from natural vanilla extracts or vanilla capsules. Both were shown to be stable in dairy products for subvention, such as butter and cream.

Kinetics of vanillin oxidation

AbstractVanillin can be produced by oxidation of kraft lignin, with air, in alkaline medium. The optimal conditions for vanillin production strongly depend on pH and temperature. This paper addresses the effect of temperature and pH on vanillin degradation by oxidation. Experiments were carried out in a wide range of vanillin concentration, oxygen partial pressure, temperature and pH. Simple models are proposed to explain the observed rate of vanillin consumption under conditions of high alkalinity (pH>12) and lower alkalinity (pH<12). At pH>12,the reaction rate of vanillin oxidation is first order in dissolved oxygen concentration [O2] and in vanillin concentration [C], i.e., (−rC) ∝[O2][C]; at pH<12, the rate is zero order in oxygen concentration and second order in vanillin concentration, i.e., (−rC)∝f(pH)[C]2.

Oxidation of vanillin by Serratia marcescens

A Serratia marcescens strain quantitatively converted vanillin (0·1%, w/v) to vanillic acid, which exerted an inhibitory effect on bacterial growth. Vanillic acid producing activity was found in cell-free extracts of cultures grown in media with and without vanillin, when glucose was the substrate.