Vanilla Flavour Production Through Biotransformation Using Capsicum frutescens Root Cultures

Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml^-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed products. Moreover, vanillin accumulation was recorded as 24.7 μM on day 6 after precursor addition, which was 1.5 times higher than in cultures fed with ferulic acid (2 mM) alone. When ferulic acid was fed along with β-cyclodextrin (1 mM each) to cultures growing in a three-litre bubble column bioreactor, the maximum vanillin production of 10.7 μM was obtained; other vanilla flavour metabolites were also formed after 9 days of precursor addition. Root cultures could also biotransform protocatechuic aldehyde wherein a maximum vanillin production of 7.9 μM was recorded on day 6 after precursor addition. The bioconversion efficiency was observed to be 5–7% in case of ferulic acid fed cultures and 3.2% in case of protocatechuic aldehyde fed cultures suggesting the possible channelling of precursors to alternate biosynthetic pathways such as lignin.     

Biotransformation of protocatechuic aldehyde and caffeic acid to vanillin and capsaicin in freely suspended and immobilized cell cultures of Capsicum frutescens

Freely suspended cells and immobilized cell cultures of Capsicum frutescens Mill. were treated with phenylpropanoid intermediates--protocatechuic aldehyde and caffeic acid to study their biotransformation ability. It was found that externally fed protocatechuic aldehyde and caffeic acids were biotransformed to vanillin and capsaicin. It was noted that this culture biotransformed externally fed protocatechuic aldehyde to vanillin more than its conversion to capsaicin, whereas, caffeic acid-treated cultures accumulated more capsaicin than vanillin. The maximum accumulation of vanillin (5.63 mg l(-1)) and capsaicin (3.83 mg l(-1)) was recorded on the 6th and 15th day, respectively in immobilized C. frutescens cell cultures treated with protocatechuic aldehyde, which was 1.8 and 1.4 times higher than in protocatechuic aldehyde-treated freely suspended cell cultures. Caffeic acid-treated immobilized C. frutescens cell cultures accumulated maximum vanillin and capsaicin at 2.68 and 3.03 mg l(-1) culture, respectively, on the 9th and 12th day, which was 1.65 and 1.33 times over freely suspended cultures treated with caffeic acid. The addition of S-adenosyl-L-methionine, a methyl donor, to protocatechuic aldehyde-treated immobilized C. frutescens cell cultures, resulted in accumulation of vanillin (14.08 mg l(-1)) on the 4th day, which was 2.5-fold higher than that in cultures treated with protocatechuic aldehyde alone, suggesting the influence of S-adenosyl-L-methionine on O-methylation of protocatechuic aldehyde, resulting in more vanillin accumulation. The increase in vanillin accumulation was well correlated with an increase in specific activity of caffeic acid O-methyltransferase in protocatechuic aldehyde and S-adenosyl-L-methionine-treated immobilized C. frutescens cell cultures. This study also provides an example for an alternative route to formation of vanillin by C. frutescens cell cultures.     

Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.     

In vitro capsaicin production by immobilized cells and placental tissues of Capsicum annuum L. grown in liquid medium

Capsaicin, from green pepper fruits is used in formulated foods and in pharmaceuticals. Cell cultures of Capsicum annuum L. were obtained from seedlings on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. In vitro-grown cells and placental tissues from fruits were immobilized in calcium alginate. Immobilized cells and placental tissues produced capsaicin which leached out into the medium. Immobilized placental tissue exhibited greater potentiality for capsaicin synthesis than immobilized cells. Production reached a level of 1345 μg capsaicin g⁻¹ of immobilized placenta on the 14th day of culture. Production of capsaicin, on replenished nutrient medium in immobilized placenta was 2400 μg on the 30th day. Ferulic acid fed to immobilized placenta at 2.5 mM level increased capsaicin production by 2-fold by the 5th day of the culture period. Of the elicitors used, curdlan was effective on capsaicin production in immobilized cells. Extracts of Aspergillius niger and Rhizopus oligosporus stimulated capsaicin production in immobilized placental tissues.     

Vanillin production using metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> under non-growing conditions

Background  

Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details.     

Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with β-cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- β-cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 μg 100 ml -1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- β-cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- β-cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 μg 100 ml -1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- β-cyclodextrin alone.     

Metabolism of cinnamic, p-coumaric, and ferulic acids by Streptomyces setonii

Streptomyces setonii strain 75Vi2 was grown at 45 degrees C in liquid media containing yeast extract and trans-cinnamic acid, p-coumaric acid, ferulic acid, or vanillin. Gas chromatography, thin-layer chromatography, and mass spectrometry showed that cinnamic acid was catabolized via benzaldehyde, benzoic acid, and catechol; p-coumaric acid was catabolized via p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and protocatechuic acid; ferulic acid was catabolized via vanillin, vanillic acid, and protocatechuic acid. When vanillin was used as the initial growth substrate, it was catabolized via vanillic acid, guaiacol, and catechol. The inducible ring-cleavage dioxygenases catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected with an oxygen electrode in cell-free extracts of cultures grown in media with aromatic growth substrates and yeast extract.     

Dynamics of phenolic acids and lignin accumulation in metal-treated Matricaria chamomilla roots

Phenylalanine ammonia-lyase (PAL) activity, 11 phenolic acids and lignin accumulation in Matricaria chamomilla roots exposed to low (3 μM) and high (60 and 120 μM) levels of cadmium (Cd) or copper (Cu) for 7 days were investigated. Five derivatives of cinnamic acid (chlorogenic, p-coumaric, caffeic, ferulic and sinapic acids) and six derivatives of benzoic acid (protocatechuic, vanillic, syringic, p-hydroxybenzoic, salicylic acids and protocatechuic aldehyde) were detected. Accumulation of glycoside-bound phenolics (revealed by acid hydrolysis) was enhanced mainly towards the end of the experiment, being more expressive in Cu-treated roots. Interestingly, chlorogenic acid was extremely elevated by the highest Cu dose (21-fold higher than control) suggesting its involvement in antioxidative protection. All compounds, with the exception of chlorogenic acid, were detected in the cell wall bound fraction, but only benzoic acids were found in the ester-bound fraction (revealed by alkaline hydrolysis). Soluble phenolics were present in substantially higher amounts in Cu-treated roots and more Cu was retained there in comparison to Cd. Cu strongly elevated PAL activity (by 5.4- and 12.1-fold in 60 and 120 μM treatment, respectively) and lignin content (by 71 and 148%, respectively) after one day of treatment, indicating formation of a barrier against metal entrance. Cd had slighter effects, supporting its non-redox active properties. Taken together, different forms of phenolic metabolites play an important role in chamomile tolerance to metal excess and participate in active antioxidative protection.     

Enzymatic oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared Guinea pig liver slices.

BACKGROUND/AIMS: The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared liver slices has not been previously reported. The present investigation compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activities in the oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared liver slices. METHODS: Vanillin, isovanillin or protocatechuic aldehyde was incubated with liver slices in the presence/absence of specific inhibitors of each enzyme, followed by HPLC. RESULTS: Vanillin was rapidly converted to vanillic acid. Vanillic acid formation was completely inhibited by isovanillin (aldehyde oxidase inhibitor), whereas disulfiram (aldehyde dehydrogenase inhibitor) inhibited acid formation by 16% and allopurinol (xanthine oxidase inhibitor) had no effect. Isovanillin was rapidly converted to isovanillic acid. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. Protocatechuic aldehyde was converted to protocatechuic acid at a lower rate than that of vanillin or isovanillin. Allopurinol only slightly inhibited protocatechuic aldehyde oxidation, isovanillin had little effect, whereas disulfiram inhibited protocatechuic acid formation by 50%. CONCLUSIONS: In freshly prepared liver slices, vanillin is rapidly oxidized by aldehyde oxidase with little contribution from xanthine oxidase or aldehyde dehydrogenase. Isovanillin is not a substrate for aldehyde oxidase and therefore it is metabolized to isovanillic acid predominantly by aldehyde dehydrogenase. All three enzymes contribute to the oxidation of protocatechuic aldehyde to its acid.     

Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of Bacillus coagulans

A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and ¹H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.     

Kainate-induced stimulation of amino acid release from primary astroglial cultures of the rat hippocampus

The effects of the excitatory amino acid analogs kainate (KA) and N-methyl- -aspartate (NMDA) on release of amino acids from astrocytes in primary culture were investigated. Under basal conditions, glutamine was present in the medium at 15 μM. The levels of serine and taurine were 1.5 and 2.0 μM, respectively, while the concentration of other amino acids was below 1 μM. At 10 μM, KA did not affect amino acid release, whereas 100 μM KA enhanced glutamine release by 34% and taurine release by 85%. At 1 mM, KA stimulated the release of all amino acids measured. However, while most amino acids increased by 50–150%, glutamate and aspartate were elevated by more than 3000%. The effect of KA was greatly reduced by 1 mM kynurenate, an excitatory amino acid receptor antagonist. 1 mM NMDA did not stimulate amino acid release from the cultures. The results indicate that astrocytes are endowed with KA-receptive sites, but they do not seem to possess NMDA receptors.     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Dissimilation of ferulic acid byBacillus subtilis

Bacillus subtilis utilized ferulic acid and its intermediates vanillin, vanillic acid, and protocatechuic acid as sole carbon source. The enzymes of the ferulic acid degradative pathway such as deacetylase, vanillin oxidase, vanillate-o-demethylase, and protocatechuate 3,4-dioxygenase were inducible in nature. Concentration of the inducer profoundly influenced the induction of the enzymes involved in ferulic acid dissimilation.     

Towards a high-yield bioconversion of ferulic acid to vanillin

Natural vanillin is of high interest in the flavor market. Microbial routes to vanillin have so far not been economical as the medium concentrations achieved have been well below 1 g l⁻¹. We have now screened microbial isolates from nature and known strains for their ability to convert eugenol or ferulic acid into vanillin. Ferulic acid, in contrast to the rather toxic eugenol, was found to be an excellent precursor for the conversion to vanillin, as doses of several g l⁻¹ could be fed. One of the isolated microbes, later identified as Pseudomonas putida, very efficiently converted ferulic acid to vanillic acid. As vanillin was oxidized faster than ferulic acid, accumulation of vanillin as an intermediate was not observed. A completely different metabolic flux was observed with Streptomyces setonii. During the metabolism of ferulic acid, this strain accumulated vanillic acid only to a level of around 200 mg l⁻¹ and then started to accumulate vanillin as the principal metabolic overflow product. In shake-flask experiments, vanillin concentrations of up to 6.4 g l⁻¹ were achieved with a molar yield of 68%. This high level now forms the basis for an economical microbial production of vanillin that can be used for flavoring purposes. Received: 15 October 1998 / Received revision: 13 January 1999 / Accepted: 18 January 1999     

Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of bacillus coagulans

A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and ¹H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.     

Bioengineering of the Plant Culture of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> with Vanillin Synthase Gene for the Production of Vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.     

刺楸中酚性成分研究

采用常压柱色谱、半制备型高效液相色谱以及制备型薄层色谱等色谱技术结合重结晶等分离纯化手段,从刺楸根中分离得到6个酚性化学成分,通过理化性质和波谱分析鉴定所得化合物分别为苔色酸乙酯(1),2-羟基-4-甲氧基-3,6-二甲基苯甲酸(2),香草醛(3),原儿茶醛(4),原儿茶酸(5)和3-甲氧基苯甲醛(6).所有化合物均为首次从该属植物中获得.     

Biotransformation of ferulic acid to acetovanillone using Rhizopus oryzae

Microbial transformation of ferulic acid to acetovanillone was studied using growing cells of Rhizopus oryzae. Ferulic acid was added to the growing medium (0.5 g L^-1) and incubated for 12 days. The progress of formation of metabolites was monitored by GC and GC-MS after extraction with ethyl acetate. The major metabolite was acetovanillone with minor metabolites formed, such as dihydroferulic acid, coniferyl alcohol and dihydroconiferyl alcohol. Traces of metabolites (≤1-3%), such as vanillin, vanillyl alcohol, vanillic acid and phenyl ethyl alcohol, were also produced. Formation of 4-vinyl guaiacol increased from day 1 (12.4%), reaching a maximum on day 4 (31.7%), and reducing to a minimum on day 12 (3.1%). The formation of acetovanillone increased only from day 2 onward, and reached a maximum (49.2%) on day 12. The optimum concentration of ferulic acid to be added into the medium was found to be only 0.5 g L^-1, as any increase in concentration (0.75 and 1.0 g L^-1) precipitated the precursor, resulting in no further degradation.     

Potential of Rhodococcus strains for biotechnological vanillin production from ferulic acid and eugenol

The potential of two Rhodococcus strains for biotechnological vanillin production from ferulic acid and eugenol was investigated. Genome sequence data of Rhodococcus sp. I24 suggested a coenzyme A-dependent, non-β-oxidative pathway for ferulic acid bioconversion, which involves feruloyl–CoA synthetase (Fcs), enoyl–CoA hydratase/aldolase (Ech), and vanillin dehydrogenase (Vdh). This pathway was proven for Rhodococcus opacus PD630 by physiological characterization of knockout mutants. However, expression and functional characterization of corresponding structural genes from I24 suggested that degradation of ferulic acid in this strain proceeds via a β-oxidative pathway. The vanillin precursor eugenol facilitated growth of I24 but not of PD630. Coniferyl aldehyde was an intermediate of eugenol degradation by I24. Since the genome sequence of I24 is devoid of eugenol hydroxylase homologous genes (ehyAB), eugenol bioconversion is most probably initiated by a new step in this bacterium. To establish eugenol bioconversion in PD630, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was expressed in PD630 together with coniferyl alcohol dehydrogenase (calA) and coniferyl aldehyde dehydrogenase (calB) genes from Pseudomonas sp. HR199. The recombinant strain converted eugenol to ferulic acid. The obtained data suggest that genetically engineered strains of I24 and PD630 are suitable candidates for vanillin production from eugenol.     

Ginkgo Biloba Extract EGB 761 or Trolox C Prevent the Ascorbic Acid/FE2+ Induced Decrease in Synaptosomal Membrane Fluidity

The ability of synaptosomes, prepared from striata, to take up ³H-dopamine declined rapidly during incubation at 37°C, in an oxygenated Krebs-Ringer medium with 0.1 mM ascorbic acid. Ascorbic acid was responsible for this decrease. Its effectiveness after a 60 min incubation was concentration dependent from 1 μM and virtually complete for 0.1 mM. Furthermore, a decrease of synaptosomal membrane fluidity was revealed by measurements of fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene. This decrease was potentiated by Fe²⁺ ions (1 μM). In contrast, it was prevented by the Fe²⁺ ion chelator, desferrioxamine (0.1 mM), by the Ginkgo biloba extract EGb 761 [2-16 μg/ml], as well as by the flavonoid quercetin (0.1 μM). This preventive effect was shared by trolox C (from 0.1 mM). It is concluded that peroxidation of neuronal membrane lipids induced by ascorbic acid/Fe²⁺ is associated with a decrease in membrane fluidity which, in turn, reduces the ability of the dopamine transporter to take up dopamine.